Journal: Frontiers in Microbiology

Article Title: Evaluation of the probiotic, technological, safety attributes, and GABA-producing capacity of microorganisms isolated from Iranian milk kefir beverages

doi: 10.3389/fmicb.2024.1385301

Figure Lengend Snippet: The percentage of co-aggregation of the isolates with E. coli and L. monocytogenes as the average of three replicates with standard deviation at 2, 4, 6, 12, and 24 h of incubation at 37°C. L. casei was considered the standard probiotic strain. *indicates that L. casei had significantly the highest co-aggregation activity with both food-borne pathogenes among the strains and the standard strain after 24 h ( p -value < 0.05).

Article Snippet: A 0 = Absorption at 0 h. A 1 = Absorption at 2, 4, 6, 8, and 24 h. To evaluate the co-aggregation ability, an equal amount of isolates and pathogenic bacteria, including Escherichia coli (PTCC 1338) and Listeria monocytogenes (ATCC 7644) with the concentration of 1.5 × 10 8 CFU/mL were prepared, mixed, and vortexed for 10 s. The absorbance of the mixture suspensions was measured at 600 nm at 0, 2, 4, 6, 8, and 24 h by a spectrophotometer ( Lactobacillus casei PTTC 1608 was used as the standard probiotic strain).

Techniques: Standard Deviation, Incubation, Activity Assay

Journal: Frontiers in Microbiology

Article Title: Evaluation of the probiotic, technological, safety attributes, and GABA-producing capacity of microorganisms isolated from Iranian milk kefir beverages

doi: 10.3389/fmicb.2024.1385301

Figure Lengend Snippet: The antimicrobial activity of the isolates against S. typhimurium , E. coli , B. cereus , and L. monocytogenes. The letters B, E, L, and P represent blank (Sterile distilled water), E. faecalis , L. lactis , and P. fermentans , respectively.

Article Snippet: A 0 = Absorption at 0 h. A 1 = Absorption at 2, 4, 6, 8, and 24 h. To evaluate the co-aggregation ability, an equal amount of isolates and pathogenic bacteria, including Escherichia coli (PTCC 1338) and Listeria monocytogenes (ATCC 7644) with the concentration of 1.5 × 10 8 CFU/mL were prepared, mixed, and vortexed for 10 s. The absorbance of the mixture suspensions was measured at 600 nm at 0, 2, 4, 6, 8, and 24 h by a spectrophotometer ( Lactobacillus casei PTTC 1608 was used as the standard probiotic strain).

Techniques: Activity Assay, Sterility

Journal: mBio

Article Title: Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii

doi: 10.1128/mBio.01660-15

Figure Lengend Snippet: G. mellonella differentiates pathogenic and nonpathogenic Acinetobacter strains. (A) G. mellonella larvae were inoculated with 10 6 CFU of the strains indicated . Larvae were homogenized and bacteria were quantified immediately following infection ( t = 0) and after 4 h at 37°C ( t = 4). Data from a representative experiment are presented as the ratio of the number of CFU recovered at t = 4 to the number of CFU recovered at t = 0 (error bars, 1 standard deviation). (B) G. mellonella larvae were inoculated with 10 6 CFU of the strains indicated . Survival was monitored daily for 6 days. ****, P < 0.0001; ***, P < 0.001. ADP1, A. baylyi ADP1; 17978, A. baumannii ATCC 17978; AB5075, A. baumannii AB5075.

Article Snippet: For example, shows that Acinetobacter baylyi , a well-characterized, nonpathogenic Acinetobacter species, does not survive for 4 h within larvae, whereas the human-pathogenic isolates ATCC 17978 and AB5075 survive and grow within the larvae during the same time period.

Techniques: Bacteria, Infection, Standard Deviation

Journal: BMC Oral Health

Article Title: Selenium nanoparticles as a biological safe endodontic irrigant improving root canal sealing

doi: 10.1186/s12903-025-07535-5

Figure Lengend Snippet: Cytological and antibacterial activity of SeNPs. The dose-dependent curves of ( a ) SeNPs and ( b ) NaOCl on OEC and HGFs reveal the safer cytological range of SeNPs over the acute toxicity of NaOCl on both cell lineages, with slight resistance of fibroblasts over epithelial cells. ( c ) The agar diffusion assay shows the inhibition zones (red lines) after 24 h of E. faecalis incubation with SeNPs, taking ampicillin as a positive control and sterile qH2O as a negative control. One-way ANOVA followed by Tukey post hoc test reveals a significant difference in bacterial inhibition zones between SeNPs and the negative control (*** p < 0.001), in contrast to the non-significant difference (ns, p > 0.05) observed between SeNPs and the positive antibacterial control

Article Snippet: A standardized bacterial suspension of E. faecalis (ATCC 47077; Rockville, MD, USA) was prepared by culturing the strain in brain heart infusion (BHI) broth (HiMedia, India) for 24 h at 37 °C, followed by adjustment to a concentration of 1 × 108 CFU/mL using a spectrophotometer (Fisher Scientific, USA, OD600) [ ].

Techniques: Activity Assay, Diffusion-based Assay, Inhibition, Incubation, Positive Control, Sterility, Negative Control, Control

Journal: BMC Oral Health

Article Title: Selenium nanoparticles as a biological safe endodontic irrigant improving root canal sealing

doi: 10.1186/s12903-025-07535-5

Figure Lengend Snippet: Assessment of dentinal surface morphology and bacterial presence following root canal irrigation. a - c Representative SEM micrographs (Scale bar = 5 μm for ×4000). a The control unirrigated group demonstrates bacterial colonization and exposure of the dentinal tubules. The presence of rod-shaped E. faecalis bacteria adherent to the dentinal surface and within tubules is evident (red arrows). b The NaOCl group reveals numerous open and clean dentinal tubules free of smear layer and debris in the middle and apical regions, indicating effective smear layer removal and deproteinization. c The SeNPs group at the middle and apical regions, respectively, exhibits a partially covered surface with granular aggregates of SeNPs (green arrows). The partially occluded tubules show clean surfaces with no observed bacterial structures. d A violin plot for the bacterial score ( n = 8/group) analyzed by Kruskal-Wallis, followed by Dunn–Bonferroni post hoc analysis, reveals a significant reduction in the median score in the NaOCl*** ( p < 0.001) and SeNPs* ( p < 0.05) groups compared separately with the control unirrigated group. Meanwhile, ns ( p > 0.05) marks the equivalent capacity in bacterial reduction between NaOCl and SeNPs

Article Snippet: A standardized bacterial suspension of E. faecalis (ATCC 47077; Rockville, MD, USA) was prepared by culturing the strain in brain heart infusion (BHI) broth (HiMedia, India) for 24 h at 37 °C, followed by adjustment to a concentration of 1 × 108 CFU/mL using a spectrophotometer (Fisher Scientific, USA, OD600) [ ].

Techniques: Control, Bacteria

Journal: Saudi Journal of Biological Sciences

Article Title: Isolation and characterization of lytic bacteriophages from sewage at an egyptian tertiary care hospital against methicillin-resistant Staphylococcus aureus clinical isolates

doi: 10.1016/j.sjbs.2022.03.019

Figure Lengend Snippet: OD 600 records of S.aureus (reference strain & 3 MRSA isolates).

Article Snippet: Under aseptic conditions, twenty µl of bacterial glycerol stocks (MRSA clinical strain and standard strain of S. aureus ATCC 25923) were inoculated into 2 ml Luria-Bertani (LB) broth (10 g tryptone, 10 g NaCl, and 5 g yeast extract per liter) and incubated overnight at 37 °C.

Techniques: Infection

Journal:

Article Title: Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

doi: 10.1128/IAI.71.5.2365-2372.2003

Figure Lengend Snippet: Bacterial species and strains used in this study and their invasion of HUVEC monolayers

Article Snippet: Streptococcus gordonii strain CH1 (Challis) was chosen as the paradigm for most of the experiments reported here because it is virulent in the rat model of infection ( 59 ), several adhesion-deficient mutants were available for comparison ( 14 , 39 , 40 ), and it produces large amounts of the alpha-hemolysin (hydrogen peroxide) ( 3 , 4 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Description Source or reference HUVEC invasion a S. gordonii DL1 (Challis) Wild-type reference strain 52 17 ± 5 EM230 DL1 hsa :: ermAM 52 4 ± 2 OB219 DL1 sspA ′ sspB ′:: ermAM 14 20 ± 4 OB220 DL1 sspA :: ermAM 14 19 ± 6 OB235 DL1 cshA3 :: ermAM 40 2 ± 0.4 OB271 DL1 cshB2 :: ermAM 40 2 ± 1 OB277 DL1 cshA31 :: cat cshB2 :: ermAM 40 2 ± 0.6 CH1 (Challis) Wild-type reference strain 59 82 ± 15 AMS12 CH1 with 1.7-kb Hin d fragment of gtfG replaced with lacZ /Erm r 59 14 ± 0.5 M5 Wild type from human dental plaque 28 5 ± 2.5 S. sanguis L22 Wild type from human dental plaque 28 76 ± 11 L52 Wild type from human dental plaque 28 9 ± 2 L74 Wild type from human dental plaque 28 52 ± 8 L79 Wild type from human dental plaque 28 14 ± 5 133-79 Wild type from human endocarditis 28 21 ± 5 2017-78 Wild type from human endocarditis 28 25 ± 3 10556 Wild type from human endocarditis ATCC 98 ± 18 S. oralis KS32AR Wild type from dental plaque 8 9 ± 8 16532AR Wild type from dental plaque 8 3 ± 2 S. mitis NCTC 10712 Wild type from dental plaque 8 23 ± 6 OP51 Wild type from dental plaque 8 35 ± 9 S. mutans UA159 Wild type from dental plaque 60 26 ± 7 S. salivarius 13419 Wild type from human saliva ATCC 2 ± 1 E. coli HB101 Hybrid of strains K-12 and B, transformable strain 7 <0.01 Open in a separate window a Average number of intracellular bacteria (× 10 4 ) per HUVEC monolayer ± standard deviation of the mean ( n = 6). b ATCC, American Type Culture Collection.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: Structure and analysis of E. coli O157 LPS molecules. ( A ) Schematic representation of E. coli O157 LPS molecule, showing O-antigen, outer core, inner core, and lipid A. Dotted lines indicate the level of LPS truncation resulting from each mutation (Kdo, 3-deoxy-D-manno-octulosonic acid; PPEtN, pyrophosphorylethanolamine; Hep, heptose; GlcNAc, N-acetylglucosamine; Glc, glucose; Gal, galactose). ( B ) LPS was isolated from wild-type and mutant cells of E. coli O157 43895 and analyzed by silver-stained Tricine–SDS-PAGE, as described in the Materials and Methods section. The expected location of O-antigen regions and the core is indicated on the left side of the gel.

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Mutagenesis, Isolation, Staining, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: Biofilm formation of E. coli O157 43895 WT and mutant derivatives. ( A ) Pellicle formation at 37 °C. Pellicle rings of a 48 h old floating biofilm of E. coli O157 43895. Mutants were detected by crystal violet (CV) staining. ( B ) Biofilm measurement using a standard microtiter assay. E. coli O157 43895 WT and mutants were incubated at 37 °C in MSM with 4% glucose. Bound CV was solubilized with 95% ethanol and quantified by absorbance at 595 nm. Assays were performed six times for each strain. Mean values with standard deviation are shown. Statistical analysis used one-way ANOVA test and Dunnett post-test. * p < 0.01, ** p < 0.001, compared with the wild type (WT).

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Mutagenesis, Staining, Incubation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: The effect of LPS-core truncation on the swimming motility and flagella biosynthesis of E. coli O157 43895. Swimming motility of wild-type and LPS mutants was assessed using soft-agar swarm plates. After 40 h incubation, the diameter of the motility halo was measured. Experiments were repeated three times. Presence of flagella was detected by immunoblot (lower panel). Mean values with standard deviation are shown. Statistical analysis used two-tailed unpaired t -test. * p < 0.01, ** p < 0.001, compared with the wild type (WT).

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Incubation, Western Blot, Standard Deviation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: The effect of LPS-core truncation on the curli biosynthesis of E. coli O157 43895. E. coli O157 43895 and mutants were grown on Congo-red (CR) indicator plates at 37 °C for 24 h. Presence of curli on the cell surface was detected by CR binding.

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: The effect of LPS core-OS truncation on cell adherence and invasion. ( A ) The adherence of LPS-core and flagella mutants of E. coli O157 43895 to epithelial cells was examined. The monolayers of MAC-T cells on coverslips in 24-well plates were co-cultured with the bacteria at 37 °C for 3 h. The cells were washed five times with PBS and fixed with 4% paraformaldehyde for 15 min, washed again with PBS, stained with Giemsa stain, and viewed under the microscope (magnification ×1000). ( B ) E. coli O157 43895 and the mutants were tested for epithelial-cell invasion using a MAC-T gentamicin protection assay, as described in the Materials and Methods section. Bacteria were co-cultured with MAC-T monolayers at a multiplicity of infection (MOI) of 10. The experiment was repeated three times with three replicates per strain, and CFUs were determined by plate count and were transformed to log10 value. Mean values with standard deviation are shown. Statistical analysis used one-way ANOVA test and Dunnett post-test. * p < 0.01, ** p < 0.001, compared with the wild type (WT).

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Cell Culture, Bacteria, Staining, Giemsa Stain, Microscopy, Infection, Transformation Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability

doi: 10.3390/ijms25179224

Figure Lengend Snippet: E. coli O157 strains and plasmids.

Article Snippet: Unlike non-invasive E. coli O157 strains, ATCC 43895 forms a robust pellicle biofilm at 37 °C.

Techniques: Plasmid Preparation, Mutagenesis

Journal: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology

Article Title: Effective photoinactivation of Gram-positive and Gram-negative bacterial strains using an HIV-1 Tat peptide-porphyrin conjugate.

doi: 10.1039/c0pp00146e

Figure Lengend Snippet: Fig. 3 Effect of drug concentration on bacterial photoinactivation with Tat-porphyrin. Survival curves of S. aureus (A), S. pyogenes (B), P. aeruginosa (C) and E. coli (D) incubated with 0.5 or 1 mM of Tat-porphyrin, irradiation time of 43 s giving a fluence of 10 J cm-2. Dark control (-L), light control (+L). Error bars indicate the standard deviation. * P < 0.05.

Article Snippet: The Tat–porphyrin compound has a good singlet oxygen quantum yield of 0.54 (T.-W. Wang and A. J. MacRobert, unpublished data) as would be expected for a porphyrin derivative, and for comparison the porphyrin itself has a singlet oxygen quantum yield of 0.56 as reported by Tanielian et al.41 Bacterial strains and growth conditions Two Gram (+) positive organisms were used in this study; Staphylococcus aureus strain 8325-4 and Streptococcus pyogenes strain 12202, and two Gram (-) bacteria Pseudomonas aeruginosa strain PA01 and Escherichia coli ATCC 25922 strain were used.

Techniques: Concentration Assay, Incubation, Irradiation, Control, Standard Deviation

Journal: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology

Article Title: Effective photoinactivation of Gram-positive and Gram-negative bacterial strains using an HIV-1 Tat peptide-porphyrin conjugate.

doi: 10.1039/c0pp00146e

Figure Lengend Snippet: Fig. 2 Tat-porphyrin fluorescence after 30 min (A) or 5 h (B) incubation with S. pyogenes (□), S. aureus ( ), E. coli (⊡) and P. aeruginosa (■).

Article Snippet: The Tat–porphyrin compound has a good singlet oxygen quantum yield of 0.54 (T.-W. Wang and A. J. MacRobert, unpublished data) as would be expected for a porphyrin derivative, and for comparison the porphyrin itself has a singlet oxygen quantum yield of 0.56 as reported by Tanielian et al.41 Bacterial strains and growth conditions Two Gram (+) positive organisms were used in this study; Staphylococcus aureus strain 8325-4 and Streptococcus pyogenes strain 12202, and two Gram (-) bacteria Pseudomonas aeruginosa strain PA01 and Escherichia coli ATCC 25922 strain were used.

Techniques: Incubation

Journal: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology

Article Title: Effective photoinactivation of Gram-positive and Gram-negative bacterial strains using an HIV-1 Tat peptide-porphyrin conjugate.

doi: 10.1039/c0pp00146e

Figure Lengend Snippet: Fig. 4 Effect of light dose on bacterial photoinactivation with Tat-por- phyrin. Survival curves of S. aureus (A), S. pyogenes (B), P. aeruginosa (C) and E. coli (D) incubated with 0.5 mM of Tat-porphyrin and irradiated for 30, 43, 60 and 120 s corresponding respectively to 7, 10, 14 and 28 J cm-2. Dark control (-L), light control (+L), with Tat-porphyrin (P+), without Tat-porphyrin (P-). Error bars indicate the standard deviation. * P < 0.05.

Article Snippet: The Tat–porphyrin compound has a good singlet oxygen quantum yield of 0.54 (T.-W. Wang and A. J. MacRobert, unpublished data) as would be expected for a porphyrin derivative, and for comparison the porphyrin itself has a singlet oxygen quantum yield of 0.56 as reported by Tanielian et al.41 Bacterial strains and growth conditions Two Gram (+) positive organisms were used in this study; Staphylococcus aureus strain 8325-4 and Streptococcus pyogenes strain 12202, and two Gram (-) bacteria Pseudomonas aeruginosa strain PA01 and Escherichia coli ATCC 25922 strain were used.

Techniques: Incubation, Irradiation, Control, Standard Deviation

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 1 Identification of Chlamydia trachomatis strains deficient in CPAF secretion or expression. (a–b). CPAF fails to accumulate in the cytoplasm of cells infected with a T2S-deficient mutant and is not detectable in a CPAF-truncation mutant (M169). The subcellular localization of CPAF in HeLa cells infected with the indicated C. trachomatis mutant strains was assessed by indirect immunofluorescence with polyclonal anti-CPAF antibodies (green), DNA with Hoescht (blue), and the inclusion membrane with anti-CT813 antibodies (red). Immunoreactive anti-CPAF material could not be detected in cells infected with a CPAF-deficient strain, or in the cytoplasm of cells infected with a T2S-deficient strain. The relative distribution of CPAF at 32 hpi in infected cells (n = 100) is shown in (b). (c) T2S-deficient mutants fail to process CPAF into its active form. Immunoblot analysis of total protein lysates from HeLa cells infected with the indicated strains at 48 hpi. Strains containing mutations in cpa that do not express CPAF- and T2S-deficient strains exhibit accumulation of a larger molecular weight band consistent with the size of unprocessed CPAF (proCPAF). The C-terminal fragment and N-terminal fragment of CPAF are indicated by ‘CPAFc’ and ‘CPAFn’, respectively. (d) Lysates from cells infected with CPAF- or T2S-deficient C. trachomatis mutants do not process CPAF substrates in vitro. Purified recombinant CPAF (rCPAF) or lysates from HeLa cells that were mock-infected or infected with the indicated strain were harvested at 40 hpi and incubated with recombinant GST-CT695, an in vitro CPAF substrate. Cleavage products were monitored by SDS-PAGE and staining with Coomassie Blue. FL: full-length GST-CT695; C: cleavage product. (e) M169-derived recombinant C. trachomatis strains retaining the cpa mutation do not express CPAF. Immunoblot analysis of total protein lysates of HeLa cells infected with RST17 (CPAF-) or its nearly coisogenic recombinant sibling RST5 (CPAF+) for 48 h. CPAF expression is restored in RST5 (CPAF+) bacteria but still absent in the RST17 (CPAF) strain. The genotypes of these strains are shown in Table S2.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Expressing, Infection, Mutagenesis, Membrane, Western Blot, Molecular Weight, In Vitro, Recombinant, Incubation, SDS Page, Staining, Derivative Assay, Bacteria

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 2 Chlamydia trachomatis mutants deficient in CPAF expression are impaired for the generation of infectious progeny. Vero cells were infected with the indicated strains at an MOI = 0.3, and infectious progeny released per input IFU was calculated at 30 hpi and 48 hpi. RST17 (CPAF) mutants produce a threefold lower yield of infectious units compared with RST5 (CPAF+) bacteria at 48 hpi. Three indepen- dent experiments, SEM, two-way ANOVA with Bonferroni post hoc, ***P < 0.001.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Expressing, Infection, Bacteria

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 3 CPAF is not required for many of the cellular phenotypes associated with Chlamydia trachomatis infection. (a–c) Chlamydia trachomatis-induced Golgi fragmentation occurs in a z-WEHD-fmk-dependent manner in the absence of CPAF. HeLa cells were infected with the indicated strains for 24 hpi and processed for immunofluorescence to visualize the Golgi apparatus (GM130, red), C. trachomatis (MOMP, green), and DNA (Hoescht, blue). No difference was observed in Golgi fragmentation as assessed visually and by postacquisition processing to assess size and number of Golgi elements between wild-type and CPAF-deficient strain infections (a and b). In the same experiment, cells were treated with 75 lM z-WEHD-fmk from 9 hpi, which inhibited Golgi fragmentation in cells infected with wild-type and a CPAF-deficient strain (a and c). Three independent experiments, SEM. (d) HeLa cells stably expressing an NFjB luciferase reporter were infected with the indicated strains at an MOI of 10, 30, or 50 for 24 h and were simultaneously treated with 10 ng mL1 IL-1b. Luciferase activity was measured in cell lysates and normalized to the treated mock-infected control. CPAF-deficient strains and RifR (WT) strains show an MOI-dependent decrease in luciferase activity. (e) Infected or mock-infected HeLa cells were treated with 2 lM staurosporine for 6 h prior to the indicated time points, and the percentage of infected cells with condensed nuclei was determined. (f) HeLa cells were either infected with the indicated strain or left uninfected for 29 h prior to being infected at an MOI = 25 with GFP-expressing LGV-L2 for 1 h. The number of phosphotyrosine foci per cell was determined for 35 cells. HeLa cells infected with the CPAF-deficient strain (M169) remain protected from superinfection. **P < 0.01, SEM, n = 3.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Infection, Stable Transfection, Expressing, Luciferase, Activity Assay, Control

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 4 Evidence for CPAF-mediated processing of vimentin in intact cells. (a) CPAF is not active in 1% SDS buffer. HeLa cell lysates prepared under denaturing conditions in 1% SDS buffer were incubated with 0.5, 2.5, and 5 lg recombinant CPAF (rCPAF) for 20 min at 37 °C. As a positive control, rCPAF was also incubated with HeLa lysates prepared under nondenaturing conditions in TNEX buffer. Where indicated, 100 lM CPAF inhibitor peptide Pep2 was included as a control. CPAF activity was assessed by monitoring the generation of vimentin cleavage products by immunoblot analysis. (b) Proteolytic processing of vimentin at later stages of infection is dependent on CPAF. The bottom panel represents an increased exposure (OE) of the middle panel. FL: full-length vimentin; C: cleavage product (c) LDH release by cells infected with wild-type and CPAF-deficient Chlamydia trachomatis strains. The supernatants of infected cells were collected at the indicated time points, and the amount of LDH release was measured and compared to total LDH levels. Error bars represent standard deviation, n = 3.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Incubation, Recombinant, Positive Control, Control, Activity Assay, Western Blot, Infection, Standard Deviation

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 5 The filament-forming properties of vimentin are altered in a CPAF-dependent manner at late stages of infection. (a) A subset of infected cells contain vimentin filaments that are sensitive to detergent extraction. Top panel: Immunofluorescence images of HeLa cells infected with RifR (WT) at 52 hpi. Infected cells were either untreated or treated with 0.5% Tx100 for 5 min on ice, prior to fixation and immunostaining for vimentin localization. A vimentin cage forms around the inclusion of infected cells (arrowhead), which are sensitive to Tx100 extraction (arrow). Bottom panel: Infected HeLa cells were treated with 0.5% Tx100 in the presence of 100 lM CPAF-inhibitory peptide (Pep2). Note the presence of inclusions with both detergent-resistant (arrow- head) and detergent-sensitive (arrow) vimentin filaments. Blue: Hoes- cht; green: vimentin. (b) The proportion of inclusions with detergent-extractable vimentin filaments increases with infection times in a CPAF-dependent manner. Cells were infected with RifR (WT) or CPAF-mutant strains, and the percentage of infected cells with Tx100 extractable vimentin (a, bottom right) was quantitated at 44 hpi, 52 hpi, and 60 hpi. All extractions were carried out in the presence of 100 lM Pep2. Infected cells with extractable intermediate filaments were not apparent in HeLa cells infected with CPAF-deficient mutants. ***P < 0.001, SEM, n = 3.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Infection, Extraction, Immunostaining, Mutagenesis

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 6 CPAF cleaves LAP1 during infection. (a) CPAF cleaves EGFP-LAP1. HeLa lysates transfected (+) or not () with EGFP-LAP1 were incubated with (r) or without () recombinant CPAF or with lysates from L2-infected HeLa cells (L2) or mock-infected (m) HeLa cells. Blots were probed with anti-EGFP (EGFP) or anti-LAP1 (LAP1). (b) LAP1 cleavage during infection is CPAF dependent. HeLa cells infected with either the CPAF strain or RifR (WT) strain or mock-infected were harvested in 1% SDS buffer at the indicated times postinfection. Blots were probed with anti-LAP1 and anti-RpoB. Arrows indicate LAP1 cleavage products.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Infection, Transfection, Incubation, Recombinant

Journal: Pathogens and Disease

Article Title: Reassessing the role of the secreted protease CPAF inChlamydia trachomatisinfection through genetic approaches

doi: 10.1111/2049-632x.12179

Figure Lengend Snippet: Fig. 7 Cleavage of vimentin and LAP1 in live infected cells occurs after inclusion rupture and is dependent on CPAF and T2S. (a) CPAF-dependent modification of vimentin filaments occurs immediately after inclusion rupture. HeLa cells were infected with the indicated strains and transfected with N-terminally EGFP-tagged vimentin and tdTomato vectors and imaged using widefield deconvolution live cell microscopy every 7 min after 54 hpi for 14 h (Video S1). Inclusion rupture was assessed by the influx of dTomato signal into the inclusion lumen. Images acquired 7 min before and after inclusion rupture are shown. In cells infected with RifR (WT) LGV-L2, the filamentous EGFP-vimentin signal became diffuse immediately after inclusion rupture. EGFP-vimentin remained in a filamentous form after loss of inclusion integrity in cells infected with CPAF- or T2S-deficient strains (arrows). (b) CPAF-dependent processing of LAP1 occurs after inclusion rupture. HeLa cells were transfected with EGFP-LAP1 and tdTomato expression vectors and infected and imaged as in (a) (Video S2). Loss of EGFP-LAP1 localization to the nuclear membrane occurs rapidly following inclusion rupture in cells infected with RifR (WT), but not in cells infected with CPAF- or T2S-deficient strains.

Article Snippet: Cell culture and Chlamydia infections HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech).

Techniques: Infection, Transfection, Microscopy, Expressing, Membrane

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) Growth curves of wild-type Salmonella and its isogenic sifA mutant in LB media in the presence of pEVOL. The error bars represent the mean ± standard deviation (n = 3). ( B ) Survival assay of wild-type Salmonella and wild-type Salmonella transformed with pEVOL in HeLa cells. Strains of Salmonella wild-type and the wild-type transformant with pEVOL were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. At 2 hr and 16 hr post infection, cells were lysed, and the intracellular bacteria were enumerated. The percentage of survival of the transformant was normalized to wildtype (100%). Figure 3—figure supplement 1—source data 1. Source data for . Figure 3—figure supplement 1—source data 2. Source data for .

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Mutagenesis, Standard Deviation, Clonogenic Cell Survival Assay, Transformation Assay, Infection, Bacteria

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with a sifA null strain of Salmonella expressing SifA-E52AzF and incubated for 16 hr. Infected cells were fixed with 4% PFA and immuno-stained for Salmonella (yellow) and LAMP1 (red). Images were acquired by spinning-disk SIM. Cells infected with sifA harboring p sifA 52TAG bacteria were capable of forming SIFs that resembled those of wild-type infected cells. ( B ) SPAAC labeling of secreted SifA-E52AzF with BDP-DBCO. HeLa cells were infected with a sifA null strain expressing SifA-E52AzF in the presence of 1 mM AzF. AzF-tagged secreted SifA-E52AzF was labeled with BDP-DBCO (green). Host nuclei and Salmonella nucleoids were labeled with DAPI (blue) and images were acquired using spinning-disk SIM. ( C ) SifA-E52AzF labeling was absent in the absence of ncAA. HeLa cells were infected with the sifA null mutant of Salmonella expressing SifA-E52AzF in the absence of AzF for 12 hr. After 12 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive washing to remove excess dye as described in Methods. At 16 hr post-infection, cells were fixed. SifA was absent in the infected cells that lacked AzF. Images were acquired by confocal microscopy. See for statistical analysis for the average fraction of infected cells that contain LAMP1 positive SIFs and SIFs containing labeled SifA. ( D ) Colocalization of SifA with LAMP1. Salmonella secreted SifA-E52AzF in HeLa cells was labeled with BDP-DBCO (green). Cells were also immuno-stained for the endosomal membrane marker LAMP1 (red, middle panel). Images were acquired by confocal microscopy. From the merged image (right), it is evident that SifA is present within the SIFs. Statistical analysis is shown in . The data are representative of at least three independent experiments. Scale bar, 10 µm ( A–D ).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Expressing, Incubation, Staining, Bacteria, Labeling, Mutagenesis, Confocal Microscopy, Membrane, Marker

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: HeLa cells were infected with Salmonella for 16 hr, fixed and immuno-stained for LAMP1 (red), LPS (yellow), and DAPI (blue). As expected, the formation of SIFs was observed in cells infected with WT Salmonella ( A ), but not in cells infected with the sifA null strain ( B ). Images were acquired on a spinning-disk SIM, as described in Materials and methods (n = >10 independent experiments). Scale bar, 10 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Staining

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( Ai ) SifA colocalizes with SseJ. Spinning-disk SIM image of an infected HeLa cell, 16 hr after invasion, fixed with PFA and stained for SifA (green) and SseJ-HA (red). ( Aii ) Magnified view of SifA and SseJ in the boxed region at the far left (arrows indicate colocalization). ( iii ) Colocalization was analyzed by calculating Mander’s M1 coefficient (SifA colocalizing with SseJ) for the original dual-color image (x = y = 0) and for control images generated by translating the spectral channels with respect to each other in x and y directions. This analysis generated a colocalization map, with a peak intensity at x = y = 0, indicating true colocalization (n = 3 cells). ( iv ) Colocalization analysis of mock-infected cells (n = 3 cells) stained for SifA-E52AzF and SseJ-HA showed no peak intensity at x = y = 0, indicating no colocalization of anti-HA antibodies and SifA. Bars, 10 µm ( i ), 5 µm (ii). ( B ) SifA colocalizes with kinesin. ( i ) Spinning-disk SIM image of an infected HeLa cell fixed with PFA at 16 hr post-infection and stained for kinesin (red) and SifA (green). (ii) Magnified view of SifA and kinesin in the boxed region (arrows indicate colocalization). (iii and iv) Colocalization analysis was performed as described in ( A ). The same color map scaling was used for comparison. px, pixels. See for statistical analysis. Scale bar, 5 µm ( i ), 2 µm (ii). ( C ) Bioorthogonal fluorescence imaging of SseJ-Y10TCO-HA with tetrazine fluorophores versus immunofluorescence staining. HeLa cells expressing SseJ-Y10TCO-HA in the presence of TCO*Lys were labeled with BDP-Tz under physiological conditions, briefly washed, and subjected to anti-HA immunofluorescence staining. Images were acquired by confocal microscopy. The data are representative of at least three independent experiments. Scale bar, 10 µm. Figure 4—source data 1. Source data for .. Figure 4—source data 2. Source data for .

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Staining, Control, Generated, Comparison, Fluorescence, Imaging, Immunofluorescence, Expressing, Labeling, Confocal Microscopy

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: HeLa cells were infected with WT -Salmonella harboring SseJ-HA and pEVOL in the presence of AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF for 2 hr. At 14 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive removal of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were fixed and washed with PBS, then immunostained for SseJ (red). The absence of any noticeable fluorescence signal in the green channel (BDP-DBCO) indicated that there was virtually no off-target labeling of secreted effectors within host cells. The results presented in each panel were obtained in at least three independent experiments. Scale bar, 10 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Incubation, Fluorescence, Labeling

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with sifA mutants harboring p SifA 52TAG and pEVOL-AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF for 2 hr. At 14 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive removal of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were extracted for 1 min using a microtubule-stabilizing buffer (MTSB). The buffer was exchanged with MTSB containing 0.2% GA, and cells were fixed for 10 min at room temperature. Excess GA was quenched for 7 min using 0.2% sodium borohydride in PBS. After several washes with PBS, cells were immunostained for microtubules (green). Scale bar, 10 µm. ( B ) Prior to immunostaining, GA-fixed HeLa cells were imaged for SifA labeled with BDP-DBCO using Spinning-disk SIM. Note the punctate labeling of SifA, indicating that while SseJ forms continuous filaments, SifA does not. The results presented in each panel were obtained in at least three independent experiments. Scale bar, 5 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Incubation, Immunostaining, Labeling

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with the sifA null strain of Salmonella expressing SifA-E52Hco and SseJ-HA for 16 hr, fixed and immuno-stained for LAMP1 (green), SseJ (red). The SifA-E52Hco strain was capable of forming SIFs and colocalized with SseJ. Images were acquired on a spinning-disk SIM as described in Materials and methods. ( B ) Direct visualization of secreted SifA. HeLa cells were infected with a sifA null strain of Salmonella expressing SifA-E52Hco in the presence of 1 mM Hco and fixed 16 hr post infection with 4% PFA. Hco-tagged secreted SifA-E52Hco was directly imaged by two photon microscopy. Immuno-stained SseJ-HA was also imaged in the same cell. The data are representative of at least three independent experiments. Scale bar, 10 µm ( A ), 20 µm ( B ).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Expressing, Staining, Microscopy

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with the ssaP null mutant for 16 hr, fixed and immuno-stained for LPS (green), SseJ (red) and DAPI (blue). In the absence of ssaP , SseJ is not secreted. ( B ) TCO*Lys-incorporated SsaP was functional. HeLa cells were infected with Salmonella complemented with p ssaP 65TAG- sseJ -HA, fixed and immunostained for LPS (red), LAMP1 (green) and DAPI (blue). SIF formation was observed in cells infected with Salmonella complemented with p ssaP 65TAG- sseJ -HA. ( C ) Engineered SsaP rescued SPI-2 secretion. HeLa cells were infected with Salmonella complemented with p ssaP 65TAG- sseJ -HA, fixed and immunostained for LPS (Green), SseJ (red), and DAPI (blue). SseJ (red) was secreted in the complemented strain, but not in the ssaP null strain (n = >10, independent experiments). Scale bar, 10 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Mutagenesis, Staining, Functional Assay

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: Strains of Salmonella wild-type, ssaP null, ssaP/ λ ssaP (complemented with ssaP integrated a λ att ), and ssaP/ λ ssaP-55HA ( ssaP with an HA tag at amino acid 55) were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. At 2 hr and 14 hr, cells were lysed and the intracellular bacteria were enumerated. The normalized fold changes were relative to those of the wildtype (set to 1). The error bars represent the mean ± standard deviation (n = 3).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Bacteria, Standard Deviation

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: Strains of Salmonella containing ssaP/λssaP-55HA (left) ssaP/ λssaP-110HA (middle and right) were grown in LB to late exponential phase and added to HeLa cells at an MOI of 100:1 for 30 min. The cells were fixed and immuno-stained for SsaP (red). A representative image at 14 hr post infection is shown. DAPI-stained DNA is in blue and GFP-expressing Salmonella are shown in green. The middle and right panels are of SsaP at different z positions.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Staining, Infection, Expressing

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) SPIEDAC labeling of secreted SifA-Y65TCO with fluorogenic SiR-Tz. HeLa cells were infected with the ssaP null mutant of Salmonella expressing SsaP-Y65TCO in the presence of TCO*Lys for 12 hr. After 12 hr post infection, HeLa cells were incubated with 1.5 μM SiR-Tz in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive wash out of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were fixed. Cells were also immuno-stained for the endosomal membrane marker LAMP1 (green), LPS (Red) and DAPI (Blue) and imaged by spinning-disk SIM. From the merged image (right), it is evident that SsaP is present within the LAMP1-positive endosomes. A higher magnification spinning-disk SIM image of a SIF-positive HeLa cell from the boxed region ( i ) is shown on the right-most panel without the green channel displayed ( i ). It clearly shows secreted SsaP from Salmonella (boxed region). ( B ) SsaP was absent in infected cells that lacked ssaP . HeLa cells that were infected with an ssaP null mutant in the presence of TOC*Lys lack an SsaP signal. Note the absence of SIFs in the merged image (right) in the ssaP null strain. The data are representative of at least three independent experiments. Scale bar, 5 µm (A and i) and 10 µm ( B ).

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Labeling, Infection, Mutagenesis, Expressing, Incubation, Staining, Membrane, Marker

Journal: eLife

Article Title: Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

doi: 10.7554/eLife.67789

Figure Lengend Snippet: ( A ) HeLa cells were infected with an ssaP null mutant of Salmonella expressing SsaP-YTCO*Lys in the presence of TCO*Lys. After 6 hr post infection, HeLa cells were incubated with 1.5 μM SiR-Tz in DMEM with 10% FBS for another 1 hr, followed by extenstive washing of excess dye with fresh growth media. At 8 hr post-infection, cells were fixed and immunostained for the endosomal membrane marker LAMP1 (green, left panel), LPS (red) and DAPI (blue). Scale bar, 5 µm. ( B ) Zoomed view of inset. Yellow arrow indicates polarly localized SsaP. Images were acquired on a spinning-disk SIM, as described in Materials and methods. Scale bar, 2 µm. ( C ) Not every pole where SsaP was located had an injectisome. Salmonella cells expressing SsaP-Y65TCO in the presence of TCO*Lys were labeled with SiR-Tz as described in Materials and methods. The same cells were immuno-stained for SseB using rabbit anti-SseB antibody (green) and imaged along with SiR fluorescence (magenta). The arrows indicate cells that have both SsaP and SseB. The data are representative of at least three independent experiments. Scale bar, 2 µm.

Article Snippet: The HeLa cell line was authenticated by ATCC (STR analysis) and the absence of mycoplasma contamination was confirmed using Hoechst DNA stain (indirect), agar culture (direct), a PCR-based assay and Lonza’s MycoAlert PLUS Mycoplasma detection kit.

Techniques: Infection, Mutagenesis, Expressing, Incubation, Membrane, Marker, Labeling, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The extended CTT of WalK is unique in S. mutans and required for its interaction with WalR. (A) Phylogenetic analysis of S. mutans HKs. Evolutionary relationship of all 14 HKs is shown in a circular tree, which are grouped into two based on six key residues following their catalytic histidine with conservation for each group below. HKs with a conserved HisKA motif are colored in green. HKs with HisKA_3 motif are grouped in orange. HKs are named with four digits in their protein ID: NP_72****.1. (B) Alignment of streptococcus WalK C-terminal sequences. Completely conserved residues are shown in white with a red background and boxed in blue. Highly conserved residues are in red with a white background and boxed in blue. Marked on top are protein secondary structures and residue numbers in S. mutans . (C) Mutations in the CTT disrupt the WalRK interaction. A GST fusion protein with full-length S. mutans WalR was used to pull-down S. mutans WalK (196–450) WT and mutant proteins (top gel). As a negative control, GST alone was used to pull down WalK WT and mutant proteins (middle gel). Shown in the bottom gel are 10% of the WalK protein levels used above. CK shows GST-WalR or GST used in the pull-down. (D–F) Quantification of the WalRK interaction by ITC experiments. WalK (196–450) WT and mutant proteins were titrated against the full-length WalR, resulting in raw titration curves at the top and their global fittings at the bottom. The derived thermodynamic parameters are shown within.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Residue, Mutagenesis, Negative Control, Titration, Derivative Assay

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK is indispensable for its enzymatic activities. (A) Autokinase activity of WalK (31–450) and its tail mutants. The WalK loadings were shown in the lower gel stained by CBB, while the phosphorylation of WalK was detected by ATPγS and anti-thiophosphate antibodies shown in the upper gel. H217A is an autokinase inactive mutant. (B) Phosphotransferase of WalK. The phosphotransferase activity was examined by the reduced phosphorylation of WalK incubated with WalR and detected using ATPγS and anti-thiophosphate antibody over time. Quantitative analysis of phosphotransferase activity normalized to 0 min is shown below the gel. (C) Phosphatase of WalK. Phosphorylated WalR was incubated with WalK and its derivatives at 1:5 (WalK:WalR), separated from the dephosphorylated WalR in a Phos-tag gel and stained with CBB. Quantitative analysis of phosphatase activity is below the gel. The phosphorylated/dephosphorylated WalR was estimated, normalized to its initial amount at 0 s of the gel. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare mutants to WT at each time point (*** p < 0.001 and **** p < 0.0001).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Activity Assay, Staining, Phospho-proteomics, Mutagenesis, Incubation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK contributes to the interaction with the WalR DBD. (A) The domain architectures of WalK and WalR. (B) WalK (196–450) interacts with WalR in GST pull-down experiments. The 10% loading controls for WalK, GST, GST-WalR full-length (FL), GST-RD, and GST-DBD are shown in lanes 1, 2, 4, 6, and 8, respectively. (C) Mutations in the WalK CTT disrupt the interaction with the WalR DBD. As a negative control, GST alone was used to pull down WalK (middle gel). Shown in the bottom gel are 10% of the WalK protein used above. CK shows GST-DBD or GST used in the pull-down. (D) Phosphotransferase activity of WalK is diminished toward the RD of WalR alone. Phosphorylated WalK detected by anti-thiophosphate antibody was incubated with WalR full-length, RD, DBD, and D52A, shown from top to bottom, respectively.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Negative Control, Activity Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK is important for WalK in competition with promoter DNA. (A,B) WalK (196–450) competes off fluorescein labeled 25-mer promoter DNA from binding to WalR or DBD in a dose-dependent manner. (C) Comparison of the relative ability of CTT mutants (W443A, Δtail) of WalK to compete with DBD in promoter binding. All samples were mixed and incubated for 15 min at RT before loading onto gels. Final concentrations of proteins and DNA used in the reactions were marked above the panels. All EMSA gels were imaged under UV to show DNA in the upper panel and stained with CBB to visualize protein loading in the lower panel.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Labeling, Binding Assay, Comparison, Incubation, Staining

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: Effect of WalK mutations on biofilm development of S. mutans . (A–C) SEM analyses of mature biofilms grown for 72 h. (D–F) Quantification of biofilms by fluorescent staining. All biofilms were quantified for their thickness and horizontal growth shown below each 3D image. (G) Phosphorylation state of WalR in vivo . Phosphorylated WalR was separated from its non-phosphorylated state in a Phos-tag gel and detected using an anti-WalR antibody. The non-phosphorylated (CK) and phosphorylated WalR (His-tagged, treated with AcP) were loaded in the first two lanes. The cytoplasmic phosphorylation ratio of WalR shown in the bar chart was determined by band intensities and averaged from three independent experiments with error bars showing standard deviation.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Staining, Phospho-proteomics, In Vivo, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: Protein profiling in S. mutans biofilms. (A) Protein profiling of S mutans WT and Δtail strains from a quantitative mass spectroscopy experiment. The x -axis indicates the fold change of LFQ in the Δtail strain. The y -axis of log ( P ) indicates a significance level of the t- test. The black curves separate those proteins at a level of false discovery rate (FDR) = 0.01 and minimal fold changes (S0) = 0.1. Below the curve in gray are those unchanged proteins. Those proteins that were upregulated are colored in red, and those that were downregulated are colored in blue. Total and altered proteins are listed in MS Dataset. (B) Proteins that were most altered in the Δtail strain. Proteins were selected at a difference cutoff of > 1.5 except GbpA and GbpB. Five short, functionally unknown, peptides were excluded. The functional annotations of the proteins are shown in . (C,D) qRT-PCR analyses of key genes known to be regulated by WalRK. Transcriptional profiles of the genes gtfBCD and gbpABC were normalized to 16S RNA. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare Δ tail strain to WT strain (** p < 0.005 and *** p < 0.001).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Mass Spectrometry, Functional Assay, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of WalK is required for S. aureus WalRK interaction and enzymatic activity. (A) Conservation of the WalK CTT across Gram-positive bacteria. Representative sequences of WalK C-termini were aligned and boxed. The residues are numbered according to S. mutans WalK. Completely conserved residues are colored in white in a red background. The less conserved residues are highlighted in yellow. (B) Alignment of staphylococcus WalK tail sequences. Completely conserved residues are shown in white in a red background and boxed in blue. Highly conserved residues are in red in a white background and boxed in blue. Marked on top are protein secondary structures and residue numbers in S. aureus . (C) Mutations in the CTT disrupt WalRK interaction. A GST fusion protein with full-length S. aureus WalR was used to pull-down S. aureus WalK (364–608) WT and mutant derivatives shown in the top panel. As a negative control, GST alone was used to pull down WalK WT and its mutants shown on the center panel. Shown in the bottom panel are 10% levels of WalK proteins used in each lane. CK shows GST-WalR or GST used in the pull-down. (D) Phosphotransferase of WalK (364–608) was disrupted by mutations in the WalK CTT. The phosphotransferase activity was represented by the reduction in WalK phosphorylation relative to T0, the initial phos-WalK. After the addition of WalR, the mixtures were incubated for the indicated time and stopped by the addition of SDS-loading buffer. Loading controls are shown in the bottom gel. The enzymatic activity was quantified shown below. (E) Phosphatase of WalK (364–608) was disrupted by mutations in the WalK CTT. Phos-tag gel is shown in the top, and a regular SDS-PAGE below shows the total protein used. The phosphatase activity was quantified by the reduction in the phos-WalR relative to the amount of T0, the initial phos-WalR. The reactions were incubated for the indicated time and stopped by the addition of SDS-loading buffer. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare mutants to WT at each time point (** p < 0.005 and * p < 0.05).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Activity Assay, Bacteria, Residue, Mutagenesis, Negative Control, Phospho-proteomics, Incubation, SDS Page