p gingivalis  (ATCC)


Bioz Manufacturer Symbol ATCC manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    ATCC p gingivalis
    COR388 target engagement and dose-dependent effects on brain P. <t>gingivalis</t> , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p gingivalis/product/ATCC
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    p gingivalis - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    2) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    3) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    4) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    5) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    6) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    7) Product Images from "Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function ▿"

    Article Title: Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00225-10

    Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo . Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis ). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.
    Figure Legend Snippet: Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo . Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis ). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Fluorescence, Microscopy, Staining

    Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.
    Figure Legend Snippet: Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Isolation

    Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis ). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m / z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m / z ). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m / z ) are not recovered in these lipid isolates.
    Figure Legend Snippet: Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis ). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m / z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m / z ). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m / z ) are not recovered in these lipid isolates.

    Techniques Used: Mass Spectrometry, Dominant Negative Mutation

    Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10 3 versus [263.0 ± 25.3] × 10 3 ; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79 ), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71 ).
    Figure Legend Snippet: Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10 3 versus [263.0 ± 25.3] × 10 3 ; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79 ), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71 ).

    Techniques Used: Concentration Assay, Ethidium Homodimer Assay, Staining, Flow Cytometry, Cytometry, Quantitation Assay

    Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids ( ) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.
    Figure Legend Snippet: Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids ( ) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.

    Techniques Used: Inhibition, Quantitation Assay, Staining, Cell Culture

    Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2 −/− ) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.
    Figure Legend Snippet: Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2 −/− ) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.

    Techniques Used: Mouse Assay, Staining, Inhibition

    8) Product Images from "Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function ▿"

    Article Title: Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00225-10

    Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo . Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis ). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.
    Figure Legend Snippet: Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo . Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis ). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Fluorescence, Microscopy, Staining

    Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.
    Figure Legend Snippet: Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Isolation

    Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis ). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m / z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m / z ). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m / z ) are not recovered in these lipid isolates.
    Figure Legend Snippet: Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis ). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m / z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m / z ). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m / z ) are not recovered in these lipid isolates.

    Techniques Used: Mass Spectrometry, Dominant Negative Mutation

    Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10 3 versus [263.0 ± 25.3] × 10 3 ; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79 ), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71 ).
    Figure Legend Snippet: Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10 3 versus [263.0 ± 25.3] × 10 3 ; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79 ), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71 ).

    Techniques Used: Concentration Assay, Ethidium Homodimer Assay, Staining, Flow Cytometry, Cytometry, Quantitation Assay

    Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids ( ) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.
    Figure Legend Snippet: Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids ( ) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.

    Techniques Used: Inhibition, Quantitation Assay, Staining, Cell Culture

    Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2 −/− ) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.
    Figure Legend Snippet: Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2 −/− ) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.

    Techniques Used: Mouse Assay, Staining, Inhibition

    9) Product Images from "Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function ▿"

    Article Title: Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00225-10

    Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo . Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis ). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.
    Figure Legend Snippet: Effects of P. gingivalis lipid preparations on osteoblast function and gene expression in vivo . Mice that express the osteoblastic GFP reporter, pOBCol3.6GFPcyan, were lightly sedated, and the indicated lipid preparations (5 μg in 50 μl of PBS) were administered as a subcutaneous injection to the surfaces of the calvaria (two mice were treated with each lipid preparation, including the total lipid extract, PG DHC lipids and PE DHC lipids of P. gingivalis ). Control mouse calvaria received only PBS. Mice were administered Alizarin complexone on day 6 and were sacrificed on day 7. Calvaria were cryosectioned and evaluated by fluorescence microscopy for osteoblast reporter (blue) and alizarin complexone (red) fluorescence (upper row). The right side of each calvarium section represents the cerebral cavity, and the left side represents the surface treated with the indicated lipid preparation. The inserts within each frame of the upper row depict magnified images of lipid-treated (left) and untreated (right) surfaces of each calvarium section. The exact same sections depicted in the upper row were then stained with hematoxylin and eosin (H E), and photomicrographs were obtained (lower row). Multiple sections of each calvarium specimen were evaluated for fluorescence changes, and the images depicted above are representative of those observed in the replicate sections.

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Fluorescence, Microscopy, Staining

    Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.
    Figure Legend Snippet: Effects of P. gingivalis total lipids on gene expression determined by real-time RT-PCR. Cells were cultured with P. gingivalis total lipids (1,250 ng/ml) or control medium for the entire 21-day culture period. Real-time RT-PCR was performed on total RNA isolated from day 21 cultures. Changes in gene expression, either as increased or decreased gene expression, in lipid-treated cultures are expressed as the fold change versus control cultures. Significant up or down expression of each gene was evaluated against parallel control cultures by using the Student t test, and P values are depicted opposite each histogram bar. Only the expression of the Col1a1 and RANKL genes was not significantly affected by lipid treatment of osteoblast cultures.

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Isolation

    Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis ). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m / z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m / z ). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m / z ) are not recovered in these lipid isolates.
    Figure Legend Snippet: Electrospray-MS characterization of total lipids of P. gingivalis and the highly enriched phosphoethanolamine dihydroceramide (PE DHC) or phosphoglycerol dihydroceramide (PG DHC) lipid fractions. Lipids were prepared and characterized by electrospray-MS as described in Materials and Methods. The upper frame shows the negative ions recovered in the total lipid extract of P. gingivalis ). The PG DHC lipids include three component lipids that produce negative ions of 960, 946, and 932 m / z and, by analogy, the PE DHC lipids produce negative ions of 705, 691, and 677 m / z ). Note that the dominant-negative ions previously reported for lipid A species of P. gingivalis (1,450, 1,690, and 1,770 m / z ) are not recovered in these lipid isolates.

    Techniques Used: Mass Spectrometry, Dominant Negative Mutation

    Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10 3 versus [263.0 ± 25.3] × 10 3 ; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79 ), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71 ).
    Figure Legend Snippet: Effects of P. gingivalis total lipids on cell viability, apoptosis, and proliferation. The concentration of P. gingivalis total lipids was 1,250 ng/ml in the indicated cultures. (A) Cell viability indicated by EthD-1 staining at day 7 showed no significant difference in cell death between lipid-treated and control osteoblast cultures (126.5 ± 15.6 versus 107.6 ± 12.0; P = 0.35). (B) Apoptosis observed at day 7 with annexin V staining, followed by flow cytometry, showed no significant difference between lipid-treated and control osteoblast cultures (5.9% ± 0.5% versus 6.3% ± 0.5%; P = 0.59). (C) Total cell counts per well at day 7 showed no significant difference in total cell recovery between lipid-treated and control osteoblast cultures ([306.6 ± 15.8] × 10 3 versus [263.0 ± 25.3] × 10 3 ; P = 0.18). (D) DNA quantitation showed no significant differences in total DNA recovered from control and lipid treated osteoblasts for day 7 cultures (4.3 ± 0.2 μg/ml versus 4.5 ± 0.5 μg/ml; P = 0.79 ), as well as day 21 cultures (8.5 ± 0.3 μg/ml versus 8.3 ± 0.4 μg/ml; P = 0.71 ).

    Techniques Used: Concentration Assay, Ethidium Homodimer Assay, Staining, Flow Cytometry, Cytometry, Quantitation Assay

    Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids ( ) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.
    Figure Legend Snippet: Inhibition of the mineralized nodule formation by P. gingivalis total lipids depending on the week of lipid treatment in culture. (A) Cells were exposed to P. gingivalis total lipids (1,250 ng/ml) only during the specified culture intervals, including the first week only, the second week only, the third week only, the first and second week, the second and third week, or all 3 weeks. Control cultures were not exposed to P. gingivalis lipids. (B) Quantitation of mineralized nodule formation stained by von Kossa of day 21 cultures. The magnitude of inhibitory action of P. gingivalis total lipids was dependent on the stage of growth in cultures. Control cultures and cultures treated for the first week with lipids ( ) were not significantly different from each other but both were significantly different from all other treatment groups, as determined by Scheffe contrasts among pairs of means. Cultures treated with lipids for the second week, the third week, or the combined first and second weeks (*) were not significantly different from each other but were significantly different from the remaining cell culture categories. Cultures treated for the second and third weeks or for all weeks (#) were not significantly different from each other but were significantly different from the remaining cell culture categories.

    Techniques Used: Inhibition, Quantitation Assay, Staining, Cell Culture

    Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2 −/− ) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.
    Figure Legend Snippet: Engagement of TLR2 by P. gingivalis total lipids and major fractions of P. gingivalis lipids. Calvarial osteoblast cultures were established from the WT CD-1 or TLR2-null (TLR2 −/− ) mice and treated with P. gingivalis total lipids, LPS, or two major P. gingivalis lipid fractions (PG DHC or PE DHC lipids) for the entire 21-day culture period. The von Kossa staining was performed at day 21 to reveal mineralized nodule formation in cultures. Control cultures did not receive P. gingivalis lipids, LPS, or lipid fractions. The inhibitory action of P. gingivalis total lipids was TLR2 dependent, and the PG DHC fraction, but not the PE DHC fraction, demonstrated TLR2-dependent inhibition of osteoblast mineralized nodule formation.

    Techniques Used: Mouse Assay, Staining, Inhibition

    10) Product Images from "Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria"

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    Journal: Mucosal Immunology

    doi: 10.1038/mi.2010.83

    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    Figure Legend Snippet: mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Techniques Used: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    mRNA expression of innate immune markers CC chemokine ligand 20 (CCL20) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of ( b ) CCL20 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of CCL20 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    Figure Legend Snippet: mRNA expression of innate immune markers CC chemokine ligand 20 (CCL20) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of ( b ) CCL20 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of CCL20 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Techniques Used: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    Differential decreased mRNA expression of HDAC1, HDAC2 and DNMT1 in gingival epithelial cells in response to various doses of oral bacteria. mRNA expression of ( a ) DNA methyltransferase (DNMT1), ( b ) histone deacetylase 1 (HDAC1), and ( c ) histone deacetylase 2 (HDAC2) are differentially decreased in gingival epithelial cells (GECs) in response to various doses of Porphyromonas gingivalis vs. Fusobacterium nucleatum . GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, 100:1, and 200:1 for 24 h. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the unstimulated control after normalization with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH ). The data are derived from three different cell donors tested in duplicate. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    Figure Legend Snippet: Differential decreased mRNA expression of HDAC1, HDAC2 and DNMT1 in gingival epithelial cells in response to various doses of oral bacteria. mRNA expression of ( a ) DNA methyltransferase (DNMT1), ( b ) histone deacetylase 1 (HDAC1), and ( c ) histone deacetylase 2 (HDAC2) are differentially decreased in gingival epithelial cells (GECs) in response to various doses of Porphyromonas gingivalis vs. Fusobacterium nucleatum . GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, 100:1, and 200:1 for 24 h. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the unstimulated control after normalization with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH ). The data are derived from three different cell donors tested in duplicate. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Techniques Used: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, CTL Assay

    Protein levels of histone H3 methylated at Lys4 were evaluated with PathScan enzyme-linked immunosorbent assay (ELISA). Gingival epithelial cells (GECs) were exposed to Porphyromonas gingivalis or Fusobacterium nucleatum for 24 h at multiplicity of infection (MOI) of 100:1, and then nuclear protein was extracted followed by sonication. The H3 tri-methylated at Lys4 was captured by coated antibody after incubation with cell lysates, and histone H3 protein level was quantified according to the absorbance readings at 450 nm. Protein expression was expressed as the ratio of absorbance readings normalized to relative protein amount. The data are average from three different donor cells tested with standard error deviation. The asterisks indicate the significant difference vs. the respective unstimulated control (** P
    Figure Legend Snippet: Protein levels of histone H3 methylated at Lys4 were evaluated with PathScan enzyme-linked immunosorbent assay (ELISA). Gingival epithelial cells (GECs) were exposed to Porphyromonas gingivalis or Fusobacterium nucleatum for 24 h at multiplicity of infection (MOI) of 100:1, and then nuclear protein was extracted followed by sonication. The H3 tri-methylated at Lys4 was captured by coated antibody after incubation with cell lysates, and histone H3 protein level was quantified according to the absorbance readings at 450 nm. Protein expression was expressed as the ratio of absorbance readings normalized to relative protein amount. The data are average from three different donor cells tested with standard error deviation. The asterisks indicate the significant difference vs. the respective unstimulated control (** P

    Techniques Used: Methylation, Enzyme-linked Immunosorbent Assay, Infection, Sonication, Incubation, Expressing

    Protein levels of histone deacetylases 1 and 2 (HDAC1 and HDAC2) and DNA methyltransferase (DNMT1) are differentially expressed in gingival epithelial cells (GECs) in response to Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 100:1 for 24 h. Nuclear proteins were extracted, denatured at 70 °C for 10 min, and separated by NuPAGE electrophoresis system. Nuclear extracts of Hela cells probed with individual primary antibody were used as positive controls. The data are derived from two different cell donors tested in duplicate.
    Figure Legend Snippet: Protein levels of histone deacetylases 1 and 2 (HDAC1 and HDAC2) and DNA methyltransferase (DNMT1) are differentially expressed in gingival epithelial cells (GECs) in response to Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 100:1 for 24 h. Nuclear proteins were extracted, denatured at 70 °C for 10 min, and separated by NuPAGE electrophoresis system. Nuclear extracts of Hela cells probed with individual primary antibody were used as positive controls. The data are derived from two different cell donors tested in duplicate.

    Techniques Used: Infection, Electrophoresis, Derivative Assay

    Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of ( a ) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and ( b ) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum . Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    Figure Legend Snippet: Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of ( a ) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and ( b ) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum . Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    Interleukin-8 (IL-8) secretion is increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Secretion of IL-8 in response to various oral bacteria is evaluated by enzyme-linked immunosorbent assay (ELISA). Cell-free supernatant was collected and the amount of IL-8 secreted is shown in pg ml –1 . Unstimulated cells (UN) are used as controls in each experiment. Data from duplicates with cells from three different donors are shown. No significant changes were found in the secretion of IL-8 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    Figure Legend Snippet: Interleukin-8 (IL-8) secretion is increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Secretion of IL-8 in response to various oral bacteria is evaluated by enzyme-linked immunosorbent assay (ELISA). Cell-free supernatant was collected and the amount of IL-8 secreted is shown in pg ml –1 . Unstimulated cells (UN) are used as controls in each experiment. Data from duplicates with cells from three different donors are shown. No significant changes were found in the secretion of IL-8 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Techniques Used: Histone Deacetylase Assay, Infection, Enzyme-linked Immunosorbent Assay, CTL Assay

    11) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    12) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    13) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    14) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    15) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    16) Product Images from "A 55-Kilodalton Immunodominant Antigen of Porphyromonas gingivalis W50 Has Arisen via Horizontal Gene Transfer"

    Article Title: A 55-Kilodalton Immunodominant Antigen of Porphyromonas gingivalis W50 Has Arisen via Horizontal Gene Transfer

    Journal: Infection and Immunity

    doi:

    PCR analysis of subgingival paper point samples from periodontal disease patients to detect the presence of P. gingivalis (A) and ragB (B). The primers used were P. gingivalis 16S RNA primers to amplify a 404-bp product and rag B.2 primers to amplify a 435-bp product. Lanes 1, 1-kb ladder; lanes 2 to 17, subgingival paper point PCRs; lanes 18, negative control (distilled H 2 O as template); lanes 19, positive control (200 ng of P. gingivalis W50 chromosomal DNA as template). Lanes 12, 15, and 17 represent three of the eight samples which are PCR positive for ragB but PCR negative for P. gingivalis . Ten microliters from a total volume of 100 μl of the PCR mixtures was run on a 1% agarose gel.
    Figure Legend Snippet: PCR analysis of subgingival paper point samples from periodontal disease patients to detect the presence of P. gingivalis (A) and ragB (B). The primers used were P. gingivalis 16S RNA primers to amplify a 404-bp product and rag B.2 primers to amplify a 435-bp product. Lanes 1, 1-kb ladder; lanes 2 to 17, subgingival paper point PCRs; lanes 18, negative control (distilled H 2 O as template); lanes 19, positive control (200 ng of P. gingivalis W50 chromosomal DNA as template). Lanes 12, 15, and 17 represent three of the eight samples which are PCR positive for ragB but PCR negative for P. gingivalis . Ten microliters from a total volume of 100 μl of the PCR mixtures was run on a 1% agarose gel.

    Techniques Used: Polymerase Chain Reaction, Negative Control, Positive Control, Agarose Gel Electrophoresis

    Percentages of P. gingivalis and ragB PCR-positive subgingival samples from periodontal disease patients. The y axis represents the percentages of P. gingivalis - and ragB -positive samples. The x axis represents both the periodontal pocket depth and the total sample number ( n ) from each pocket depth.
    Figure Legend Snippet: Percentages of P. gingivalis and ragB PCR-positive subgingival samples from periodontal disease patients. The y axis represents the percentages of P. gingivalis - and ragB -positive samples. The x axis represents both the periodontal pocket depth and the total sample number ( n ) from each pocket depth.

    Techniques Used: Polymerase Chain Reaction

    Northern analysis of RagA and RagB expression in exponentially grown P. gingivalis W50. Lane 1, P. gingivalis W50 RNA probed with a PCR-amplified 1.6-kb ragA probe; lane 2, P. gingivalis W50 RNA probed with a PCR-amplified 1.5-kb ragB probe. The arrows indicate the position of the ragAB mRNA transcript of approximately 4.7 kb and the positions of the 23S and 16S mRNAs.
    Figure Legend Snippet: Northern analysis of RagA and RagB expression in exponentially grown P. gingivalis W50. Lane 1, P. gingivalis W50 RNA probed with a PCR-amplified 1.6-kb ragA probe; lane 2, P. gingivalis W50 RNA probed with a PCR-amplified 1.5-kb ragB probe. The arrows indicate the position of the ragAB mRNA transcript of approximately 4.7 kb and the positions of the 23S and 16S mRNAs.

    Techniques Used: Northern Blot, Expressing, Polymerase Chain Reaction, Amplification

    (A) Protein composition of the outer membrane of P. gingivalis W50 in SDS–10% polyacrylamide gel electrophoresis. Lane 1, low-molecular-weight standards; lane 2, outer membrane preparation of P. gingivalis W50. Arrows indicate the positions of the immunodominant 115- and 55-kDa outer membrane proteins. (B) Western blot analysis of whole-cell preparations of E. coli XL-Blue containing pUC18 (lane 1), E. coli clone PM1 containing a 9.5-kb fragment of P. gingivalis W50 DNA cloned in pUC18 (lane 2), E. coli subclone SM containing a 7.5-kb fragment of P. gingivalis W50 DNA (lane 3), and an outer membrane preparation of P. gingivalis W50 (lane 4). Western analysis was performed with the monoclonal antibody DRU55.5.
    Figure Legend Snippet: (A) Protein composition of the outer membrane of P. gingivalis W50 in SDS–10% polyacrylamide gel electrophoresis. Lane 1, low-molecular-weight standards; lane 2, outer membrane preparation of P. gingivalis W50. Arrows indicate the positions of the immunodominant 115- and 55-kDa outer membrane proteins. (B) Western blot analysis of whole-cell preparations of E. coli XL-Blue containing pUC18 (lane 1), E. coli clone PM1 containing a 9.5-kb fragment of P. gingivalis W50 DNA cloned in pUC18 (lane 2), E. coli subclone SM containing a 7.5-kb fragment of P. gingivalis W50 DNA (lane 3), and an outer membrane preparation of P. gingivalis W50 (lane 4). Western analysis was performed with the monoclonal antibody DRU55.5.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Molecular Weight, Western Blot, Clone Assay

    Schematic diagram of the genetic organization of P. gingivalis W50 DNA in pPM1 and pSM. The horizontal bar represents the 9.5- and 7.5-kb P. gingivalis DNA fragments cloned in pPM1 and pSM, respectively; the vertical bars indicate positions of restriction enzyme sites. Restriction enzyme sites: S, Sma I; Bg, Bgl II; H, Hin dIII; B, Bam HI; P, Pst I. The arrows indicate the locations and directions of ORFs, with known motifs shaded: ■, TonB box sites; , signal sequence motif of lipoproteins. The heavier outlined arrows of ragA and ragB represent the difference in the G+C contents of these genes in comparison to the G+C content of P. gingivalis genes.
    Figure Legend Snippet: Schematic diagram of the genetic organization of P. gingivalis W50 DNA in pPM1 and pSM. The horizontal bar represents the 9.5- and 7.5-kb P. gingivalis DNA fragments cloned in pPM1 and pSM, respectively; the vertical bars indicate positions of restriction enzyme sites. Restriction enzyme sites: S, Sma I; Bg, Bgl II; H, Hin dIII; B, Bam HI; P, Pst I. The arrows indicate the locations and directions of ORFs, with known motifs shaded: ■, TonB box sites; , signal sequence motif of lipoproteins. The heavier outlined arrows of ragA and ragB represent the difference in the G+C contents of these genes in comparison to the G+C content of P. gingivalis genes.

    Techniques Used: Clone Assay, Sequencing

    Nucleotide and amino acid sequences of the coding regions of the P. gingivalis W50 insert in pPM1. Sequences shown are those of IS 1126 , ragA , ragB , and the incomplete orf3 . The putative RBS, the E. coli −35 and −10 promoter sites, and the methionine ATG start codons are shown in boldface. Restriction enzyme and primer sites are shown above the sequence and underlined. The 12-bp inverted repeats of IS 1126 are depicted in boldface. In the RagA sequence the TonB boxes and conserved TonB C terminus are labelled and underlined, and the conserved hexapeptide motif involved in membrane anchoring is boxed. The signal peptidase II cleavage sites are indicated by arrows at the cysteine residues in the RagB and Orf3 deduced amino acid sequences.
    Figure Legend Snippet: Nucleotide and amino acid sequences of the coding regions of the P. gingivalis W50 insert in pPM1. Sequences shown are those of IS 1126 , ragA , ragB , and the incomplete orf3 . The putative RBS, the E. coli −35 and −10 promoter sites, and the methionine ATG start codons are shown in boldface. Restriction enzyme and primer sites are shown above the sequence and underlined. The 12-bp inverted repeats of IS 1126 are depicted in boldface. In the RagA sequence the TonB boxes and conserved TonB C terminus are labelled and underlined, and the conserved hexapeptide motif involved in membrane anchoring is boxed. The signal peptidase II cleavage sites are indicated by arrows at the cysteine residues in the RagB and Orf3 deduced amino acid sequences.

    Techniques Used: Sequencing

    Southern blot analysis of the distributions of ragA , ragB , and orf3 in laboratory P. gingivalis strains. (A) P. gingivalis chromosomal DNA was digested with Cla I (C) and probed with a 32 P-labelled 1.6-kb PCR-amplified product of ragA . (B) P. gingivalis chromosomal DNA was digested with Hin dIII (H) and probed with a 32 P-labelled 1.6-kb amplified product of ragB . (C) P. gingivalis chromosomal DNA was digested with Eco RV (E) and probed with a 0.47-kb 32 P-labelled Eco RV-restricted fragment of orf3 . Lanes 1 to 10, P. gingivalis W50, W50Be1, W50Br1, W83, LB13D-3, 381, 11834, 23A4, WPH35, and WPH34, respectively; lane 11, E. coli XL-Blue; lane 12, P. asaccharolytica ATCC 25260.
    Figure Legend Snippet: Southern blot analysis of the distributions of ragA , ragB , and orf3 in laboratory P. gingivalis strains. (A) P. gingivalis chromosomal DNA was digested with Cla I (C) and probed with a 32 P-labelled 1.6-kb PCR-amplified product of ragA . (B) P. gingivalis chromosomal DNA was digested with Hin dIII (H) and probed with a 32 P-labelled 1.6-kb amplified product of ragB . (C) P. gingivalis chromosomal DNA was digested with Eco RV (E) and probed with a 0.47-kb 32 P-labelled Eco RV-restricted fragment of orf3 . Lanes 1 to 10, P. gingivalis W50, W50Be1, W50Br1, W83, LB13D-3, 381, 11834, 23A4, WPH35, and WPH34, respectively; lane 11, E. coli XL-Blue; lane 12, P. asaccharolytica ATCC 25260.

    Techniques Used: Southern Blot, Polymerase Chain Reaction, Amplification

    Multiple alignments of TonB boxes with the highest similarity scores compared to P. gingivalis RagA. The receptors are SusC (starch utilization), FepA (ferric enterochelin), BfeA (ferrichrome-iron), BfrA (exogenous ferric siderophore), IrgA (iron-regulated outer membrane), CirA (colicin 1), HemR (hemin-regulated protein), Tla (TonB linked adhesion), and FyuA (yersiniabactin siderophore). Abbreviations for bacteria: pg, P. gingivalis ; bt, B. thetaiotamicron ; pa, Pseudomonas aeruginosa ; ec, E. coli ; bp, Bordetella pertussis ; bb, Bordetella bronchiseptica ; vc, V. cholerae ; and ye, Y. enterocolitica . Sequences are aligned in descending order of relatedness. Boldface amino acid residues correspond to residues conserved throughout the alignment, with asterisks indicating the consensus sequence, while the underlined residues are conserved in more than 50% of the sequences. Amino acid sequence positions are shown at the beginning and end of each sequence. For boxIII the percentage of similarity is calculated with reference to RagA.
    Figure Legend Snippet: Multiple alignments of TonB boxes with the highest similarity scores compared to P. gingivalis RagA. The receptors are SusC (starch utilization), FepA (ferric enterochelin), BfeA (ferrichrome-iron), BfrA (exogenous ferric siderophore), IrgA (iron-regulated outer membrane), CirA (colicin 1), HemR (hemin-regulated protein), Tla (TonB linked adhesion), and FyuA (yersiniabactin siderophore). Abbreviations for bacteria: pg, P. gingivalis ; bt, B. thetaiotamicron ; pa, Pseudomonas aeruginosa ; ec, E. coli ; bp, Bordetella pertussis ; bb, Bordetella bronchiseptica ; vc, V. cholerae ; and ye, Y. enterocolitica . Sequences are aligned in descending order of relatedness. Boldface amino acid residues correspond to residues conserved throughout the alignment, with asterisks indicating the consensus sequence, while the underlined residues are conserved in more than 50% of the sequences. Amino acid sequence positions are shown at the beginning and end of each sequence. For boxIII the percentage of similarity is calculated with reference to RagA.

    Techniques Used: Sequencing

    17) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    18) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    19) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    20) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    21) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    22) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    23) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    24) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    25) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    26) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    27) Product Images from "Sialidase Deficiency in Porphyromonas gingivalis Increases IL-12 Secretion in Stimulated Macrophages Through Regulation of CR3, IncRNA GAS5 and miR-21"

    Article Title: Sialidase Deficiency in Porphyromonas gingivalis Increases IL-12 Secretion in Stimulated Macrophages Through Regulation of CR3, IncRNA GAS5 and miR-21

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00100

    The levels of IL-12p70 in macrophages stimulated by different P. gingivalis strains after CR3 receptor suppressed by CD11b antibody. * P
    Figure Legend Snippet: The levels of IL-12p70 in macrophages stimulated by different P. gingivalis strains after CR3 receptor suppressed by CD11b antibody. * P

    Techniques Used:

    The expression of lncRNA GAS5 after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P
    Figure Legend Snippet: The expression of lncRNA GAS5 after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P

    Techniques Used: Expressing

    The expression of lncRNA GAS5 in macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352 after CR3 suppressed by CD11b antibody. * P
    Figure Legend Snippet: The expression of lncRNA GAS5 in macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352 after CR3 suppressed by CD11b antibody. * P

    Techniques Used: Expressing

    The expression of lncRNA MEG3, GAS5, FASLG, BTG2, SPRY2 , and TAGAP genes after macrophages stimulated by P. gingivalis W83 and ΔPG0352. * P
    Figure Legend Snippet: The expression of lncRNA MEG3, GAS5, FASLG, BTG2, SPRY2 , and TAGAP genes after macrophages stimulated by P. gingivalis W83 and ΔPG0352. * P

    Techniques Used: Expressing

    The mechanism of sialidase deficiency increasing IL-12 secretion in P. gingivalis -stimulated macrophages. P. gingivalis can activate CR3 in macrophages, inhibit the expression of lncRNA GAS5, increase the expression of miR-21, decrease the level of IL-12 and subvert phagocytosis by macrophages. The sialidase-deficiency in P. gingivalis attenuates CR3 activation in macrophages, reduces the inhibition of lncRNA GAS5 , induces less miR-21 and more IL-12 in macrophages.
    Figure Legend Snippet: The mechanism of sialidase deficiency increasing IL-12 secretion in P. gingivalis -stimulated macrophages. P. gingivalis can activate CR3 in macrophages, inhibit the expression of lncRNA GAS5, increase the expression of miR-21, decrease the level of IL-12 and subvert phagocytosis by macrophages. The sialidase-deficiency in P. gingivalis attenuates CR3 activation in macrophages, reduces the inhibition of lncRNA GAS5 , induces less miR-21 and more IL-12 in macrophages.

    Techniques Used: Expressing, Activation Assay, Inhibition

    The expression of IL-12p35 (A) and IL-12p40 (B) genes after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P
    Figure Legend Snippet: The expression of IL-12p35 (A) and IL-12p40 (B) genes after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P

    Techniques Used: Expressing

    The levels of IL-12p70 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P
    Figure Legend Snippet: The levels of IL-12p70 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P

    Techniques Used:

    The CR3 expression levels after stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352. (A) The U937-differentiated macrophages membranes stained with DiI appear red, while the nuclei appeared blue (200×). (B) Analysis of fluorescent levels using IMAGEJ software revealed elevated CD11b levels in P. gingivalis W83, ΔPG0352 and comΔPG0352 groups compared with control group. After 6 h post-infection, the fluorescence intensity of CR3 in P. gingivalis W83 group was about 2 times higher than that in the control group, and about 1.5 times higher than that in ΔPG0352 group. There were no significant differences between P. gingivalis W83 and comΔPG0352 groups. * P
    Figure Legend Snippet: The CR3 expression levels after stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352. (A) The U937-differentiated macrophages membranes stained with DiI appear red, while the nuclei appeared blue (200×). (B) Analysis of fluorescent levels using IMAGEJ software revealed elevated CD11b levels in P. gingivalis W83, ΔPG0352 and comΔPG0352 groups compared with control group. After 6 h post-infection, the fluorescence intensity of CR3 in P. gingivalis W83 group was about 2 times higher than that in the control group, and about 1.5 times higher than that in ΔPG0352 group. There were no significant differences between P. gingivalis W83 and comΔPG0352 groups. * P

    Techniques Used: Expressing, Staining, Software, Infection, Fluorescence

    The expression of miR-21 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P
    Figure Legend Snippet: The expression of miR-21 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P

    Techniques Used: Expressing

    Transmission electron microscope observation of macrophages stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352 respectively (15,000 ×, bar: 2 μm). (A) control group; (B) P. gingivalis W83 group; (C) ΔPG0352 group; (D) comΔPG0352 group. White arrow: P. gingivalis internalized by macrophage; black arrow: P. gingivalis adhering to macrophage surface.
    Figure Legend Snippet: Transmission electron microscope observation of macrophages stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352 respectively (15,000 ×, bar: 2 μm). (A) control group; (B) P. gingivalis W83 group; (C) ΔPG0352 group; (D) comΔPG0352 group. White arrow: P. gingivalis internalized by macrophage; black arrow: P. gingivalis adhering to macrophage surface.

    Techniques Used: Transmission Assay, Microscopy

    28) Product Images from "Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis"

    Article Title: Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00424-15

    TLR9 KO (TLR9 −/− ) mice are resistant to P. gingivalis -instigated periodontal bone loss. Groups of mice (WT [ n = 17] and TLR9 −/− [ n = 47]) were infected with P. gingivalis (Pg) or sham infected and euthanized 42 days later. Measurements were performed in defleshed maxillae. The data are represented as the mean results ± SD ( n = 64 mice). (A) Distance (in millimeters) between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) in each group of animals. (B) Amount of bone change in WT and TLR9 −/− mice. Negative values indicate bone loss in P. gingivalis -inoculated mice relative to the results for vehicle-inoculated (sham) controls. (C) Representative micro-CT images of maxillae from each group of mice. *, P
    Figure Legend Snippet: TLR9 KO (TLR9 −/− ) mice are resistant to P. gingivalis -instigated periodontal bone loss. Groups of mice (WT [ n = 17] and TLR9 −/− [ n = 47]) were infected with P. gingivalis (Pg) or sham infected and euthanized 42 days later. Measurements were performed in defleshed maxillae. The data are represented as the mean results ± SD ( n = 64 mice). (A) Distance (in millimeters) between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) in each group of animals. (B) Amount of bone change in WT and TLR9 −/− mice. Negative values indicate bone loss in P. gingivalis -inoculated mice relative to the results for vehicle-inoculated (sham) controls. (C) Representative micro-CT images of maxillae from each group of mice. *, P

    Techniques Used: Mouse Assay, Infection, Micro-CT

    Inflammatory molecule expression in gingival tissues. WT and TLR9 −/− mice were orally inoculated with P. gingivalis or vehicle only (sham) and euthanized 42 days later. Gingival tissues around maxillary molars were excised and processed for qPCR analyses to determine mRNA expression of TNF (A), IL-6 (B), and RANKL (C). Results are reported as fold induction after normalization to GAPDH. The data shown are the mean results ± SD ( n = 5 or 6 mice per group) and were analyzed using the unpaired t test. *, P
    Figure Legend Snippet: Inflammatory molecule expression in gingival tissues. WT and TLR9 −/− mice were orally inoculated with P. gingivalis or vehicle only (sham) and euthanized 42 days later. Gingival tissues around maxillary molars were excised and processed for qPCR analyses to determine mRNA expression of TNF (A), IL-6 (B), and RANKL (C). Results are reported as fold induction after normalization to GAPDH. The data shown are the mean results ± SD ( n = 5 or 6 mice per group) and were analyzed using the unpaired t test. *, P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Comparison of proinflammatory cytokine production in WT versus TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to P. gingivalis challenge after 24 h. The cells were stimulated with heat-killed P. gingivalis (MOI of 1:100), P. gingivalis DNA (100 ng/μl), and ODN 1668 (TLR9 agonist), and cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. Comparisons between the results for WT and TLR9 KO cells were performed using the unpaired student t test. The levels of IL-6 and TNF production were significantly reduced in TLR9 −/− macrophages (A, B) and splenocytes (C, D) compared to the levels in WT cells. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). *, P
    Figure Legend Snippet: Comparison of proinflammatory cytokine production in WT versus TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to P. gingivalis challenge after 24 h. The cells were stimulated with heat-killed P. gingivalis (MOI of 1:100), P. gingivalis DNA (100 ng/μl), and ODN 1668 (TLR9 agonist), and cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. Comparisons between the results for WT and TLR9 KO cells were performed using the unpaired student t test. The levels of IL-6 and TNF production were significantly reduced in TLR9 −/− macrophages (A, B) and splenocytes (C, D) compared to the levels in WT cells. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Comparison of P. gingivalis levels within the periodontal tissues (A) and P. gingivalis -specific antibody responses in serum (B) in TLR9 −/− and WT mice. Each strain was orally inoculated with P. gingivalis or vehicle only (sham). The P. gingivalis levels were determined by qPCR of the ISPg1 gene ( P. gingivalis ) at 2 weeks postinfection. There was no statistically significant difference in P. gingivalis levels among groups. P. gingivalis -specific antibody responses were determined at the termination of the experiment. The antibody titers in TLR9 −/− mice and WT mice infected with P. gingivalis were significantly higher than the titers in the uninfected mice ( P
    Figure Legend Snippet: Comparison of P. gingivalis levels within the periodontal tissues (A) and P. gingivalis -specific antibody responses in serum (B) in TLR9 −/− and WT mice. Each strain was orally inoculated with P. gingivalis or vehicle only (sham). The P. gingivalis levels were determined by qPCR of the ISPg1 gene ( P. gingivalis ) at 2 weeks postinfection. There was no statistically significant difference in P. gingivalis levels among groups. P. gingivalis -specific antibody responses were determined at the termination of the experiment. The antibody titers in TLR9 −/− mice and WT mice infected with P. gingivalis were significantly higher than the titers in the uninfected mice ( P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Infection

    Comparison of proinflammatory cytokine production in WT and TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to different TLR agonists. The cells were stimulated with ODN 1668 (TLR9 agonist; 100 ng/μl), P. gingivalis LPS (TLR4 agonist; 10 ng/μl), E. coli LPS (TLR4 agonist; 10 ng/μl), or Pam3Cys (TLR2 agonist; 1 ng/μl) for 24 h. Cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). Comparisons between WT and KO cells were performed using the unpaired Student t test. *, P
    Figure Legend Snippet: Comparison of proinflammatory cytokine production in WT and TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to different TLR agonists. The cells were stimulated with ODN 1668 (TLR9 agonist; 100 ng/μl), P. gingivalis LPS (TLR4 agonist; 10 ng/μl), E. coli LPS (TLR4 agonist; 10 ng/μl), or Pam3Cys (TLR2 agonist; 1 ng/μl) for 24 h. Cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). Comparisons between WT and KO cells were performed using the unpaired Student t test. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    29) Product Images from "Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis"

    Article Title: Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00424-15

    TLR9 KO (TLR9 −/− ) mice are resistant to P. gingivalis -instigated periodontal bone loss. Groups of mice (WT [ n = 17] and TLR9 −/− [ n = 47]) were infected with P. gingivalis (Pg) or sham infected and euthanized 42 days later. Measurements were performed in defleshed maxillae. The data are represented as the mean results ± SD ( n = 64 mice). (A) Distance (in millimeters) between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) in each group of animals. (B) Amount of bone change in WT and TLR9 −/− mice. Negative values indicate bone loss in P. gingivalis -inoculated mice relative to the results for vehicle-inoculated (sham) controls. (C) Representative micro-CT images of maxillae from each group of mice. *, P
    Figure Legend Snippet: TLR9 KO (TLR9 −/− ) mice are resistant to P. gingivalis -instigated periodontal bone loss. Groups of mice (WT [ n = 17] and TLR9 −/− [ n = 47]) were infected with P. gingivalis (Pg) or sham infected and euthanized 42 days later. Measurements were performed in defleshed maxillae. The data are represented as the mean results ± SD ( n = 64 mice). (A) Distance (in millimeters) between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) in each group of animals. (B) Amount of bone change in WT and TLR9 −/− mice. Negative values indicate bone loss in P. gingivalis -inoculated mice relative to the results for vehicle-inoculated (sham) controls. (C) Representative micro-CT images of maxillae from each group of mice. *, P

    Techniques Used: Mouse Assay, Infection, Micro-CT

    Inflammatory molecule expression in gingival tissues. WT and TLR9 −/− mice were orally inoculated with P. gingivalis or vehicle only (sham) and euthanized 42 days later. Gingival tissues around maxillary molars were excised and processed for qPCR analyses to determine mRNA expression of TNF (A), IL-6 (B), and RANKL (C). Results are reported as fold induction after normalization to GAPDH. The data shown are the mean results ± SD ( n = 5 or 6 mice per group) and were analyzed using the unpaired t test. *, P
    Figure Legend Snippet: Inflammatory molecule expression in gingival tissues. WT and TLR9 −/− mice were orally inoculated with P. gingivalis or vehicle only (sham) and euthanized 42 days later. Gingival tissues around maxillary molars were excised and processed for qPCR analyses to determine mRNA expression of TNF (A), IL-6 (B), and RANKL (C). Results are reported as fold induction after normalization to GAPDH. The data shown are the mean results ± SD ( n = 5 or 6 mice per group) and were analyzed using the unpaired t test. *, P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Comparison of proinflammatory cytokine production in WT versus TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to P. gingivalis challenge after 24 h. The cells were stimulated with heat-killed P. gingivalis (MOI of 1:100), P. gingivalis DNA (100 ng/μl), and ODN 1668 (TLR9 agonist), and cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. Comparisons between the results for WT and TLR9 KO cells were performed using the unpaired student t test. The levels of IL-6 and TNF production were significantly reduced in TLR9 −/− macrophages (A, B) and splenocytes (C, D) compared to the levels in WT cells. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). *, P
    Figure Legend Snippet: Comparison of proinflammatory cytokine production in WT versus TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to P. gingivalis challenge after 24 h. The cells were stimulated with heat-killed P. gingivalis (MOI of 1:100), P. gingivalis DNA (100 ng/μl), and ODN 1668 (TLR9 agonist), and cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. Comparisons between the results for WT and TLR9 KO cells were performed using the unpaired student t test. The levels of IL-6 and TNF production were significantly reduced in TLR9 −/− macrophages (A, B) and splenocytes (C, D) compared to the levels in WT cells. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Comparison of P. gingivalis levels within the periodontal tissues (A) and P. gingivalis -specific antibody responses in serum (B) in TLR9 −/− and WT mice. Each strain was orally inoculated with P. gingivalis or vehicle only (sham). The P. gingivalis levels were determined by qPCR of the ISPg1 gene ( P. gingivalis ) at 2 weeks postinfection. There was no statistically significant difference in P. gingivalis levels among groups. P. gingivalis -specific antibody responses were determined at the termination of the experiment. The antibody titers in TLR9 −/− mice and WT mice infected with P. gingivalis were significantly higher than the titers in the uninfected mice ( P
    Figure Legend Snippet: Comparison of P. gingivalis levels within the periodontal tissues (A) and P. gingivalis -specific antibody responses in serum (B) in TLR9 −/− and WT mice. Each strain was orally inoculated with P. gingivalis or vehicle only (sham). The P. gingivalis levels were determined by qPCR of the ISPg1 gene ( P. gingivalis ) at 2 weeks postinfection. There was no statistically significant difference in P. gingivalis levels among groups. P. gingivalis -specific antibody responses were determined at the termination of the experiment. The antibody titers in TLR9 −/− mice and WT mice infected with P. gingivalis were significantly higher than the titers in the uninfected mice ( P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Infection

    Comparison of proinflammatory cytokine production in WT and TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to different TLR agonists. The cells were stimulated with ODN 1668 (TLR9 agonist; 100 ng/μl), P. gingivalis LPS (TLR4 agonist; 10 ng/μl), E. coli LPS (TLR4 agonist; 10 ng/μl), or Pam3Cys (TLR2 agonist; 1 ng/μl) for 24 h. Cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). Comparisons between WT and KO cells were performed using the unpaired Student t test. *, P
    Figure Legend Snippet: Comparison of proinflammatory cytokine production in WT and TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to different TLR agonists. The cells were stimulated with ODN 1668 (TLR9 agonist; 100 ng/μl), P. gingivalis LPS (TLR4 agonist; 10 ng/μl), E. coli LPS (TLR4 agonist; 10 ng/μl), or Pam3Cys (TLR2 agonist; 1 ng/μl) for 24 h. Cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). Comparisons between WT and KO cells were performed using the unpaired Student t test. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    30) Product Images from "Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis"

    Article Title: Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00424-15

    TLR9 KO (TLR9 −/− ) mice are resistant to P. gingivalis -instigated periodontal bone loss. Groups of mice (WT [ n = 17] and TLR9 −/− [ n = 47]) were infected with P. gingivalis (Pg) or sham infected and euthanized 42 days later. Measurements were performed in defleshed maxillae. The data are represented as the mean results ± SD ( n = 64 mice). (A) Distance (in millimeters) between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) in each group of animals. (B) Amount of bone change in WT and TLR9 −/− mice. Negative values indicate bone loss in P. gingivalis -inoculated mice relative to the results for vehicle-inoculated (sham) controls. (C) Representative micro-CT images of maxillae from each group of mice. *, P
    Figure Legend Snippet: TLR9 KO (TLR9 −/− ) mice are resistant to P. gingivalis -instigated periodontal bone loss. Groups of mice (WT [ n = 17] and TLR9 −/− [ n = 47]) were infected with P. gingivalis (Pg) or sham infected and euthanized 42 days later. Measurements were performed in defleshed maxillae. The data are represented as the mean results ± SD ( n = 64 mice). (A) Distance (in millimeters) between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) in each group of animals. (B) Amount of bone change in WT and TLR9 −/− mice. Negative values indicate bone loss in P. gingivalis -inoculated mice relative to the results for vehicle-inoculated (sham) controls. (C) Representative micro-CT images of maxillae from each group of mice. *, P

    Techniques Used: Mouse Assay, Infection, Micro-CT

    Inflammatory molecule expression in gingival tissues. WT and TLR9 −/− mice were orally inoculated with P. gingivalis or vehicle only (sham) and euthanized 42 days later. Gingival tissues around maxillary molars were excised and processed for qPCR analyses to determine mRNA expression of TNF (A), IL-6 (B), and RANKL (C). Results are reported as fold induction after normalization to GAPDH. The data shown are the mean results ± SD ( n = 5 or 6 mice per group) and were analyzed using the unpaired t test. *, P
    Figure Legend Snippet: Inflammatory molecule expression in gingival tissues. WT and TLR9 −/− mice were orally inoculated with P. gingivalis or vehicle only (sham) and euthanized 42 days later. Gingival tissues around maxillary molars were excised and processed for qPCR analyses to determine mRNA expression of TNF (A), IL-6 (B), and RANKL (C). Results are reported as fold induction after normalization to GAPDH. The data shown are the mean results ± SD ( n = 5 or 6 mice per group) and were analyzed using the unpaired t test. *, P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Comparison of proinflammatory cytokine production in WT versus TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to P. gingivalis challenge after 24 h. The cells were stimulated with heat-killed P. gingivalis (MOI of 1:100), P. gingivalis DNA (100 ng/μl), and ODN 1668 (TLR9 agonist), and cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. Comparisons between the results for WT and TLR9 KO cells were performed using the unpaired student t test. The levels of IL-6 and TNF production were significantly reduced in TLR9 −/− macrophages (A, B) and splenocytes (C, D) compared to the levels in WT cells. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). *, P
    Figure Legend Snippet: Comparison of proinflammatory cytokine production in WT versus TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to P. gingivalis challenge after 24 h. The cells were stimulated with heat-killed P. gingivalis (MOI of 1:100), P. gingivalis DNA (100 ng/μl), and ODN 1668 (TLR9 agonist), and cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. Comparisons between the results for WT and TLR9 KO cells were performed using the unpaired student t test. The levels of IL-6 and TNF production were significantly reduced in TLR9 −/− macrophages (A, B) and splenocytes (C, D) compared to the levels in WT cells. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Comparison of P. gingivalis levels within the periodontal tissues (A) and P. gingivalis -specific antibody responses in serum (B) in TLR9 −/− and WT mice. Each strain was orally inoculated with P. gingivalis or vehicle only (sham). The P. gingivalis levels were determined by qPCR of the ISPg1 gene ( P. gingivalis ) at 2 weeks postinfection. There was no statistically significant difference in P. gingivalis levels among groups. P. gingivalis -specific antibody responses were determined at the termination of the experiment. The antibody titers in TLR9 −/− mice and WT mice infected with P. gingivalis were significantly higher than the titers in the uninfected mice ( P
    Figure Legend Snippet: Comparison of P. gingivalis levels within the periodontal tissues (A) and P. gingivalis -specific antibody responses in serum (B) in TLR9 −/− and WT mice. Each strain was orally inoculated with P. gingivalis or vehicle only (sham). The P. gingivalis levels were determined by qPCR of the ISPg1 gene ( P. gingivalis ) at 2 weeks postinfection. There was no statistically significant difference in P. gingivalis levels among groups. P. gingivalis -specific antibody responses were determined at the termination of the experiment. The antibody titers in TLR9 −/− mice and WT mice infected with P. gingivalis were significantly higher than the titers in the uninfected mice ( P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Infection

    Comparison of proinflammatory cytokine production in WT and TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to different TLR agonists. The cells were stimulated with ODN 1668 (TLR9 agonist; 100 ng/μl), P. gingivalis LPS (TLR4 agonist; 10 ng/μl), E. coli LPS (TLR4 agonist; 10 ng/μl), or Pam3Cys (TLR2 agonist; 1 ng/μl) for 24 h. Cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). Comparisons between WT and KO cells were performed using the unpaired Student t test. *, P
    Figure Legend Snippet: Comparison of proinflammatory cytokine production in WT and TLR9 −/− macrophages (A, B) and splenocytes (C, D) in response to different TLR agonists. The cells were stimulated with ODN 1668 (TLR9 agonist; 100 ng/μl), P. gingivalis LPS (TLR4 agonist; 10 ng/μl), E. coli LPS (TLR4 agonist; 10 ng/μl), or Pam3Cys (TLR2 agonist; 1 ng/μl) for 24 h. Cell-free supernatants were analyzed for the presence of IL-6 and TNF using ELISA. The results shown are representative of at least 3 independent experiments that were run in triplicates. The data shown are the mean results ± SD ( n = 9). Comparisons between WT and KO cells were performed using the unpaired Student t test. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    31) Product Images from "Novel Assay To Characterize Neutrophil Responses to Oral Biofilms"

    Article Title: Novel Assay To Characterize Neutrophil Responses to Oral Biofilms

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00790-18

    Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Incubation, Marker

    Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .
    Figure Legend Snippet: Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .

    Techniques Used: Activation Assay, Incubation, Flow Cytometry, Cytometry, Marker, Expressing

    P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.
    Figure Legend Snippet: P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.

    Techniques Used: Incubation, Mutagenesis

    S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

    Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Isolation, Incubation

    S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Labeling, Incubation, Flow Cytometry, Cytometry

    32) Product Images from "Novel Assay To Characterize Neutrophil Responses to Oral Biofilms"

    Article Title: Novel Assay To Characterize Neutrophil Responses to Oral Biofilms

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00790-18

    Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Incubation, Marker

    Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .
    Figure Legend Snippet: Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .

    Techniques Used: Activation Assay, Incubation, Flow Cytometry, Cytometry, Marker, Expressing

    P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.
    Figure Legend Snippet: P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.

    Techniques Used: Incubation, Mutagenesis

    S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

    Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Isolation, Incubation

    S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Labeling, Incubation, Flow Cytometry, Cytometry

    33) Product Images from "Novel Assay To Characterize Neutrophil Responses to Oral Biofilms"

    Article Title: Novel Assay To Characterize Neutrophil Responses to Oral Biofilms

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00790-18

    Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Incubation, Marker

    Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .
    Figure Legend Snippet: Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .

    Techniques Used: Activation Assay, Incubation, Flow Cytometry, Cytometry, Marker, Expressing

    P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.
    Figure Legend Snippet: P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.

    Techniques Used: Incubation, Mutagenesis

    S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

    Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Isolation, Incubation

    S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Labeling, Incubation, Flow Cytometry, Cytometry

    34) Product Images from "Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88"

    Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00359

    TLR2-PI3K plays a non-redundant role in the murine and human macrophage response to P. gingivalis . WT murine BMM (A) , RAW264.7 macrophages (B) , and PMA-differentiated human THP-1 cells (C) were primed with IFN-γ. TLR2 and TLR4 were inhibited with blocking antibodies vs. isotype control (I.C.) for 1 h and PI3K was blocked with LY294 prior to challenge with P. gingivalis (MOI 10). Supernatants were collected after overnight incubation and TNF was measured by ELISA. Background (BG) represents IFN-γ primed cells not challenged with P. gingivalis . Cells challenged with P. gingivalis without any blocker are referred to in the graphs as (–). Representative graphs of > 3 repeats are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    Figure Legend Snippet: TLR2-PI3K plays a non-redundant role in the murine and human macrophage response to P. gingivalis . WT murine BMM (A) , RAW264.7 macrophages (B) , and PMA-differentiated human THP-1 cells (C) were primed with IFN-γ. TLR2 and TLR4 were inhibited with blocking antibodies vs. isotype control (I.C.) for 1 h and PI3K was blocked with LY294 prior to challenge with P. gingivalis (MOI 10). Supernatants were collected after overnight incubation and TNF was measured by ELISA. Background (BG) represents IFN-γ primed cells not challenged with P. gingivalis . Cells challenged with P. gingivalis without any blocker are referred to in the graphs as (–). Representative graphs of > 3 repeats are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

    Techniques Used: Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay

    TLR2-PI3K signaling enhances intracellular survival by blocking phago-lysosomal maturation. RAW 264.7 cells were seeded at 3 × 104 cells/ well in Ibidi 8 well m-slides. The cells were untreated (A) or treated with anti-TLR2 (B) , anti-TLR4 (C) , or with the PI3K inhibitor LY 294002 (D) for an hour. Cells were then infected with FITC-labeled P. gingivalis at MOI 10 for 1 h. LysoTracker red was added at 50 nM for the last 10 min of infection. Cells were washed and fixed with 2% formaldehyde and mounted with mounting media. Images were captured using a NIKON confocal microscope at 60X magnification. Yellow color indicates co-localization of P. gingivalis (green) with lysosomes (red). In each field (A–D) the cell in the box is further magnified and shown in the upper right corner. (E) The percent of co-localization was determined by counting cells that demonstrate co-localization as a percentage of all FITC positive cells. (F) Schematic representation of the pathway used by P. gingivalis to evade bactericidal activity without preventing inflammation. *** P ≤ 0.005.
    Figure Legend Snippet: TLR2-PI3K signaling enhances intracellular survival by blocking phago-lysosomal maturation. RAW 264.7 cells were seeded at 3 × 104 cells/ well in Ibidi 8 well m-slides. The cells were untreated (A) or treated with anti-TLR2 (B) , anti-TLR4 (C) , or with the PI3K inhibitor LY 294002 (D) for an hour. Cells were then infected with FITC-labeled P. gingivalis at MOI 10 for 1 h. LysoTracker red was added at 50 nM for the last 10 min of infection. Cells were washed and fixed with 2% formaldehyde and mounted with mounting media. Images were captured using a NIKON confocal microscope at 60X magnification. Yellow color indicates co-localization of P. gingivalis (green) with lysosomes (red). In each field (A–D) the cell in the box is further magnified and shown in the upper right corner. (E) The percent of co-localization was determined by counting cells that demonstrate co-localization as a percentage of all FITC positive cells. (F) Schematic representation of the pathway used by P. gingivalis to evade bactericidal activity without preventing inflammation. *** P ≤ 0.005.

    Techniques Used: Blocking Assay, Infection, Labeling, Microscopy, Activity Assay

    Myd88 −/− neutrophils respond to challenge with P. gingivalis . (A) Total Myd88 −/− bone marrow cells and neutrophil-enriched bone marrow cells were plated 4 * 10 5 cells/well in a 96-well plate and incubated with P. gingivalis (MOI 100). (B) Glycogen-induced Myd88 −/− PECs, or naïve bone marrow Myd88 −/− Ly6G+ neutrophils isolated by positive selection (C) produce TNF-α in response to P. gingivalis challenge but not in response to S. minnesota LPS (1 μg/ml) or Pam3CSK4 (10 μg/ml), or buffer control. (D) The response of Myd88 −/− total and neutrophil enriched BM was compared to the response of WT BM cells. (E) BM cells were collected from WT and Myd88 −/− mice and primed with GM-CSF or IFN-γ for 2 h prior to challenge with P. gingivalis (E) . The percent increase in TNF produced in response to P. gingivalis challenge in primed cells vs. unprimed cells is shown. (A–E) Supernatants were collected after overnight challenge and the level of TNF in the supernatants was measured by ELISA. One representative experiment is shown in each case. Experiments were repeated 3–5 times. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    Figure Legend Snippet: Myd88 −/− neutrophils respond to challenge with P. gingivalis . (A) Total Myd88 −/− bone marrow cells and neutrophil-enriched bone marrow cells were plated 4 * 10 5 cells/well in a 96-well plate and incubated with P. gingivalis (MOI 100). (B) Glycogen-induced Myd88 −/− PECs, or naïve bone marrow Myd88 −/− Ly6G+ neutrophils isolated by positive selection (C) produce TNF-α in response to P. gingivalis challenge but not in response to S. minnesota LPS (1 μg/ml) or Pam3CSK4 (10 μg/ml), or buffer control. (D) The response of Myd88 −/− total and neutrophil enriched BM was compared to the response of WT BM cells. (E) BM cells were collected from WT and Myd88 −/− mice and primed with GM-CSF or IFN-γ for 2 h prior to challenge with P. gingivalis (E) . The percent increase in TNF produced in response to P. gingivalis challenge in primed cells vs. unprimed cells is shown. (A–E) Supernatants were collected after overnight challenge and the level of TNF in the supernatants was measured by ELISA. One representative experiment is shown in each case. Experiments were repeated 3–5 times. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

    Techniques Used: Incubation, Isolation, Selection, Mouse Assay, Produced, Enzyme-linked Immunosorbent Assay

    IFN-γ priming enables TLR2-dependent, MYD88-independent signaling in macrophages in response to P. gingivalis challenge. (A) Naïve Myd88 −/− BMM vs. BMM primed with IFN-γ (100 ng/ml) for 2 h were challenged with P. gingivalis (MOI 100) vs. Pam3CSK4 (10 μg/ml). In (B) Myd88 −/− BMM primed with IFN-γ were challenged with increasing MOI of P. gingivalis . (A,B) Supernatants were collected after overnight stimulation and tested for TNF by ELISA. ** P ≤ 0.01, *** P ≤ 0.005.
    Figure Legend Snippet: IFN-γ priming enables TLR2-dependent, MYD88-independent signaling in macrophages in response to P. gingivalis challenge. (A) Naïve Myd88 −/− BMM vs. BMM primed with IFN-γ (100 ng/ml) for 2 h were challenged with P. gingivalis (MOI 100) vs. Pam3CSK4 (10 μg/ml). In (B) Myd88 −/− BMM primed with IFN-γ were challenged with increasing MOI of P. gingivalis . (A,B) Supernatants were collected after overnight stimulation and tested for TNF by ELISA. ** P ≤ 0.01, *** P ≤ 0.005.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Kinase involvement in the Myd88 −/− response to P. gingivalis . (A) Ly6C+ BM neutrophils or (B–D) BMM were prepared from Myd88 −/− mice and primed with IFN-γ (100 ng/ml for 2 h). (A,B) Inhibitors for PI3K (LY 2940002 100 μM), p38 MAPK (SB 202190 50 μM), mTORC1 (RaPamycin 60 nM) and RAC1 inhibitor (NSC 23766, 50 μM) were added 30 min before challenge with P. gingivalis (MOI 100). DMSO was used as a control at the highest concentration used in the inhibitor wells. Supernatants were collected after overnight stimulation and the percent inhibition of TNF production is shown. (C) Myd88 −/− BMM were similarly primed and Ly294 was added at increasing concentrations 30 min prior to challenge with P. gingivalis . (D) Myd88 −/− BMM were primed with IFN-γ and antibodies (20 μg/ml anti-TLR2 or TLR4 vs. isotype control, I.C.) or LY294 were added prior to challenge with P. gingivalis . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    Figure Legend Snippet: Kinase involvement in the Myd88 −/− response to P. gingivalis . (A) Ly6C+ BM neutrophils or (B–D) BMM were prepared from Myd88 −/− mice and primed with IFN-γ (100 ng/ml for 2 h). (A,B) Inhibitors for PI3K (LY 2940002 100 μM), p38 MAPK (SB 202190 50 μM), mTORC1 (RaPamycin 60 nM) and RAC1 inhibitor (NSC 23766, 50 μM) were added 30 min before challenge with P. gingivalis (MOI 100). DMSO was used as a control at the highest concentration used in the inhibitor wells. Supernatants were collected after overnight stimulation and the percent inhibition of TNF production is shown. (C) Myd88 −/− BMM were similarly primed and Ly294 was added at increasing concentrations 30 min prior to challenge with P. gingivalis . (D) Myd88 −/− BMM were primed with IFN-γ and antibodies (20 μg/ml anti-TLR2 or TLR4 vs. isotype control, I.C.) or LY294 were added prior to challenge with P. gingivalis . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

    Techniques Used: Mouse Assay, Concentration Assay, Inhibition

    TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. gingivalis at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.
    Figure Legend Snippet: TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. gingivalis at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

    Techniques Used: Blocking Assay, Labeling, Fluorescence, Serial Dilution

    Experimental periodontitis induced by oral challenge with P. gingivalis . Groups of mice were administered P. gingivalis in CMC vs. CMC alone by repeated oral gavage. Six weeks later, the maxillae were harvested and alveolar bone volume was measured by μCT from the cemento-enamel junction to a reference line. The residual bone volume of P. gingivalis infected mice was compared to vehicle-treated mice ( n = 8–10 per group). (A) WT, Tlr2 −/− , Myd88 −/− , and Tlr2/Myd88 double knock-out mice (DKO) infected with P. gingivalis ATCC 381. (B) An independent experiment examined bone loss in WT vs. Myd88 −/− mice infected with P. gingivalis ATCC 381 vs. P. gingivalis ATCC 53977. Ns, non-significant. ** P ≤ 0.01, *** P ≤ 0.005.
    Figure Legend Snippet: Experimental periodontitis induced by oral challenge with P. gingivalis . Groups of mice were administered P. gingivalis in CMC vs. CMC alone by repeated oral gavage. Six weeks later, the maxillae were harvested and alveolar bone volume was measured by μCT from the cemento-enamel junction to a reference line. The residual bone volume of P. gingivalis infected mice was compared to vehicle-treated mice ( n = 8–10 per group). (A) WT, Tlr2 −/− , Myd88 −/− , and Tlr2/Myd88 double knock-out mice (DKO) infected with P. gingivalis ATCC 381. (B) An independent experiment examined bone loss in WT vs. Myd88 −/− mice infected with P. gingivalis ATCC 381 vs. P. gingivalis ATCC 53977. Ns, non-significant. ** P ≤ 0.01, *** P ≤ 0.005.

    Techniques Used: Mouse Assay, Infection, Knock-Out

    35) Product Images from "Novel Assay To Characterize Neutrophil Responses to Oral Biofilms"

    Article Title: Novel Assay To Characterize Neutrophil Responses to Oral Biofilms

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00790-18

    Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Neutrophil CD markers are upregulated differentially by monospecies biofilms of commensal and pathogenic bacteria. Human blood was incubated with commensal and pathogenic bacterial biofilms for 1 h at 37°C. The fold increase of surface CD marker expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for each CD marker ( n = 9). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Incubation, Marker

    Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .
    Figure Legend Snippet: Mixed biofilms of S. oralis and S. salivarius result in a lower level of neutrophil activation than the monospecies biofilm of S. oralis , whereas the biofilm mixture of S. oralis and P. gingivalis results in a higher level of neutrophil activation than the monospecies biofilm of S. oralis . Each sample was incubated with 100 μl human whole blood for 1 h. Flow cytometry was performed, and the mean fold increase ± SEM is shown for each CD marker ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (a) The activation of neutrophils through CD16 and CD64 is higher by S. oralis than by S. salivarius . However, in mixed biofilms, this expression is reduced. (b) There is greater neutrophil activation through CD63, CD66, and CD18 by a mixed biofilm of S. oralis and P. gingivalis than by the anaerobically grown monospecies S. oralis . Abbreviations: So, S. oralis ; Ssal, S. salivarius ; Pg, P. gingivalis .

    Techniques Used: Activation Assay, Incubation, Flow Cytometry, Cytometry, Marker, Expressing

    P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.
    Figure Legend Snippet: P. gingivalis lacking gingipains activates neutrophils through CD14, whereas P. gingivalis does not. This suggests that gingipains play an important role in cleaving CD14. Human blood was incubated with P. gingivalis and P. gingivalis gingipain mutant (KDP131 and KDP132) biofilms for 1 h at 37°C. The fold increase in the level of surface CD14 expressions of gated neutrophil populations relative to that for unstimulated controls was determined. The mean fold increase ± SEM is shown for CD14 ( n = 4). ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; Pg, P. gingivalis ; KDP131, P. gingivalis Arg-gingipain A mutant; KDP132, P. gingivalis Arg-gingipain B mutant.

    Techniques Used: Incubation, Mutagenesis

    S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce expression of the NETosis marker H3Cit. Only S. sanguinis results in a significant release of MPO. Human blood neutrophils were incubated with biofilms for 1 h at 37°C. Using flow cytometry, the fold increase in the levels of surface H3Cit (a) and MPO (b) on gated neutrophil populations relative to that for unstimulated controls was determined ( n = 6). *, P ≤ 0.05; ***, P ≤ 0.001. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

    Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: Only the S. oralis biofilm induces neutrophil ROS production. Isolated human blood neutrophils were incubated with the biofilms for 1 h at 37°C. The fold increase in the level of intracellular ROS production relative to that for unstimulated controls was determined ( n = 4). *, P ≤ 0.05. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Isolation, Incubation

    S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .
    Figure Legend Snippet: S. oralis and S. sanguinis induce neutrophil phagocytosis. Biofilms of monospecies were labeled with pHrodo Red for 1 h. Neutrophils were then incubated with labeled bacteria for 1 h at 37°C. The mean fluorescent intensity of pHrodo Red was measured using flow cytometry. S. oralis and S. sanguinis resulted in more neutrophil phagocytosis ( n = 3). *, P ≤ 0.05; **, P ≤ 0.01. Abbreviations: unstim, unstimulated; So, S. oralis ; Ssang, S. sanguinis ; Ssal, S. salivarius ; Sm, S. mutans ; Aa, A. actinomycetemcomitans ; Pg, P. gingivalis .

    Techniques Used: Labeling, Incubation, Flow Cytometry, Cytometry

    36) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    37) Product Images from "Sialidase Deficiency in Porphyromonas gingivalis Increases IL-12 Secretion in Stimulated Macrophages Through Regulation of CR3, IncRNA GAS5 and miR-21"

    Article Title: Sialidase Deficiency in Porphyromonas gingivalis Increases IL-12 Secretion in Stimulated Macrophages Through Regulation of CR3, IncRNA GAS5 and miR-21

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00100

    The expression of lncRNA GAS5 after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P
    Figure Legend Snippet: The expression of lncRNA GAS5 after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P

    Techniques Used: Expressing

    The expression of lncRNA GAS5 in macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352 after CR3 suppressed by CD11b antibody. * P
    Figure Legend Snippet: The expression of lncRNA GAS5 in macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352 after CR3 suppressed by CD11b antibody. * P

    Techniques Used: Expressing

    The expression of lncRNA MEG3, GAS5, FASLG, BTG2, SPRY2 , and TAGAP genes after macrophages stimulated by P. gingivalis W83 and ΔPG0352. * P
    Figure Legend Snippet: The expression of lncRNA MEG3, GAS5, FASLG, BTG2, SPRY2 , and TAGAP genes after macrophages stimulated by P. gingivalis W83 and ΔPG0352. * P

    Techniques Used: Expressing

    The mechanism of sialidase deficiency increasing IL-12 secretion in P. gingivalis -stimulated macrophages. P. gingivalis can activate CR3 in macrophages, inhibit the expression of lncRNA GAS5, increase the expression of miR-21, decrease the level of IL-12 and subvert phagocytosis by macrophages. The sialidase-deficiency in P. gingivalis attenuates CR3 activation in macrophages, reduces the inhibition of lncRNA GAS5 , induces less miR-21 and more IL-12 in macrophages.
    Figure Legend Snippet: The mechanism of sialidase deficiency increasing IL-12 secretion in P. gingivalis -stimulated macrophages. P. gingivalis can activate CR3 in macrophages, inhibit the expression of lncRNA GAS5, increase the expression of miR-21, decrease the level of IL-12 and subvert phagocytosis by macrophages. The sialidase-deficiency in P. gingivalis attenuates CR3 activation in macrophages, reduces the inhibition of lncRNA GAS5 , induces less miR-21 and more IL-12 in macrophages.

    Techniques Used: Expressing, Activation Assay, Inhibition

    The expression of IL-12p35 (A) and IL-12p40 (B) genes after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P
    Figure Legend Snippet: The expression of IL-12p35 (A) and IL-12p40 (B) genes after macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. * P

    Techniques Used: Expressing

    The levels of IL-12p70 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P
    Figure Legend Snippet: The levels of IL-12p70 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P

    Techniques Used:

    The CR3 expression levels after stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352. (A) The U937-differentiated macrophages membranes stained with DiI appear red, while the nuclei appeared blue (200×). (B) Analysis of fluorescent levels using IMAGEJ software revealed elevated CD11b levels in P. gingivalis W83, ΔPG0352 and comΔPG0352 groups compared with control group. After 6 h post-infection, the fluorescence intensity of CR3 in P. gingivalis W83 group was about 2 times higher than that in the control group, and about 1.5 times higher than that in ΔPG0352 group. There were no significant differences between P. gingivalis W83 and comΔPG0352 groups. * P
    Figure Legend Snippet: The CR3 expression levels after stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352. (A) The U937-differentiated macrophages membranes stained with DiI appear red, while the nuclei appeared blue (200×). (B) Analysis of fluorescent levels using IMAGEJ software revealed elevated CD11b levels in P. gingivalis W83, ΔPG0352 and comΔPG0352 groups compared with control group. After 6 h post-infection, the fluorescence intensity of CR3 in P. gingivalis W83 group was about 2 times higher than that in the control group, and about 1.5 times higher than that in ΔPG0352 group. There were no significant differences between P. gingivalis W83 and comΔPG0352 groups. * P

    Techniques Used: Expressing, Staining, Software, Infection, Fluorescence

    The expression of miR-21 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P
    Figure Legend Snippet: The expression of miR-21 after macrophages stimulated by P. gingivalis W83, ΔPG0352, or comΔPG0352. * P

    Techniques Used: Expressing

    Transmission electron microscope observation of macrophages stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352 respectively (15,000 ×, bar: 2 μm). (A) control group; (B) P. gingivalis W83 group; (C) ΔPG0352 group; (D) comΔPG0352 group. White arrow: P. gingivalis internalized by macrophage; black arrow: P. gingivalis adhering to macrophage surface.
    Figure Legend Snippet: Transmission electron microscope observation of macrophages stimulated by P. gingivalis W83, ΔPG0352 and comΔPG0352 respectively (15,000 ×, bar: 2 μm). (A) control group; (B) P. gingivalis W83 group; (C) ΔPG0352 group; (D) comΔPG0352 group. White arrow: P. gingivalis internalized by macrophage; black arrow: P. gingivalis adhering to macrophage surface.

    Techniques Used: Transmission Assay, Microscopy

    38) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    39) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    40) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    41) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    42) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    43) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    44) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    45) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    46) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    47) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    48) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    49) Product Images from "Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis"

    Article Title: Characterization of Heat-Inducible Expression and Cloning of HtpG (Hsp90 Homologue) of Porphyromonas gingivalis

    Journal: Infection and Immunity

    doi:

    Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.
    Figure Legend Snippet: Comparison of HtpG expression by five strains of P. gingivalis (ATCC 33277, 381, A7A1-28, and ATCC 53978 [W50] and W83). (A) Expression of HtpG protein following heat shock of P. gingivalis . Immunoreactivity with the rabbit anti-human Hsp90 antibody in Western blot analysis is shown in the upper panel. All cultures were grown to early log phase and heat stressed as described in the text. (B) Expression of htpG mRNA transcript following heat shock of P. gingivalis . Northern blot of RNA from heat-stressed (S, 45°C) and unstressed (U, 37°C) RNA from cultures of P. gingivalis strains is shown. Ten micrograms of total RNA was electrophoresed and probed with the 32 P-labeled 0.6-kb heat-denatured DNA fragment encoding the N-terminal region of P. gingivalis HtpG. The location of the 23S and 16S rRNAs are indicated.

    Techniques Used: Expressing, Western Blot, Northern Blot, Labeling

    Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.
    Figure Legend Snippet: Effect of sample processing on PAGE analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.23) as described in Materials and Methods. The bacterial suspensions were split under anaerobic conditions and cultured for an additional hour at 37°C (U) or 45°C (S). The cells were harvested by centrifugation for 10 min at 12,000 × g and then placed in LDS sample buffer, cell lysis buffer or 10% TCA, followed by a final resuspension in LDS sample buffer. Proteins in the gels were then stained with Coomassie blue. Lanes labeled with a superscript “R” were reduced with dithiothreitol. Those without the superscript were not reduced.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Cell Culture, Centrifugation, Lysis, Staining, Labeling

    Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Comparison of anti-Hsp90 and anti-HtpG reactivities with P. gingivalis in Western blot analysis. P. gingivalis ATCC 33277 was grown to mid-log phase ( A 600 = 0.32) at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Membranes were probed with one of three antibodies (monoclonal anti- A. ambisexualis Hsp90 ( A. a. ), rabbit anti- E. coli HtpG ( E. c. ), and rabbit anti-human Hsp90 ( H. s. ). Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Western Blot, Cell Culture

    Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).
    Figure Legend Snippet: Effect of growth phase of P. gingivalis on expression of HtpG. P. gingivalis ATCC 33277 was grown from early log ( A 600 = 0.07) to stationary phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated (upper panel).

    Techniques Used: Expressing, Cell Culture

    Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Kinetics of expression of HtpG following heat shock of P. gingivalis. P. gingivalis ATCC 33277 was grown to mid-log ( A 600 = 0.34) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). Pairs of cell cultures (stressed and unstressed) were harvested by centrifugation at 0, 15, 30, 60, 120, and 240 min poststress. Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Expressing, Cell Culture, Centrifugation

    Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).
    Figure Legend Snippet: Alignments of deduced amino acid sequences of P. gingivalis , E. coli , and A. actinomycetemcomitans HtpG. Optimal alignment of the deduced HtpG peptide sequences of E. coli ), A. actinomycetemcomitans ), and P. gingivalis ).

    Techniques Used:

    Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.
    Figure Legend Snippet: Location of HtpG in subcellular fractions of P. gingivalis. P. gingivalis ATCC 33277 was grown to early log ( A 600 = 0.15) phase at 37°C, split under anaerobic conditions, and cultured for an additional hour at 37°C (U) or 45°C (S). The bacteria were fractionated as described in Materials and Methods. The fractions (left to right) included a whole-cell culture (Culture), a culture supernatant (Sup1), whole cells (Cells), French press product (Lysate), a supernatant fraction of French press product (Cleared Lysate), an ultracentrifuge-pelleted membrane fraction of cleared lysate (Memb), an ultracentrifuge supernatant of cleared lysate (Sup2), and an ultracentrifuge-pelleted vesicle fraction from culture supernate (Vesicles). Membranes were probed with rabbit anti-human Hsp90. Molecular masses of the three major bands (68, 44, and 40 kDa) are indicated.

    Techniques Used: Cell Culture

    50) Product Images from "Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein"

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    Journal: Journal of Periodontal & Implant Science

    doi: 10.5051/jpis.2018.48.1.60

    TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P
    Figure Legend Snippet: TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P

    Techniques Used: Incubation

    Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P
    Figure Legend Snippet: Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P

    Techniques Used: TBARS Assay

    Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P
    Figure Legend Snippet: Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P

    Techniques Used: Activity Assay, Incubation, Produced

    Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.
    Figure Legend Snippet: Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.

    Techniques Used: Staining, Light Microscopy

    51) Product Images from "Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein"

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    Journal: Journal of Periodontal & Implant Science

    doi: 10.5051/jpis.2018.48.1.60

    TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P
    Figure Legend Snippet: TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P

    Techniques Used: Incubation

    Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P
    Figure Legend Snippet: Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P

    Techniques Used: TBARS Assay

    Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P
    Figure Legend Snippet: Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P

    Techniques Used: Activity Assay, Incubation, Produced

    Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.
    Figure Legend Snippet: Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.

    Techniques Used: Staining, Light Microscopy

    52) Product Images from "Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein"

    Article Title: Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

    Journal: Journal of Periodontal & Implant Science

    doi: 10.5051/jpis.2018.48.1.60

    TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P
    Figure Legend Snippet: TBARS analysis of LDL incubated with CuSO 4 and HDL in the presence and absence of Porphyromonas gingivalis . LDL: low-density lipoprotein, HDL: high-density lipoprotein, Control: sample without LDL, HDL, and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis , TBARS: thiobarbituric acid-reactive substances. a) Statistically significant ( P

    Techniques Used: Incubation

    Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P
    Figure Legend Snippet: Quantification of Porphyromonas gingivalis -induced HDL oxidation using a TBARS assay. HDL: high-density lipoprotein, TBARS: thiobarbituric acid-reactive substances, Control: sample without HDL and Porphyromonas gingivalis , P.g. : Porphyromonas gingivalis . a) Statistically significant ( P

    Techniques Used: TBARS Assay

    Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P
    Figure Legend Snippet: Proinflammatory activity by Porphyromonas gingivalis -induced oxidized HDL. (A) Monocytes incubated with Porphyromonas gingivalis in the presence of HDL produced significantly higher levels of TNF-α than monocytes treated with CuSO 4 or with HDL alone. (B) Monocytes incubated with Porphyromonas gingivalis and HDL showed higher MMP-9 activity than cells incubated with HDL alone. HDL: high-density lipoprotein, TNF-α: tumor necrosis factor alpha, MMP: matrix metalloproteinase, P.g. : Porphyromonas gingivalis , Control: sample without Porphyromonas gingivalis . a) Statistically significant ( P

    Techniques Used: Activity Assay, Incubation, Produced

    Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.
    Figure Legend Snippet: Macrophage foam cell formation assessed by Oil Red O staining. The uptake of oxidized HDL by macrophages treated with (A) HDL only, (B) HDL oxidized with Porphyromonas gingivalis , and (C) HDL oxidized with CuSO 4 was determined by light microscopy (scale bar=200 μm). HDL: high-density lipoprotein.

    Techniques Used: Staining, Light Microscopy

    Related Articles

    Clone Assay:

    Article Title:
    Article Snippet: .. Analysis of the cloned gene yielded a sequence of 2,052 bp which we believe to contain the entire htpG gene of P. gingivalis . ..

    Real-time Polymerase Chain Reaction:

    Article Title:
    Article Snippet: .. A serial dilution of synthetic template DNA was included in the qPCR assay to calculate the copy number of P. gingivalis in saliva. qPCR was performed on 2 μl of neat eluate of subgingival plaque (no DNA extraction). .. P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber.

    Article Title:
    Article Snippet: .. A serial dilution of synthetic template DNA was included in the qPCR assay to calculate the copy number of P. gingivalis in saliva. qPCR was performed on 2 μl of neat eluate of subgingival plaque (no DNA extraction). .. Determination of Kgp-dependent growth of P. gingivalis P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber.

    Incubation:

    Article Title:
    Article Snippet: Cells were seeded at a density of 2 × 104 to 4 × 104 cells per well (200 μl of 1 × 105 to 2 × 105 cells/ml) in 96-well black/flat-bottom plates (Greiner) manually coated with collagen type I and then incubated in a CO2 incubator at 37 ° C. When cells reached 70 to 80% confluency (~6 × 104 cells per well), they were challenged with P. gingivalis with or without COR271, COR286, moxifloxacin (32477, Fluka), doxycycline (D9891-5G, Sigma-Aldrich), or semagacestat (S1594, SELLECK) at various concentrations. .. P. gingivalis (ATCC BAA-308) was inoculated from −80°C stock onto a brain heart infusion agar (BD-211065).

    Article Title:
    Article Snippet: P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above. .. Resistance was assessed by incubation of 16 serial passages of P. gingivalis in the defined medium listed previously, and resistance to COR388 was performed simultaneously with cultures incubated with the broad-spectrum antibiotic moxifloxacin.

    Article Title:
    Article Snippet: .. Resistance was assessed by incubation of 16 serial passages of P. gingivalis in the defined medium listed previously, and resistance to COR388 was performed simultaneously with cultures incubated with the broad-spectrum antibiotic moxifloxacin. .. Because COR388 does not completely inhibit P. gingivalis growth in vitro, we defined MIC as the minimum COR388 concentration that produced a partial inhibition cutoff, specifically > 50% inhibition compared to nontreated cultures.

    Article Title:
    Article Snippet: SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP Δ Ig-B , and P. gingivalis Δ K/ Δ RAB-A . .. SH-SY5Y cells and P. gingivalis strains were centrifuged at 1000 g for 10 min at RT in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 2 mM l -glutamine and BSA (200 μg/ml), followed by incubation for 1, 4, and 8 hours, respectively, in a CO2 incubator.

    Article Title:
    Article Snippet: Effects of P. gingivalis infection on tau in SH-SY5Y cells SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP ΔIg-B , and P. gingivalis ΔK/ ΔRAB-A . .. SH-SY5Y cells and P. gingivalis strains were centrifuged at 1000g for 10 min at RT in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 2 mM l -glutamine and BSA (200 μg/ml), followed by incubation for 1, 4, and 8 hours, respectively, in a CO2 incubator.

    Article Title:
    Article Snippet: Overnight cultures were diluted 1:100 in medium for S. oralis (ATCC 10577) and 1:10 in prewarmed fresh medium for S. salivarius (ATCC 13419) and P. gingivalis (ATCC 33277). .. For the mixed biofilm of S. oralis and S. salivarius , the culture of diluted bacteria was mixed equally (100 µl of each bacterium) and incubated at 37°C in 5% CO2 overnight.

    Article Title:
    Article Snippet: .. P. gingivalis was washed in PBS and incubated at 108 CFU/ml in Aβ (10, 30, and 100 μg/ml) for 1 hour at RT and ambient oxygen. .. For IHC, a 10-μl solution was dried on a SuperFrost Plus glass slide (VWR) fixed in 4% paraformaldehyde for 10 min. Slide was then rinsed with PBS and dH2 O, exposed to formic acid, and, after washing with PBS, incubated for 2 hours in PBS, 0.3% Triton X-100, and primary antibodies CAB102 (1:1000) and 4G8 (1:1000; BioLegend).

    Article Title:
    Article Snippet: .. Resistance was assessed by incubation of 16 serial passages of P. gingivalis in the defined medium listed previously, and resistance to COR388 was performed simultaneously with cultures incubated with the broad-spectrum antibiotic moxifloxacin. .. Because COR388 does not completely inhibit P. gingivalis growth in vitro, we defined MIC as the minimum COR388 concentration that produced a partial inhibition cutoff, specifically > 50% inhibition compared to nontreated cultures.

    Infection:

    Article Title:
    Article Snippet: .. M.B. and A.M. designed, performed, and analyzed experiments using a mouse model of P. gingivalis –induced periodontal disease and brain infection. ..

    Article Title:
    Article Snippet: Paragraph title: Oral infection with P. gingivalis induces alveolar bone resorption in the absence of MyD88 ... Surprisingly, in contrast to TLR2-deficient mice, the absence of MyD88 did not prevent P. gingivalis from inducing alveolar bone resorption, as shown for a non-encapsulated P. gingivalis strain (ATCC 381), and an additional strain that expresses a polysaccharide capsule (ATCC 53977; Figures ).

    Article Title:
    Article Snippet: Paragraph title: Effects of P. gingivalis infection on tau in SH-SY5Y cells ... SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP Δ Ig-B , and P. gingivalis Δ K/ Δ RAB-A .

    Article Title:
    Article Snippet: .. Effects of P. gingivalis infection on tau in SH-SY5Y cells SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP ΔIg-B , and P. gingivalis ΔK/ ΔRAB-A . ..

    Article Title:
    Article Snippet: .. M.B. and A.M. designed, performed, and analyzed experiments using a mouse model of P. gingivalis –induced periodontal disease and brain infection. ..

    Article Title:
    Article Snippet: .. We investigated the role of TLR9 in periodontal disease pathogenesis using the P. gingivalis -induced murine periodontitis model. P. gingivalis colonization, as well as P. gingivalis -specific antibody levels in the serum, were assessed to confirm that the mice were successfully infected with P. gingivalis. .. P. gingivalis was detected in WT and TLR9−/− mice at comparable levels 2 weeks postinoculation ( ).

    Article Title:
    Article Snippet: .. To further define the role of gingipains in the induction of brain Aβ1–42 , mice were infected with P. gingivalis W83 (WT) or P. gingivalis lacking Kgp (Δkgp ) or the Rgp-null P. gingivalis mutant strain (ΔRgp) ( ). .. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.

    Article Title:
    Article Snippet: .. To further define the role of gingipains in the induction of brain Aβ1–42 , mice were infected with P. gingivalis W83 (WT) or P. gingivalis lacking Kgp (Δ kgp ) or the Rgp-null P. gingivalis mutant strain (ΔRgp) ( ). .. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.

    Expressing:

    Article Title:
    Article Snippet: .. Sugano et al. also compared cytokine induction in two strains of P. gingivalis , and the results showed that P. gingivalis ATCC 49417 showed statistically higher gene expression of IL-12 induction than that of controls at 24 h post-infection. ..

    Article Title:
    Article Snippet: .. We did not see major differences in expression of the HtpG protein, either at the protein or transcript levels when different strains of P. gingivalis were examined. ..

    Article Title:
    Article Snippet: .. Sugano et al. also compared cytokine induction in two strains of P. gingivalis , and the results showed that P. gingivalis ATCC 49417 showed statistically higher gene expression of IL-12 induction than that of controls at 24 h post-infection. ..

    Modification:

    Article Title:
    Article Snippet: SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP Δ Ig-B , and P. gingivalis Δ K/ Δ RAB-A . .. SH-SY5Y cells and P. gingivalis strains were centrifuged at 1000 g for 10 min at RT in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 2 mM l -glutamine and BSA (200 μg/ml), followed by incubation for 1, 4, and 8 hours, respectively, in a CO2 incubator.

    Article Title:
    Article Snippet: Effects of P. gingivalis infection on tau in SH-SY5Y cells SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP ΔIg-B , and P. gingivalis ΔK/ ΔRAB-A . .. SH-SY5Y cells and P. gingivalis strains were centrifuged at 1000g for 10 min at RT in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 2 mM l -glutamine and BSA (200 μg/ml), followed by incubation for 1, 4, and 8 hours, respectively, in a CO2 incubator.

    Western Blot:

    Article Title:
    Article Snippet: SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP Δ Ig-B , and P. gingivalis Δ K/ Δ RAB-A . .. After the indicated incubation times, cells were collected and cell pellets were lysed with 250 μl of radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail for 10 to 15 min. Total protein (16 μg) was used for WB.

    Article Title:
    Article Snippet: Effects of P. gingivalis infection on tau in SH-SY5Y cells SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP ΔIg-B , and P. gingivalis ΔK/ ΔRAB-A . .. After the indicated incubation times, cells were collected and cell pellets were lysed with 250 μ l of radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail for 10 to 15 min. Total protein (16 μg) was used for WB.

    Derivative Assay:

    Article Title:
    Article Snippet: Sugano et al. also compared cytokine induction in two strains of P. gingivalis , and the results showed that P. gingivalis ATCC 49417 showed statistically higher gene expression of IL-12 induction than that of controls at 24 h post-infection. .. Complement receptor 3 (CR3; CD11b/CD18), which is the most common integrin expressed abundantly in monocytes and neutrophils (Bhat et al., ; Yakubenko et al., ), can recognize and interact with molecules derived from host cells and pathogens.

    Article Title:
    Article Snippet: Sugano et al. also compared cytokine induction in two strains of P. gingivalis , and the results showed that P. gingivalis ATCC 49417 showed statistically higher gene expression of IL-12 induction than that of controls at 24 h post-infection. .. Complement receptor 3 (CR3; CD11b/CD18), which is the most common integrin expressed abundantly in monocytes and neutrophils (Bhat et al., ; Yakubenko et al., ), can recognize and interact with molecules derived from host cells and pathogens.

    Hybridization:

    Article Title:
    Article Snippet: To determine whether this was reflected in a restricted distribution of the ragAB genomic locus, Southern hybridization experiments were performed under stringent conditions. .. Genomic DNAs from laboratory strains of P. gingivalis , P. asaccharolytica ATCC 25260, and E. coli XL-1 Blue were restricted and probed with a 32 P-labelled DNA fragment.

    Immunohistochemistry:

    Article Title:
    Article Snippet: .. F.E. designed, performed, and analyzed IHC and immunofluorescent experiments on human brain tissues; designed, performed, and analyzed experiments on stereotactic injection of gingipains into mouse brain; designed, performed, and analyzed experiments on the antibacterial effects of Aβ on P. gingivalis ; and analyzed brain samples from all animal experiments. .. M.B. and A.M. designed, performed, and analyzed experiments using a mouse model of P. gingivalis –induced periodontal disease and brain infection.

    Article Title:
    Article Snippet: .. F.E. designed, performed, and analyzed IHC and immunofluorescent experiments on human brain tissues; designed, performed, and analyzed experiments on stereotactic injection of gingipains into mouse brain; designed, performed, and analyzed experiments on the antibacterial effects of Aβ on P. gingivalis ; and analyzed brain samples from all animal experiments. .. M.B. and A.M. designed, performed, and analyzed experiments using a mouse model of P. gingivalis –induced periodontal disease and brain infection.

    Activation Assay:

    Article Title:
    Article Snippet: Oral infection with P. gingivalis induces alveolar bone resorption in the absence of MyD88 Repeated oral challenge of mice with P. gingivalis leads to its colonization followed by chronic inflammation, osteoclast activation, and resorption of alveolar bone surrounding the teeth (Baker et al., ). .. Surprisingly, in contrast to TLR2-deficient mice, the absence of MyD88 did not prevent P. gingivalis from inducing alveolar bone resorption, as shown for a non-encapsulated P. gingivalis strain (ATCC 381), and an additional strain that expresses a polysaccharide capsule (ATCC 53977; Figures ).

    Article Title:
    Article Snippet: Paragraph title: Neutrophil activation assay with planktonic bacteria. ... S. oralis (ATCC 10577), A. actinomycetemcomitans (SUNY465), and P. gingivalis (ATCC 33277) were grown on an agar plate.

    Protease Inhibitor:

    Article Title:
    Article Snippet: Because of our prior concerns regarding P. gingivalis proteases, we assessed several extraction protocols (protease inhibitor cocktail and TCA extraction) aimed at circumventing proteolysis. .. Figure compares the results of three extraction protocols on P. gingivalis (ATCC 33277; A 600 = 0.23).

    Article Title:
    Article Snippet: SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP Δ Ig-B , and P. gingivalis Δ K/ Δ RAB-A . .. After the indicated incubation times, cells were collected and cell pellets were lysed with 250 μl of radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail for 10 to 15 min. Total protein (16 μg) was used for WB.

    Article Title:
    Article Snippet: Effects of P. gingivalis infection on tau in SH-SY5Y cells SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP ΔIg-B , and P. gingivalis ΔK/ ΔRAB-A . .. After the indicated incubation times, cells were collected and cell pellets were lysed with 250 μ l of radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail for 10 to 15 min. Total protein (16 μg) was used for WB.

    Cell Culture:

    Article Title:
    Article Snippet: .. P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter. .. F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Passaging:

    Article Title:
    Article Snippet: P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above. .. Cultures were first prepared with drug and moxifloxacin in a range of doses passaging each time for 17 passages and monitoring MIC with each passage.

    Inhibition:

    Article Title:
    Article Snippet: P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above. .. Because COR388 does not completely inhibit P. gingivalis growth in vitro, we defined MIC as the minimum COR388 concentration that produced a partial inhibition cutoff, specifically > 50% inhibition compared to nontreated cultures.

    Polymerase Chain Reaction:

    Article Title:
    Article Snippet: Surprisingly, in contrast to TLR2-deficient mice, the absence of MyD88 did not prevent P. gingivalis from inducing alveolar bone resorption, as shown for a non-encapsulated P. gingivalis strain (ATCC 381), and an additional strain that expresses a polysaccharide capsule (ATCC 53977; Figures ). .. We next tracked the clearance of P. gingivalis by PCR from mouse tissues 24 h and 7 days following the last oral challenge.

    Article Title:
    Article Snippet: Genomic DNAs from laboratory strains of P. gingivalis , P. asaccharolytica ATCC 25260, and E. coli XL-1 Blue were restricted and probed with a 32 P-labelled DNA fragment. .. Analysis of the ragA , ragB , and orf3 distributions was performed as follows: chromosomal DNA was restricted with Cla I and probed with an internal 1.6-kb PCR-amplified fragment of ragA (Fig. A), chromosomal DNA was restricted with Hin dIII and probed with a 1.5-kb PCR-amplified product of ragB (Fig. B), and chromosomal DNA was restricted with Eco RV and probed with a 470-bp Eco RV-restricted fragment of orf3 (Fig. C).

    Injection:

    Article Title:
    Article Snippet: .. F.E. designed, performed, and analyzed IHC and immunofluorescent experiments on human brain tissues; designed, performed, and analyzed experiments on stereotactic injection of gingipains into mouse brain; designed, performed, and analyzed experiments on the antibacterial effects of Aβ on P. gingivalis ; and analyzed brain samples from all animal experiments. .. M.B. and A.M. designed, performed, and analyzed experiments using a mouse model of P. gingivalis –induced periodontal disease and brain infection.

    Article Title:
    Article Snippet: .. F.E. designed, performed, and analyzed IHC and immunofluorescent experiments on human brain tissues; designed, performed, and analyzed experiments on stereotactic injection of gingipains into mouse brain; designed, performed, and analyzed experiments on the antibacterial effects of Aβ on P. gingivalis ; and analyzed brain samples from all animal experiments. .. M.B. and A.M. designed, performed, and analyzed experiments using a mouse model of P. gingivalis –induced periodontal disease and brain infection.

    DNA Extraction:

    Article Title:
    Article Snippet: .. A serial dilution of synthetic template DNA was included in the qPCR assay to calculate the copy number of P. gingivalis in saliva. qPCR was performed on 2 μl of neat eluate of subgingival plaque (no DNA extraction). .. P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber.

    Article Title:
    Article Snippet: .. A serial dilution of synthetic template DNA was included in the qPCR assay to calculate the copy number of P. gingivalis in saliva. qPCR was performed on 2 μl of neat eluate of subgingival plaque (no DNA extraction). .. Determination of Kgp-dependent growth of P. gingivalis P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber.

    Mutagenesis:

    Article Title:
    Article Snippet: .. To further define the role of gingipains in the induction of brain Aβ1–42 , mice were infected with P. gingivalis W83 (WT) or P. gingivalis lacking Kgp (Δkgp ) or the Rgp-null P. gingivalis mutant strain (ΔRgp) ( ). .. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.

    Article Title:
    Article Snippet: .. To further define the role of gingipains in the induction of brain Aβ1–42 , mice were infected with P. gingivalis W83 (WT) or P. gingivalis lacking Kgp (Δ kgp ) or the Rgp-null P. gingivalis mutant strain (ΔRgp) ( ). .. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.

    Isolation:

    Article Title:
    Article Snippet: .. Kgp, RgpB, and RgpA were isolated from culture of P. gingivalis , as described by Potempa and Nguyen ( ). ..

    Article Title:
    Article Snippet: .. Kgp, RgpB, and RgpA were isolated from culture of P. gingivalis , as described by Potempa and Nguyen ( ). ..

    Mouse Assay:

    Article Title:
    Article Snippet: .. Surprisingly, in contrast to TLR2-deficient mice, the absence of MyD88 did not prevent P. gingivalis from inducing alveolar bone resorption, as shown for a non-encapsulated P. gingivalis strain (ATCC 381), and an additional strain that expresses a polysaccharide capsule (ATCC 53977; Figures ). .. We next tracked the clearance of P. gingivalis by PCR from mouse tissues 24 h and 7 days following the last oral challenge.

    Article Title:
    Article Snippet: .. We investigated the role of TLR9 in periodontal disease pathogenesis using the P. gingivalis -induced murine periodontitis model. P. gingivalis colonization, as well as P. gingivalis -specific antibody levels in the serum, were assessed to confirm that the mice were successfully infected with P. gingivalis. .. P. gingivalis was detected in WT and TLR9−/− mice at comparable levels 2 weeks postinoculation ( ).

    Article Title:
    Article Snippet: .. To further define the role of gingipains in the induction of brain Aβ1–42 , mice were infected with P. gingivalis W83 (WT) or P. gingivalis lacking Kgp (Δkgp ) or the Rgp-null P. gingivalis mutant strain (ΔRgp) ( ). .. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.

    Article Title:
    Article Snippet: .. To further define the role of gingipains in the induction of brain Aβ1–42 , mice were infected with P. gingivalis W83 (WT) or P. gingivalis lacking Kgp (Δ kgp ) or the Rgp-null P. gingivalis mutant strain (ΔRgp) ( ). .. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.

    Sequencing:

    Article Title:
    Article Snippet: .. That the identical sequence was determined independently from two strains of P. gingivalis argues strongly that this was not a sequencing artifact. .. No HtpG sequences available through the National Center for Biotechnology Information BLAST server appeared to share significant homology with the C-terminal domain of P. gingivalis HtpG, suggesting that this region is unique. ( ( ATCC 33277.

    Article Title:
    Article Snippet: .. The monoclonal antibody does not appear to detect this epitope in E. coli (not shown), which is supported by the lack of sequence homology with P. gingivalis in the C-terminal regions of their respective HtpG proteins. ..

    Article Title:
    Article Snippet: .. Analysis of the cloned gene yielded a sequence of 2,052 bp which we believe to contain the entire htpG gene of P. gingivalis . ..

    Polyacrylamide Gel Electrophoresis:

    Article Title:
    Article Snippet: .. In contrast, other stress proteins, such as the DnaK and the GroEL homologues, which were used in unrelated studies as controls for stress protein induction in P. gingivalis , appeared to be quite resistant to proteolysis and could be detected even when polyacrylamide gel electrophoresis (PAGE) analysis was performed in the absence of TCA or protease inhibitors. ..

    Staining:

    Article Title:
    Article Snippet: S. oralis (ATCC 10577), S. sanguinis (ATCC 10556), S. salivarius (ATCC 13419), and S. mutans (UA159) were grown aerobically for 24 h. A. actinomycetemcomitans (SUNY465) and P. gingivalis (ATCC 33277) were grown in an anaerobic chamber containing 90% nitrogen, 5% hydrogen, 5% carbon dioxide for 24 h and 48 h, respectively. .. We confirmed biofilm growth and thickness through the crystal violet staining method.

    Article Title:
    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter. .. Bacterial purity was determined by Gram staining, and numbers were estimated by absorbance measurement using the TECAN GENios Multidetection Reader, V.4.51 (Phoenix, Hayward, CA).

    In Vitro:

    Article Title:
    Article Snippet: Paragraph title: Assessment of in vitro resistance of P. gingivalis ... P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above.

    Radio Immunoprecipitation:

    Article Title:
    Article Snippet: SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP Δ Ig-B , and P. gingivalis Δ K/ Δ RAB-A . .. After the indicated incubation times, cells were collected and cell pellets were lysed with 250 μl of radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail for 10 to 15 min. Total protein (16 μg) was used for WB.

    Article Title:
    Article Snippet: Effects of P. gingivalis infection on tau in SH-SY5Y cells SH-SY5Y cells (~2.4 × 106 ) were spin-inoculated with MOIs of 10, 50, and 100 with each of the following: P. gingivalis [American Type Culture Collection (ATCC) BAA-308] [wild type (WT)], P. gingivalis KgP ΔIg-B , and P. gingivalis ΔK/ ΔRAB-A . .. After the indicated incubation times, cells were collected and cell pellets were lysed with 250 μ l of radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail for 10 to 15 min. Total protein (16 μg) was used for WB.

    Knock-Out:

    Article Title:
    Article Snippet: .. P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above. .. Resistance was assessed by incubation of 16 serial passages of P. gingivalis in the defined medium listed previously, and resistance to COR388 was performed simultaneously with cultures incubated with the broad-spectrum antibiotic moxifloxacin.

    Produced:

    Article Title:
    Article Snippet: P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above. .. Because COR388 does not completely inhibit P. gingivalis growth in vitro, we defined MIC as the minimum COR388 concentration that produced a partial inhibition cutoff, specifically > 50% inhibition compared to nontreated cultures.

    Concentration Assay:

    Article Title:
    Article Snippet: Then, 6 μl from the mother plate was transferred into a 96-deep-well plate filled with 594 μl of complete medium–penicillin/streptomycin (1:100 dilution) to 2× testing concentration (128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0 μg/ml). .. P. gingivalis (ATCC BAA-308) was inoculated from −80°C stock onto a brain heart infusion agar (BD-211065).

    Article Title:
    Article Snippet: P. gingivalis (ATCC BAA-308) and P. gingivalis Kgp knockout were thawed, and a culture of OD600 = 1.2 (equals 3 × 109 to 5 × 109 CFU/ml) was prepared as described above. .. Because COR388 does not completely inhibit P. gingivalis growth in vitro, we defined MIC as the minimum COR388 concentration that produced a partial inhibition cutoff, specifically > 50% inhibition compared to nontreated cultures.

    Article Title:
    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days. .. Bacteria were washed in PBS and prepared at a final concentration of 1 × 1010 cells/ml in PBS + 2% methylcellulose.

    Serial Dilution:

    Article Title:
    Article Snippet: .. A serial dilution of synthetic template DNA was included in the qPCR assay to calculate the copy number of P. gingivalis in saliva. qPCR was performed on 2 μl of neat eluate of subgingival plaque (no DNA extraction). .. P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber.

    Article Title:
    Article Snippet: .. A serial dilution of synthetic template DNA was included in the qPCR assay to calculate the copy number of P. gingivalis in saliva. qPCR was performed on 2 μl of neat eluate of subgingival plaque (no DNA extraction). .. Determination of Kgp-dependent growth of P. gingivalis P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber.

    Lysis:

    Article Title:
    Article Snippet: Figure compares the results of three extraction protocols on P. gingivalis (ATCC 33277; A 600 = 0.23). .. As shown, there were significant losses of protein bands in both the cell lysis buffer (protease cocktail) and in the SDS sample buffer alone compared to the use of TCA.

    other:

    Article Title:
    Article Snippet: This relationship was significant only when the individuals were colonized with P. gingivalis .

    Article Title:
    Article Snippet: P. gingivalis was purchased from ATCC.

    Article Title:
    Article Snippet: Because diseased gingival tissues are exposed to total lipids of P. gingivalis through contact with subgingival calculus and previous reports suggest that P. gingivalis can invade gingival tissues ( , , ), the total lipid extract of P. gingivalis should be the principal lipid preparation for evaluating effects on bone cells.

    Article Title:
    Article Snippet: Investigation of the nature of the HtpG protein of P. gingivalis has been driven by two compelling factors.

    Article Title:
    Article Snippet: Our previous studies examined the localization of Hsp90 cross-reactive proteins (epitopes shared with human Hsp90) in P. gingivalis ( ).

    Article Title:
    Article Snippet: Those studies revealed that P. gingivalis possessed 68-, 44-, and 40-kDa polypeptides that reacted strongly with the rabbit anti-human Hsp90 polyclonal antibodies.

    Article Title:
    Article Snippet: To support a potential role of HtpG as a virulence factor of P. gingivalis it was important to demonstrate that HtpG localized in the cell envelope and, thus, could be accessible for interaction with cytoplasmic components of the host cell.

    Article Title:
    Article Snippet: Given the similarities with our findings, we felt that further investigation of the nature of the Hsp90 homologue (HtpG) of P. gingivalis was necessary in order to study its potential contribution to the virulence of the microorganism.

    Article Title:
    Article Snippet: We used a commercially available monoclonal antibody directed against a conserved epitope of the Hsp90 protein of the water mold A. ambisexualis , which reacted with the Hsp90 proteins of numerous species , including humans and P. gingivalis .

    Article Title:
    Article Snippet: Katz J., Chegini N., Shiverick K. T., Lamont R. J., Localization of P. gingivalis in preterm delivery placenta .

    Article Title:
    Article Snippet: However, although the P. gingivalis -treated HDL sample showed higher TNF-α levels, we do not know whether this was caused by other products of P. gingivalis that were affected by P. gingivalis- induced oxidized HDL or directly by the P. gingivalis- induced oxidized HDL.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 76
    ATCC p gingivalis pigment less mutant
    Analysis of LPS to assess Wzz activity by heterologous complementation. Silver-stained polyacrylamide gel displaying the O-antigen LPS profiles of Escherichia coli W3110 (lane 1), EVV16 ( wzzB ) containing pMF19 (lane 2), EVV16/pMF19 containing pBAD vector control (lane 3), EVV16/pMF19 containing PGN_2005-expressing plasmid from Porphyromonas <t>gingivalis</t> (lane 4), EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-SF) from Shigella flexneri (lane 5), and EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-ST) from Salmonella typhimurium (lane 6) (A). Immunoblot analysis of the cell lysates was performed with anti-PGN_2005 mouse polyclonal antiserum to confirm the expression of the PGN_2005 protein in the E. coli EVV16 (pMF19) strain (B).
    P Gingivalis Pigment Less Mutant, supplied by ATCC, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p gingivalis pigment less mutant/product/ATCC
    Average 76 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p gingivalis pigment less mutant - by Bioz Stars, 2020-01
    76/100 stars
      Buy from Supplier

    99
    ATCC p gingivalis strains
    Analysis of LPS to assess Wzz activity by heterologous complementation. Silver-stained polyacrylamide gel displaying the O-antigen LPS profiles of Escherichia coli W3110 (lane 1), EVV16 ( wzzB ) containing pMF19 (lane 2), EVV16/pMF19 containing pBAD vector control (lane 3), EVV16/pMF19 containing PGN_2005-expressing plasmid from Porphyromonas <t>gingivalis</t> (lane 4), EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-SF) from Shigella flexneri (lane 5), and EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-ST) from Salmonella typhimurium (lane 6) (A). Immunoblot analysis of the cell lysates was performed with anti-PGN_2005 mouse polyclonal antiserum to confirm the expression of the PGN_2005 protein in the E. coli EVV16 (pMF19) strain (B).
    P Gingivalis Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p gingivalis strains/product/ATCC
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    p gingivalis strains - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    99
    ATCC p gingivalis
    COR388 target engagement and dose-dependent effects on brain P. <t>gingivalis</t> , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    P Gingivalis, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p gingivalis/product/ATCC
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    p gingivalis - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    Image Search Results


    Analysis of LPS to assess Wzz activity by heterologous complementation. Silver-stained polyacrylamide gel displaying the O-antigen LPS profiles of Escherichia coli W3110 (lane 1), EVV16 ( wzzB ) containing pMF19 (lane 2), EVV16/pMF19 containing pBAD vector control (lane 3), EVV16/pMF19 containing PGN_2005-expressing plasmid from Porphyromonas gingivalis (lane 4), EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-SF) from Shigella flexneri (lane 5), and EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-ST) from Salmonella typhimurium (lane 6) (A). Immunoblot analysis of the cell lysates was performed with anti-PGN_2005 mouse polyclonal antiserum to confirm the expression of the PGN_2005 protein in the E. coli EVV16 (pMF19) strain (B).

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Analysis of LPS to assess Wzz activity by heterologous complementation. Silver-stained polyacrylamide gel displaying the O-antigen LPS profiles of Escherichia coli W3110 (lane 1), EVV16 ( wzzB ) containing pMF19 (lane 2), EVV16/pMF19 containing pBAD vector control (lane 3), EVV16/pMF19 containing PGN_2005-expressing plasmid from Porphyromonas gingivalis (lane 4), EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-SF) from Shigella flexneri (lane 5), and EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-ST) from Salmonella typhimurium (lane 6) (A). Immunoblot analysis of the cell lysates was performed with anti-PGN_2005 mouse polyclonal antiserum to confirm the expression of the PGN_2005 protein in the E. coli EVV16 (pMF19) strain (B).

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: Activity Assay, Staining, Plasmid Preparation, Expressing

    Localization of the PGN_2005 protein in Porphyromonas gingivalis . The cell lysates of the wild type and the PGN_2005 mutant were subjected to immunodetection with anti-PGN_2005 (A). Cell fractionation analysis from the wild type. W, C/P, and TM indicate the whole cell lysate, cytoplasm/periplasm, and total membrane fraction, respectively (B). Five micrograms of protein from the inner membrane (IM) or outer membrane (OM) fractions that were separated by sucrose density gradient centrifugation from the membrane fraction of the wild type were subjected to immunodetection with anti-PGN_2005, anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1 (C).

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Localization of the PGN_2005 protein in Porphyromonas gingivalis . The cell lysates of the wild type and the PGN_2005 mutant were subjected to immunodetection with anti-PGN_2005 (A). Cell fractionation analysis from the wild type. W, C/P, and TM indicate the whole cell lysate, cytoplasm/periplasm, and total membrane fraction, respectively (B). Five micrograms of protein from the inner membrane (IM) or outer membrane (OM) fractions that were separated by sucrose density gradient centrifugation from the membrane fraction of the wild type were subjected to immunodetection with anti-PGN_2005, anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1 (C).

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: Mutagenesis, Immunodetection, Cell Fractionation, Gradient Centrifugation

    Immunoblot analysis of various Porphyromonas gingivalis mutants related to the O-antigen flippase with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analysis of various Porphyromonas gingivalis mutants related to the O-antigen flippase with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: SDS Page

    Immunoblot analyses of various Porphyromonas gingivalis mutants related to the first initiation enzyme of UndPP-glycan with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analyses of various Porphyromonas gingivalis mutants related to the first initiation enzyme of UndPP-glycan with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: SDS Page

    Physical map of the area around the PGN_2005 gene and pigmentation of PGN_2005 mutant. Physical map of the PGN_2005 gene region (A). A triangle indicates the Tn 4400 ' insertion site of the PGN_2005 insertion mutant. Colony pigmentation (B). Porphyromonas gingivalis cells were anaerobically grown on blood agar plates at 35°C for 2 days.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Physical map of the area around the PGN_2005 gene and pigmentation of PGN_2005 mutant. Physical map of the PGN_2005 gene region (A). A triangle indicates the Tn 4400 ' insertion site of the PGN_2005 insertion mutant. Colony pigmentation (B). Porphyromonas gingivalis cells were anaerobically grown on blood agar plates at 35°C for 2 days.

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: Mutagenesis

    Transport model of LPS and CTD proteins. The first initiation enzymes of UndPP-glycan for two LPSs in Porphyromonas gingivalis are WbaP-like proteins (PGN_1896 and PGN_1233). Assembly of UndPP-glycans is achieved at cytoplasmic side of the inner membrane, and the block is then transported onto the periplasmic side of the inner membrane by Wzx (PGN_1033). The nonrandom (modal) chain length of O-antigen is dictated by Wzy and Wzz proteins, which correspond to an O-antigen polymerase (PGN_1242) and O-antigen chain length regulator (PGN_2005), respectively. Then, O-antigen is ligated to preformed lipid A-cores by O-antigen ligase (PGN_1302), resulting in LPS. LPS is transported to the outer membrane by LPS transport proteins, which are poorly characterized in P. gingivalis . The C-terminal domain proteins are transported to the outer membrane by Sec and the Por secretion system/Type IX secretion system (PorSS/T9SS). Currently, the precise glycosylation mechanism of the CTD proteins remains uncertain.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Transport model of LPS and CTD proteins. The first initiation enzymes of UndPP-glycan for two LPSs in Porphyromonas gingivalis are WbaP-like proteins (PGN_1896 and PGN_1233). Assembly of UndPP-glycans is achieved at cytoplasmic side of the inner membrane, and the block is then transported onto the periplasmic side of the inner membrane by Wzx (PGN_1033). The nonrandom (modal) chain length of O-antigen is dictated by Wzy and Wzz proteins, which correspond to an O-antigen polymerase (PGN_1242) and O-antigen chain length regulator (PGN_2005), respectively. Then, O-antigen is ligated to preformed lipid A-cores by O-antigen ligase (PGN_1302), resulting in LPS. LPS is transported to the outer membrane by LPS transport proteins, which are poorly characterized in P. gingivalis . The C-terminal domain proteins are transported to the outer membrane by Sec and the Por secretion system/Type IX secretion system (PorSS/T9SS). Currently, the precise glycosylation mechanism of the CTD proteins remains uncertain.

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: Blocking Assay, Size-exclusion Chromatography

    Immunoblot analyses of various Porphyromonas gingivalis strains. Immunoblot analyses of cell lysates of various P. gingivalis strains were performed with mAb 1B5, mAb TDC-5-2-1, anti-HBP35, or anti-Rgp. Three sets of PGN_2005/PGN_2005+ or PGN_2005 porT/PGN_2005+ strains were obtained from each single clone. The asterisks indicate nonspecific cross-reactive bands.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analyses of various Porphyromonas gingivalis strains. Immunoblot analyses of cell lysates of various P. gingivalis strains were performed with mAb 1B5, mAb TDC-5-2-1, anti-HBP35, or anti-Rgp. Three sets of PGN_2005/PGN_2005+ or PGN_2005 porT/PGN_2005+ strains were obtained from each single clone. The asterisks indicate nonspecific cross-reactive bands.

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques:

    Immunoblot analyses of various Porphyromonas gingivalis strains. The cell lysates of various P. gingivalis strains were subjected to SDS-PAGE, and immunoblot analyses were performed with anti-HBP35, anti-Rgp, mAb1B5, or mAb TDC-5-2-1. The asterisks indicate nonspecific cross-reactive bands (A). Immunoblot analyses of various P. gingivalis A-LPS-deficient mutants. Cell lysates of various P. gingivalis A-LPS-deficient mutants were subjected to SDS-PAGE, and immunoblot analysis was performed with mAb TDC-5-2-1 (B).

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analyses of various Porphyromonas gingivalis strains. The cell lysates of various P. gingivalis strains were subjected to SDS-PAGE, and immunoblot analyses were performed with anti-HBP35, anti-Rgp, mAb1B5, or mAb TDC-5-2-1. The asterisks indicate nonspecific cross-reactive bands (A). Immunoblot analyses of various P. gingivalis A-LPS-deficient mutants. Cell lysates of various P. gingivalis A-LPS-deficient mutants were subjected to SDS-PAGE, and immunoblot analysis was performed with mAb TDC-5-2-1 (B).

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques: SDS Page

    Gingipain and hemagglutination activities of Porphyromonas gingivalis . Porphyromonas gingivalis cells were anaerobically grown in enriched BHI medium at 35°C. The Kgp and Rgp activities of the cell lysates (cell) and vesicle-containing culture supernatants (sup) of ATCC 33277 (wild type), PGN_2005, or PGN_2005/PGN2005+ were measured (A). The hemagglutination activities of various P. gingivalis strains were measured (B). Twofold serial dilutions of various P. gingivalis cells were mixed with 1% sheep red blood cells and stored for 3 h at room temperature.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Gingipain and hemagglutination activities of Porphyromonas gingivalis . Porphyromonas gingivalis cells were anaerobically grown in enriched BHI medium at 35°C. The Kgp and Rgp activities of the cell lysates (cell) and vesicle-containing culture supernatants (sup) of ATCC 33277 (wild type), PGN_2005, or PGN_2005/PGN2005+ were measured (A). The hemagglutination activities of various P. gingivalis strains were measured (B). Twofold serial dilutions of various P. gingivalis cells were mixed with 1% sheep red blood cells and stored for 3 h at room temperature.

    Article Snippet: In conclusion, we found a novel P. gingivalis pigment-less mutant by transposon mutagenesis: the gene responsible was the PGN_2005 gene of strain ATCC 33277.

    Techniques:

    Analysis of LPS to assess Wzz activity by heterologous complementation. Silver-stained polyacrylamide gel displaying the O-antigen LPS profiles of Escherichia coli W3110 (lane 1), EVV16 ( wzzB ) containing pMF19 (lane 2), EVV16/pMF19 containing pBAD vector control (lane 3), EVV16/pMF19 containing PGN_2005-expressing plasmid from Porphyromonas gingivalis (lane 4), EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-SF) from Shigella flexneri (lane 5), and EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-ST) from Salmonella typhimurium (lane 6) (A). Immunoblot analysis of the cell lysates was performed with anti-PGN_2005 mouse polyclonal antiserum to confirm the expression of the PGN_2005 protein in the E. coli EVV16 (pMF19) strain (B).

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Analysis of LPS to assess Wzz activity by heterologous complementation. Silver-stained polyacrylamide gel displaying the O-antigen LPS profiles of Escherichia coli W3110 (lane 1), EVV16 ( wzzB ) containing pMF19 (lane 2), EVV16/pMF19 containing pBAD vector control (lane 3), EVV16/pMF19 containing PGN_2005-expressing plasmid from Porphyromonas gingivalis (lane 4), EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-SF) from Shigella flexneri (lane 5), and EVV16/pMF19 containing WzzB-expressing plasmid (pWzzB-ST) from Salmonella typhimurium (lane 6) (A). Immunoblot analysis of the cell lysates was performed with anti-PGN_2005 mouse polyclonal antiserum to confirm the expression of the PGN_2005 protein in the E. coli EVV16 (pMF19) strain (B).

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: Activity Assay, Staining, Plasmid Preparation, Expressing

    Localization of the PGN_2005 protein in Porphyromonas gingivalis . The cell lysates of the wild type and the PGN_2005 mutant were subjected to immunodetection with anti-PGN_2005 (A). Cell fractionation analysis from the wild type. W, C/P, and TM indicate the whole cell lysate, cytoplasm/periplasm, and total membrane fraction, respectively (B). Five micrograms of protein from the inner membrane (IM) or outer membrane (OM) fractions that were separated by sucrose density gradient centrifugation from the membrane fraction of the wild type were subjected to immunodetection with anti-PGN_2005, anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1 (C).

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Localization of the PGN_2005 protein in Porphyromonas gingivalis . The cell lysates of the wild type and the PGN_2005 mutant were subjected to immunodetection with anti-PGN_2005 (A). Cell fractionation analysis from the wild type. W, C/P, and TM indicate the whole cell lysate, cytoplasm/periplasm, and total membrane fraction, respectively (B). Five micrograms of protein from the inner membrane (IM) or outer membrane (OM) fractions that were separated by sucrose density gradient centrifugation from the membrane fraction of the wild type were subjected to immunodetection with anti-PGN_2005, anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1 (C).

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: Mutagenesis, Immunodetection, Cell Fractionation, Gradient Centrifugation

    Immunoblot analysis of various Porphyromonas gingivalis mutants related to the O-antigen flippase with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analysis of various Porphyromonas gingivalis mutants related to the O-antigen flippase with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: SDS Page

    Immunoblot analyses of various Porphyromonas gingivalis mutants related to the first initiation enzyme of UndPP-glycan with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analyses of various Porphyromonas gingivalis mutants related to the first initiation enzyme of UndPP-glycan with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1. Cell lysates of various P. gingivalis mutants were subjected to SDS-PAGE and immunoblot analysis with anti-HBP35, anti-Rgp, mAb 1B5, and mAb TDC-5-2-1.

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: SDS Page

    Physical map of the area around the PGN_2005 gene and pigmentation of PGN_2005 mutant. Physical map of the PGN_2005 gene region (A). A triangle indicates the Tn 4400 ' insertion site of the PGN_2005 insertion mutant. Colony pigmentation (B). Porphyromonas gingivalis cells were anaerobically grown on blood agar plates at 35°C for 2 days.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Physical map of the area around the PGN_2005 gene and pigmentation of PGN_2005 mutant. Physical map of the PGN_2005 gene region (A). A triangle indicates the Tn 4400 ' insertion site of the PGN_2005 insertion mutant. Colony pigmentation (B). Porphyromonas gingivalis cells were anaerobically grown on blood agar plates at 35°C for 2 days.

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: Mutagenesis

    Transport model of LPS and CTD proteins. The first initiation enzymes of UndPP-glycan for two LPSs in Porphyromonas gingivalis are WbaP-like proteins (PGN_1896 and PGN_1233). Assembly of UndPP-glycans is achieved at cytoplasmic side of the inner membrane, and the block is then transported onto the periplasmic side of the inner membrane by Wzx (PGN_1033). The nonrandom (modal) chain length of O-antigen is dictated by Wzy and Wzz proteins, which correspond to an O-antigen polymerase (PGN_1242) and O-antigen chain length regulator (PGN_2005), respectively. Then, O-antigen is ligated to preformed lipid A-cores by O-antigen ligase (PGN_1302), resulting in LPS. LPS is transported to the outer membrane by LPS transport proteins, which are poorly characterized in P. gingivalis . The C-terminal domain proteins are transported to the outer membrane by Sec and the Por secretion system/Type IX secretion system (PorSS/T9SS). Currently, the precise glycosylation mechanism of the CTD proteins remains uncertain.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Transport model of LPS and CTD proteins. The first initiation enzymes of UndPP-glycan for two LPSs in Porphyromonas gingivalis are WbaP-like proteins (PGN_1896 and PGN_1233). Assembly of UndPP-glycans is achieved at cytoplasmic side of the inner membrane, and the block is then transported onto the periplasmic side of the inner membrane by Wzx (PGN_1033). The nonrandom (modal) chain length of O-antigen is dictated by Wzy and Wzz proteins, which correspond to an O-antigen polymerase (PGN_1242) and O-antigen chain length regulator (PGN_2005), respectively. Then, O-antigen is ligated to preformed lipid A-cores by O-antigen ligase (PGN_1302), resulting in LPS. LPS is transported to the outer membrane by LPS transport proteins, which are poorly characterized in P. gingivalis . The C-terminal domain proteins are transported to the outer membrane by Sec and the Por secretion system/Type IX secretion system (PorSS/T9SS). Currently, the precise glycosylation mechanism of the CTD proteins remains uncertain.

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: Blocking Assay, Size-exclusion Chromatography

    Immunoblot analyses of various Porphyromonas gingivalis strains. Immunoblot analyses of cell lysates of various P. gingivalis strains were performed with mAb 1B5, mAb TDC-5-2-1, anti-HBP35, or anti-Rgp. Three sets of PGN_2005/PGN_2005+ or PGN_2005 porT/PGN_2005+ strains were obtained from each single clone. The asterisks indicate nonspecific cross-reactive bands.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analyses of various Porphyromonas gingivalis strains. Immunoblot analyses of cell lysates of various P. gingivalis strains were performed with mAb 1B5, mAb TDC-5-2-1, anti-HBP35, or anti-Rgp. Three sets of PGN_2005/PGN_2005+ or PGN_2005 porT/PGN_2005+ strains were obtained from each single clone. The asterisks indicate nonspecific cross-reactive bands.

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques:

    Immunoblot analyses of various Porphyromonas gingivalis strains. The cell lysates of various P. gingivalis strains were subjected to SDS-PAGE, and immunoblot analyses were performed with anti-HBP35, anti-Rgp, mAb1B5, or mAb TDC-5-2-1. The asterisks indicate nonspecific cross-reactive bands (A). Immunoblot analyses of various P. gingivalis A-LPS-deficient mutants. Cell lysates of various P. gingivalis A-LPS-deficient mutants were subjected to SDS-PAGE, and immunoblot analysis was performed with mAb TDC-5-2-1 (B).

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Immunoblot analyses of various Porphyromonas gingivalis strains. The cell lysates of various P. gingivalis strains were subjected to SDS-PAGE, and immunoblot analyses were performed with anti-HBP35, anti-Rgp, mAb1B5, or mAb TDC-5-2-1. The asterisks indicate nonspecific cross-reactive bands (A). Immunoblot analyses of various P. gingivalis A-LPS-deficient mutants. Cell lysates of various P. gingivalis A-LPS-deficient mutants were subjected to SDS-PAGE, and immunoblot analysis was performed with mAb TDC-5-2-1 (B).

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques: SDS Page

    Gingipain and hemagglutination activities of Porphyromonas gingivalis . Porphyromonas gingivalis cells were anaerobically grown in enriched BHI medium at 35°C. The Kgp and Rgp activities of the cell lysates (cell) and vesicle-containing culture supernatants (sup) of ATCC 33277 (wild type), PGN_2005, or PGN_2005/PGN2005+ were measured (A). The hemagglutination activities of various P. gingivalis strains were measured (B). Twofold serial dilutions of various P. gingivalis cells were mixed with 1% sheep red blood cells and stored for 3 h at room temperature.

    Journal: MicrobiologyOpen

    Article Title: Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    doi: 10.1002/mbo3.84

    Figure Lengend Snippet: Gingipain and hemagglutination activities of Porphyromonas gingivalis . Porphyromonas gingivalis cells were anaerobically grown in enriched BHI medium at 35°C. The Kgp and Rgp activities of the cell lysates (cell) and vesicle-containing culture supernatants (sup) of ATCC 33277 (wild type), PGN_2005, or PGN_2005/PGN2005+ were measured (A). The hemagglutination activities of various P. gingivalis strains were measured (B). Twofold serial dilutions of various P. gingivalis cells were mixed with 1% sheep red blood cells and stored for 3 h at room temperature.

    Article Snippet: Via genome analysis, we revealed that P. gingivalis strains have 34 and 33 proteins, including a conserved C-terminal domain, in strains W83 and strain ATCC 33277, respectively.

    Techniques:

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Journal: Science Advances

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    doi: 10.1126/sciadv.aau3333

    Figure Lengend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Article Snippet: P. gingivalis [W83 (ATCC, Rockville, MD), ΔKgp (Δ kgp ), and ΔRgp (Δ rgpArgpBΔ495-B Cmr , Emr ] ( , ) was streaked on tryptic soy broth (TSB) agar plates [5% sheep blood, supplemented with l -cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K (0.5 μg/ml)] and grown under anaerobic conditions at 37°C for 5 to 7 days.

    Techniques: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing