Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Serum creatinine (Cre) levels of SPF and GF mice on low vs. high Saa+Ade diets. B. Representative H&E staining and C. Representative trichrome staining of kidneys from mice in A. D. Histology-based renal injury score. E and F. SPF and GF mice cecal sulfide levels detected by lead acetate or methylene blue assay. G. Normalized E. coli mean gene abundance in CKD patient samples compared to non-CKD controls, PTRI whole genome shotgun sequencing dataset. H. Serum Cre levels from ASF or ASF E. coli mice on low vs. high Saa+Ade diets. I. Representative H&E staining and J . Representative trichrome staining of kidneys from mice in H. K. Histology-based renal injury score. L and M. ASF and ASF E. coli cecal sulfide levels detected by lead acetate or methylene blue assay. Data represent 2 independent experiments for L and M , 3 for A , D, H and K, and 4 for E and F. Symbols represent individual mice. Bars represent mean ± SEM. * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Two-way ANOVA with Tukey’s post-hoc test for A, D , E, F, H and K , and Mann-Whitney test for L and M.

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Staining, Shotgun Sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: LEfSe analysis of 16S rRNA gene amplicon survey data from Xu et al. 2017. B. LEfSe analysis of 16S rRNA gene amplicon survey data from an unpublished CKD patient cohort (NCBI accession PRJEB5761). For clarity, taxonomy is shown from the class level. C. Volcano plot of PhyloChip analysis data from Vaziri et al. (2013). Taxa with fold change > 2 and q-value < 0.05 are labeled in color with their family level taxonomy. D. Boxplot representation of the combined averaged relative abundance of 7 E. coli strains measured in the fecal samples from CKD and non-CKD subjects using PhyloChip analysis. * P value < 0.05, ** P value < 0.01.

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Amplification, Labeling

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Colonization of ASF mice with E. coli on Saa+Ade diets. B. Colonization of ASF mice with E. coli on Saa diets. C. Relative abundances of ASF strains in cecal contents of mice on Saa+Ade diets. D. Serum Cre levels from WT ASF E. coli mice on low vs. high Saa diets. Data represent 3 independent experiments for A , B , C and D . Symbols represent individual mice. Bars represent mean ± SEM. * P value < 0.05. Mann-Whitney test for D .

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. E. coli sulfide production by lead acetate. B. E. coli sulfide production by methylene blue. C. Schematic of S-sulfhydrated protein pull-down method. D. Silver staining of E. coli lysates subjected to S-sulfhydration pull-down and eluted either with or without DTT. E. Silver staining of WT and Δ decR E. coli lysates subjected to S-sulfhydration pull-down. F. Heatmap of the relative quantity of the 212 S-sulfhydrated proteins by TMT LC-MS 3 analysis from S-sulfhydration pull-down fractions from WT E. coli samples eluted with or without DTT and ΔdecR mutant samples eluted with DTT. Proteins ordered based on q-value score for enrichment in the DTT vs non-DTT eluted samples. Data represent 2 independent experiments for E , 3 for D and F, 4 for A and 6 for B . Bars represent mean ± SEM. ** P value < 0.01. Linear model test A, two-way Kruskal-Wallis test with Dunn’s post-hoc test B and two-way ANOVA with Tukey’s post-hoc test F .

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Final OD 600 of WT and Δ decR E. coli cultures grown in LB supplemented with cysteine under aerobic conditions. B. Lead acetate detection of H2S production by WT and Δ decR E. coli cultures grown in LB supplemented with cysteine under anaerobic conditions. C. Final OD 600 of WT and Δ decR E. coli cultures grown in LB supplemented with cysteine under anaerobic conditions. D. Coomassie stain of S-sulfhydrated proteins from WT E. coli lysates treated with NaCl, H2O2 or NaHS. Lower gel shows Western blotting of RpoD in the flow-through samples, as loading control. Data are representative of 3 independent experiments. E. Coomassie stain of S-sulfhydrated proteins from WT E. coli grown in LB or LB supplemented with 0.4mM cysteine. Lower gel shows Western blotting of RpoD in the flow-through samples, as loading control. F. Quantification of Coomassie stains from E. G. Boxplot representation of the data presented in . H. Pathway enrichment analysis using the PANTHER database (Mi et al., 2019) of the 212 S-sulfhydrated proteins. Pathways with q-value < 0.05 are reported. Data are representative of 3 independent experiments for A , B , C , D , F and G . Bars represent the mean ± SEM. ** P value < 0.01, *** P value < 0.001. Mann-Whitney test for F .

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Staining, Western Blot, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Representative western blot analysis of TnaA-His from WT and Δ decR E. coli lysates subjected to S-sulfhydration pull-down. Loading controls show RpoD in the flow-through. B. Same method as in A with E. coli lysates treated with NaCl, H2O2, or NaHS. C. LC-MS/MS analysis of indoles in WT E. coli cultures with cysteine or NaHS. D-E. Kovac’s assay for indole production in ( D ) WT E. coli cultures with cysteine or NaHS and ( E ) purified TnaA enzyme supplemented with NaCl, Na2S4, or DTT. Data represent 3 independent experiments for A and E , 4 for B and D, and 5 for C . Bars represent mean ± SEM. * P value < 0.05, ** P value < 0.01, *** P value < 0.001. Mann-Whitney test A and two-way ANOVA with Tukey’s post-hoc test B and E, and two-way Kruskal-Wallis test with Dunn’s post-hoc test C and D .

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Purification, MANN-WHITNEY

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Relative indole levels of WT and Δ decR E. coli cultures grown aerobically in LB supplemented with cysteine measured by LC-MS/MS. B. Western blotting of TnaA in WT E. coli cultures grown aerobically in LB or LB supplemented with cysteine or NaHS, or Δ decR E. coli grown in LB. Lower gel shows Western blotting for RpoD as loading control. C. Relative indole levels of WT and tnaA mut E. coli cultures grown aerobically in LB measured by Kovac’s assay. D. Western blotting of the S-sulfhydration pull-down fractions of purified E. coli TnaA treated with NaCl, H2O2 or Na2S4. Flow through samples represent loading control. Data represent 3 independent experiments for A , B , C and D . Bars represent mean ± SEM. * P value < 0.05, ** P value < 0.01. Mann-Whitney test for A .

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Purification, MANN-WHITNEY

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Colonization of ASF mice with WT E. coli on Saa diets. B. Chromatogram of indole detection in cecal contents from ASF mice on Saa diets. C-D. Colonization of ASF mice with different E. coli strains on low ( C ) and high ( D ) Saa+Ade diet. E. LC-MS measurements of serum indoxyl sulfate in ASF mice on high Saa+Ade diet, colonized with E. coli strains. F. Serum creatinine levels of mice in E. G. Representative H&E staining of kidneys from mice in E. H. Representative trichrome staining of kidneys from mice in E. I. Histology-based renal injury score of mice in E. Data represent 3 independent experiments for A , B , C , D , E , F and I . Symbols represent individual mice. Bars represent mean ± SEM. ** P value < 0.01 Two-way ANOVA with Tukey’s post-hoc test for F.

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A. Western blot analysis of TnaA of S-sulfhydration pull-down and flow-through samples from cecal contents and B. Kovac’s assay measurement of indole levels in cecal contents from ASF E. coli mice on Saa diets. C. LC-MS/MS analysis of indole levels in cecal contents from ASF E. coli mice on Saa diets. Left, spectra representative of an experiment with 3 mice per group and indole standard. D. LC-MS measurements of serum indoxyl-sulfate in ASF mice on low Saa+Ade diet, colonized with E. coli strains. E. Serum Cre levels in ASF mice colonized with E. coli strains on low Saa+Ade diets. F. Representative H&E staining and G. Representative trichrome staining of kidneys from mice in E. H. Histology-based renal injury score. I. Illustration showing the effects of low and high Saa-Ade diets on gut microbial activity and the consequences for renal function. Data represent 3 independent experiments for A , B , C , D , E and H . Symbols represent individual mice. Bars represent mean ± SEM. * P value < 0.05, ** P value < 0.01. Mann-Whitney test for A , B and C, two-way ANOVA with Tukey’s post-hoc test for D , E and H .

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Staining, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Diet Post-translationally Modifies The Gut Microbial Proteome To Modulate Renal Function

doi: 10.1101/2020.02.25.964957

Figure Lengend Snippet: A multiple sequence alignment (MSA) of the 15 closest orthologs of E. coli TnaA in gut bacteria species, obtained by BLAST search with E. coli TnaA vs the microbiome reference sequence database. Identical amino acids are highlighted in blue. The top logo sequence represents the consensus sequence of the MSA with the cysteine residues labeled in red. The MSA was performed using the Clustal Omega algorithm.

Article Snippet: The PCR product was then digested with BamHI and NotI restriction enzymes, ligated into the pKOV plasmid (obtained from AddGene) and chemically transformed into E. coli MG1655.

Techniques: Sequencing, Bacteria, Labeling

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

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: MS-based quantitative profiling of E. coli Big-Six group strains secretome. A schematic illustration of E. coli Big-Six group strains. Bacteria were harvested and filtered using 0.22 µm filters and concentrated using 3 kDa cutoff filters. Equal amount of proteins from each bacteria were trypsin-digested, cleaned using Sep-Pak C18 cartridges, and TMTs labeling was carried out. Labeled peptides were pooled and fractionated using bRPLC fractions and analyzed on Orbitrap Elite mass spectrometer.

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Bacteria, Labeling, Mass Spectrometry

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: Result summary of E. coli Big-Six group strains secretome: (A) Venn diagram depicting the strains of secreted proteins from technical replicates. (B) The detailed summary of protein identifications from combined technical replicate analysis.

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques:

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: Hierarchical clustering of E. coli Big-Six group strains T3SS. The relative protein expression of T3SS system was shown in a heat map. Each row value for a given strain is a log2 value of fold change with K12. Higher expression of T3SS-secreted proteins was observed in O45:H2 and O121:H19 strains.

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Expressing

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: Partial list of proteins identified in type III secretion system in E. coli Big-Six group of strains

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Translocation Assay, Membrane, Infection

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: Hierarchical clustering of E. coli Big-Six group strains outer membrane proteins: The relative protein expression of outer membrane proteins was shown in a heat map. The fold-change value with K12 is log2-transformed.

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Membrane, Expressing, Transformation Assay

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: Hierarchical clustering of E. coli Big-Six group strains extracellular proteins: The relative protein expression of extracellular proteins was shown in a heat map. Two unique clusters were shown in clusters A and B in O103:H11 and O121:H19 strains, respectively.

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Expressing

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: The list of extracellular proteins identified in E. coli Big-Six group strains

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Control, Modification

Journal: Proteomics

Article Title: Secretome analysis of diarrhea-inducing strains of Escherichia coli

doi: 10.1002/pmic.201600299

Figure Lengend Snippet: Identification of strain-specific proteins in E. coli Big-Six group strains. (A) The strain-specific proteins in E. coli Big-Six group strains are shown. The genome of each strain was translated and mapped to each of the identified peptide. The presence of each strain-specific protein is shown as red. (B) Representative MS/MS spectrum of an identified peptide, DEAWVILEGHIVK of membrane protein (yhha) in E. coli O121:H19.

Article Snippet: 2.2 Culture and harvesting secretome The E. coli strains O111, O145, O26, O103, O45, O121, and K12 obtained from ATCC (Manassas, VA, USA).

Techniques: Tandem Mass Spectroscopy, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Contribution of −Omics Technologies in the Study of Porphyromonas gingivalis during Periodontitis Pathogenesis: A Minireview

doi: 10.3390/ijms24010620

Figure Lengend Snippet: Periodontitis pathogenesis and P. gingivalis overview. ( A ) During the onset and progression of periodontitis, P. gingivalis resides in the subgingival biofilm adhered to the tooth surface, where it interacts metabolically with other bacteria, inducing them to express different virulence factors with pathogenic potential. In parallel, P. gingivalis acts as a keystone pathogen, altering the regulation of the immune response in the susceptible host. The metabolic synergism and immune response subversion provide the nutritional and protective conditions required by the dysbiotic subgingival community to increase their diversity and abundance, with the concomitant induction of a strong, destructive inflammatory response. Together, all these activities cause irreversible connective tissue breakdown and resorption of the tooth-supporting alveolar bone, the critical hallmark of periodontitis that causes tooth loss. ( B ) To invade the periodontium, P. gingivalis uses various virulence factors that allow it to colonize, replicate, and spread in different subsets of cells to increase its progeny and generate infection. ( C ) In addition to causing tooth loss, P. gingivalis -induced periodontitis can also affect systemic health, influencing the course of other diseases and conditions. This figure was created using BioRender.com.

Article Snippet: Transcriptomics , Sequencing of cultured P. gingivalis (WT, ATCC ® 33277TM and isogenic ∆luxS strain). , Validation of the role of LuxS in regulating hemin uptake and microcolony formation with other bacteria. Both activities related to quorum sensing. , [ ] .

Techniques: Metabolic Labelling, Bacteria, Infection

Journal: International Journal of Molecular Sciences

Article Title: Contribution of −Omics Technologies in the Study of Porphyromonas gingivalis during Periodontitis Pathogenesis: A Minireview

doi: 10.3390/ijms24010620

Figure Lengend Snippet: Workflow for −omic research focused on P. gingivalis studies. Samples from subgingival plaque are used for metagenomic, metatranscriptomic, proteomic, and metabolomic analyses. Samples are processed in the data acquisition stage by using sequencing or microarray techniques for DNA and RNA, or by mass spectrometry, used to identify proteins and metabolites. Bioinformatic workflows involve quality control and several downstream analyses, such as data clustering or data classification. For example, in the case of sequencing-based procedures, the analyses start with nucleic acid extraction, purification, quality control, library preparation, and sequencing; raw data from sequencing is analyzed using bioinformatics approaches, depending on the target or strategy for sequencing, such as a marker-targeted amplicon (e.g., a region of the 16S rRNA gene) or a shotgun sequencing (for a whole metagenome). In the case of isolated genome sequencing, generated data can be used in comparative genomic analysis. Transcriptomics studies are directed to RNA samples, involving RNA extraction, isolation, and quality checking, before sequencing. Most transcriptome studies are focused on mRNAs, focusing on the identification of the upregulated and downregulated gene expression. The further analysis comprises the determination of the global expression profile, clustering profile, and community composition profile. This figure was created using BioRender.com.

Article Snippet: Transcriptomics , Sequencing of cultured P. gingivalis (WT, ATCC ® 33277TM and isogenic ∆luxS strain). , Validation of the role of LuxS in regulating hemin uptake and microcolony formation with other bacteria. Both activities related to quorum sensing. , [ ] .

Techniques: Sequencing, Microarray, Mass Spectrometry, Control, Extraction, Purification, Marker, Amplification, Shotgun Sequencing, Isolation, Generated, RNA Extraction, Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Contribution of −Omics Technologies in the Study of Porphyromonas gingivalis during Periodontitis Pathogenesis: A Minireview

doi: 10.3390/ijms24010620

Figure Lengend Snippet: Summary of deep amplicon sequencing, comparative genomics and metagenomics research studies associated to this review.

Article Snippet: Transcriptomics , Sequencing of cultured P. gingivalis (WT, ATCC ® 33277TM and isogenic ∆luxS strain). , Validation of the role of LuxS in regulating hemin uptake and microcolony formation with other bacteria. Both activities related to quorum sensing. , [ ] .

Techniques: Amplification, Sequencing, Functional Assay, Microarray, Genome Wide, Comparison, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Contribution of −Omics Technologies in the Study of Porphyromonas gingivalis during Periodontitis Pathogenesis: A Minireview

doi: 10.3390/ijms24010620

Figure Lengend Snippet: Summary of Transcriptomics and metatranscriptomics research studies associated to this review.

Article Snippet: Transcriptomics , Sequencing of cultured P. gingivalis (WT, ATCC ® 33277TM and isogenic ∆luxS strain). , Validation of the role of LuxS in regulating hemin uptake and microcolony formation with other bacteria. Both activities related to quorum sensing. , [ ] .

Techniques: Comparison, Expressing, Cell Culture, Bacteria, Binding Assay, Membrane, Quantitative Proteomics, Microarray, Isolation, In Vitro, Sequencing, Infection, Biomarker Discovery, In Silico, Control, Plasmid Preparation, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Contribution of −Omics Technologies in the Study of Porphyromonas gingivalis during Periodontitis Pathogenesis: A Minireview

doi: 10.3390/ijms24010620

Figure Lengend Snippet: Summary of metabolomics and proteomics research studies associated to this review.

Article Snippet: Transcriptomics , Sequencing of cultured P. gingivalis (WT, ATCC ® 33277TM and isogenic ∆luxS strain). , Validation of the role of LuxS in regulating hemin uptake and microcolony formation with other bacteria. Both activities related to quorum sensing. , [ ] .

Techniques: Cell Culture, Control, Mass Spectrometry, Membrane, Expressing, Mutagenesis, Binding Assay, Nuclear Magnetic Resonance, Comparison, Biomarker Discovery, Chromatography, Infection, RNA Extraction, Derivative Assay

Journal: Cell

Article Title: Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii.

doi: 10.1016/j.cell.2005.08.046

Figure Lengend Snippet: Figure 1. R. conorii Interacts with a Subset of Specific Host- Cell Proteins (A) Eluates from an in vitro bacterial affinity pull-down assay using Vero and HeLa cells were separated on SDS-PAGE and silver stained. Mass- spectrometry analysis of protein bands identified specific host-cell pro- teins, including Ku70, Ku86, PARP, and b-actin (arrows), that interact with R. conorii. (B and C) Biotin-labeled surface proteins from Vero cells were incubated with R. conorii to determine host-cell surface proteins that could potentially serve as receptors. R. conorii but not E. coli, L. monocytogenes (EGD), or S.flexneri(M90T)wasfoundtointeractwitha70–75kDaplasma-membrane protein as revealed by immunoblotting with streptavidin-HRP (arrow in [B]) that was subsequently identified by immunoblotting as Ku70 with monoclo- nal antisera directed at Ku70(N3H10) (C).Arrow marked ‘‘?’’ in (B) refers toa biotin-labeled surface protein that strongly interacts with L. monocytogenes but was not further analyzed. Biotin lysate in (B) refers to the biotin-labeled cell lysate prior to incubation with whole-cell bacteria. R. conorii lysate in (C) demonstrates that the interaction with Ku70 is specific and is not due to copurification of Ku70 during isolation of R. conorii from mammalian cells.

Article Snippet: To visualize biotin-labeled surface proteins that interacted with bacteria, eluted proteins were separated on 10% SDS-PAGE, transferred to nitrocellulose, and incubated with HRP-labeled streptavidin (1:5000) in membrane blocking buffer (Zymed).

Techniques: In Vitro, Pull Down Assay, SDS Page, Staining, Mass Spectrometry, Labeling, Incubation, Membrane, Western Blot, Bacteria, Isolation

Journal: Nature

Article Title: Commensal consortia decolonize Enterobacteriaceae via ecological control

doi: 10.1038/s41586-024-07960-6

Figure Lengend Snippet: a , GF B6 mice ( n = 3–10 per group) were monocolonized with the indicated pathogenic or antibiotic-resistant strain, and then treated with the indicated bacterial mixture. Faecal pathobiont load was examined by counting CFUs or by qPCR of bacterial DNA (for C. difficile ). b , c , GF B6 mice were colonized with faecal microbiota from a patient with Crohn’s disease (CD15) containing a high level of K. pneumoniae ( b ) or from a patient with ulcerative colitis (UC5) containing ESBL + E. coli ( c ). All mice were subsequently treated with vancomycin (VCM), and half of the mice received oral F18-mix administration four times over the next two days. Full-length 16S rRNA gene sequencing was performed on faecal samples to determine the relative abundance of detected strains. d – f , GF Il10 −/− mice ( n = 6 per group) were colonized with UC5 microbiota and then treated with F18-mix, F13-mix or vehicle control; faecal CFUs of ESBL + E. coli throughout the experiment ( d ), representative haematoxylin and eosin staining of the colon on day 28 ( e ; scale bars, 200 μm) and histological colitis scores on day 28 ( f ) are shown. Data in a , d , f , are median ± IQR and are compared by Kruskal–Wallis test using the Benjamini–Hochberg correction for multiple comparisons.

Article Snippet: To examine the effects of defined consortia on pathogenic bacteria, C57BL/6 GF mice (8–14 weeks of age, housed in separate GF isolators) were inoculated with K. pneumoniae 2H7 (Kp-2H7), carbapenem-resistant K. pneumoniae (CPM + Kp, ATCC BAA1705), K. aerogenes (strain Ka-11E12 ), extended-spectrum-β-lactamase producing E. coli (ESBL + E. coli , ATCC BAA2777), adherent-invasive E. coli (AIEC, strain LF82, provided by N. Barnich ), P. aeruginosa (ATCC 10145), vancomycin-resistant E. faecium (VRE Ef, ATCC 700221), C. upsaliensis (ATCC BAA1059), or C. difficile (strain 630, ATCC BAA1382) by oral gavage (2 × 10 8 CFU per mouse).

Techniques: Sequencing, Control, Staining

Journal: Nature

Article Title: Commensal consortia decolonize Enterobacteriaceae via ecological control

doi: 10.1038/s41586-024-07960-6

Figure Lengend Snippet: a, b , GF B6 mice were colonized with faecal microbiota from either a patient with Crohn’s disease (CD#15) containing a high level of K. pneumoniae ( a ) or from a patient with ulcerative colitis (UC#5) containing ESBL + E .coli ( b ). All mice were subsequently treated with vancomycin, and half received oral F18-mix administration four times over two days. CFUs of K. pneumoniae and E. coli (upper panels) and Shannon index (lower panels) of the faecal microbiota were examined longitudinally and compared by Mann-Whitney U test at day 28 (two-sided). c-g , GF Il10 −/− mice were monocolonized with Kp-2H7, then orally administered the indicated bacterial mix seven days later. Representative haematoxylin and eosin staining of the colon (scale bar = 100 μm) ( d ), histological colitis scores ( e ), faecal lipocalin-2 and calprotectin levels ( f ), and frequency of IFNγ + cells among colonic lamina propria CD4 + TCRβ + T cells ( g ) are shown. In panels a - c and e - g , median ± IQR are shown, representative of two independent experiments. Statistical analysis was performed using the Kruskal-Wallis test with the Benjamini-Hochberg correction for multiple comparisons.

Article Snippet: To examine the effects of defined consortia on pathogenic bacteria, C57BL/6 GF mice (8–14 weeks of age, housed in separate GF isolators) were inoculated with K. pneumoniae 2H7 (Kp-2H7), carbapenem-resistant K. pneumoniae (CPM + Kp, ATCC BAA1705), K. aerogenes (strain Ka-11E12 ), extended-spectrum-β-lactamase producing E. coli (ESBL + E. coli , ATCC BAA2777), adherent-invasive E. coli (AIEC, strain LF82, provided by N. Barnich ), P. aeruginosa (ATCC 10145), vancomycin-resistant E. faecium (VRE Ef, ATCC 700221), C. upsaliensis (ATCC BAA1059), or C. difficile (strain 630, ATCC BAA1382) by oral gavage (2 × 10 8 CFU per mouse).

Techniques: MANN-WHITNEY, Staining

Journal: Nature

Article Title: Commensal consortia decolonize Enterobacteriaceae via ecological control

doi: 10.1038/s41586-024-07960-6

Figure Lengend Snippet: Bacterial strains isolated from donors F, K, or I were cultured in mGAM broth containing 300 μM gluconate for 48 hr at 37 °C (n = 3 biological replicates). Gluconate concentration in the culture supernatant was measured by LC-MS/MS and is depicted in the middle bar graph. Data are shown as median ± IQR. Genomes of cultured strains were sequenced and examined for carriage of genes putatively involved in gluconate metabolism. For classical pathway genes, gluconate kinase ( gntK , MKMCEHOJ_02531) and gluconate transporter sequences (MKMCEHOJ_02530, MKMCEHOJ_02505) from the f37_ E. coli strain were used as the reference. For alternative pathway genes, gluconate dehydratase ( gad , EAOGLLOI_00767), gluconate transporter sequences (EAOGLLOI_00766, EAOGLLOI_00912), 2-dehydro-3-deoxygluconokinase ( kdgK , EAOGLLOI_00768), and 2-dehydro-3-deoxyphosphogluconate aldolase ( eda , EAOGLLOI_00769) from the f17_ Blautia caecimuris strain were used as the reference. Asterisk indicates that the gluconate dehydratase in the f19_ Blautia caecimuris strain is nonfunctional due to a frameshift mutation. GntK, gluconate kinase. GAD, gluconate dehydratase.

Article Snippet: To examine the effects of defined consortia on pathogenic bacteria, C57BL/6 GF mice (8–14 weeks of age, housed in separate GF isolators) were inoculated with K. pneumoniae 2H7 (Kp-2H7), carbapenem-resistant K. pneumoniae (CPM + Kp, ATCC BAA1705), K. aerogenes (strain Ka-11E12 ), extended-spectrum-β-lactamase producing E. coli (ESBL + E. coli , ATCC BAA2777), adherent-invasive E. coli (AIEC, strain LF82, provided by N. Barnich ), P. aeruginosa (ATCC 10145), vancomycin-resistant E. faecium (VRE Ef, ATCC 700221), C. upsaliensis (ATCC BAA1059), or C. difficile (strain 630, ATCC BAA1382) by oral gavage (2 × 10 8 CFU per mouse).

Techniques: Isolation, Cell Culture, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: Frontiers in Microbiology

Article Title: Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

doi: 10.3389/fmicb.2021.654193

Figure Lengend Snippet: Kinetics, viability, and morphology of Helicobacter pylori growth in liquid F-12-cholesterol. (A) Growth curve of Helicobacter pylori 26695 grown in F12 liquid medium supplemented with 1× cholesterol (continuous line), modeled using the Gompertz growth equation model (dotted line), calculated with GraphPad Prism, and based on the optical density measurements of the bacterial suspension at 600 nm (OD 600 ) from 24, 48, 64, and 72 h cultures. Data are shown as mean ± SEM of 16 biological replicates. (B) Bacterial viability evaluated by colony-forming units (CFUs) counting (left graph) and the LIVE/DEAD BacLight Bacterial Viability assay (right graph). The number of CFUs was determined at the referred time points, represented as CFUs/mL. Each dot represents a biological replicate ( n = 9) and data are shown as mean ± SEM. Statistical significance was evaluated using the one-way ANOVA with post hoc Tukey’s test, comparing all datasets with each other; only comparisons between 48, 64, and 72 h were illustrated for the sake of simplicity; ∗∗ p ≤ 0.01, n.s. – not significant. LIVE/DEAD BacLight Bacterial Viability assay at 48, 64, and 72 h of growth, by flow cytometry. After sample staining with SYTO9 and PI dies and acquisition on a FACSCanto II cytometer, live and dead bacteria were gated and defined as SYTO9 + PI – and SYTO9 + PI + , respectively. Each dot represents a biological replicate ( n = 4) and data are shown as mean ± SEM of the frequency of gated SYTO9 + PI – and SYTO9 + PI + bacteria. Statistical significance was evaluated using the two-way ANOVA with post hoc Tukey’s test; n.s. – not significant. (C) Representative negative stain TEM micrographs of bacillary, U-shaped, and coccoid forms of H. pylori from a 64 h liquid culture. Scale bars: 1 μm; 80,000× (bacillary and coccoid) and 8,000× (U-shaped) original magnifications. (D) Quantification of bacillary, U-shaped, and coccoid forms of H. pylori present in F12-cholesterol liquid cultures at 48, 64, and 72 h from negative stained TEM micrographs, using manual counting. The frequency of each bacterial form was calculated considering the total number of bacteria (741, 1,215, and 1,061) counted in micrographs taken from samples of each time point, 48 h ( n = 5), 64 h ( n = 5), and 72 h ( n = 2), respectively. The mean frequency of each form is displayed inside the respective bar. Statistical significance was evaluated using the two-way ANOVA with post hoc Tukey’s test, comparing all datasets with each other; statistical significance was only observed between 72 and 48 h for bacillary and coccoid forms; ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01.

Article Snippet: Helicobacter pylori strains 26695 (ATCC ® 700392, cagA + , vacA s1/m1), 60190 (ATCC ® 49503, cagA + , vacA s1/m1) and Tx30a (ATCC ® 51932; cagA – , vacA s2/m2) were routinely cultured in Trypticase TM Soy Agar (TSA) supplemented with 5% Sheep Blood (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) and incubated in a sealed jar with a microaerophilic atmosphere (GENBox microaer; bioMérieux S.A., Marcy l’Etoile, France) at 37°C for 48 h. Bacteria were sub-cultured for a maximum of 12 passages.

Techniques: Suspension, Viability Assay, Flow Cytometry, Staining, Cytometry, Bacteria

Journal: Frontiers in Microbiology

Article Title: Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

doi: 10.3389/fmicb.2021.654193

Figure Lengend Snippet: Morphological characterization, size distribution, and yield of OMVs secreted by Helicobacter pylori grown in F12-cholesterol medium. (A) Negative staining of OMVs isolated from H. pylori 26695 F12-cholesterol liquid cultures at 48, 64, and 72 h of growth, and (B) ultrastructure section of OMVs from 64 h H. pylori cultures. Scale bars: 200 nm; 50,000×, 100,000× [insets in panel (A) ] and 200,000× [inset in panel (B) ] original magnifications. (C) Size distribution, represented as percentage of the total number of isolated OMVs; data are shown as mean ± SEM of four biological replicates and statistical significance was evaluated using the one-way ANOVA with post hoc Tukey’s test; n.s. – not significant. (D) Number of recovered OMVs per mL of bacterial culture, determined using Nanoparticle Tracking Analysis (NTA) at 48, 64, and 72 h periods of bacterial growth; data are shown as mean ± SEM of four biological replicates and statistical significance was evaluated using the one-way ANOVA with post hoc Tukey’s test; ∗ p ≤ 0.05, n.s. – not significant. (E) Representative negative stain TEM micrographs of H. pylori 60190 and Tx30a-OMVs generated from 64 h F12-cholesterol bacterial cultures. Scale bars: 200 nm; 50,000× and 100,000× (insets) original magnification. (F) Size distribution, represented as percentage of the total number of isolated OMVs at 64 h of liquid culture, and (G) number of recovered OMVs per mL of bacterial culture determined by NTA; data are shown as mean ± SEM of 4 (for 26695) or 3 (for 60190 and Tx30a) biological replicates and statistical significance was evaluated using the Brown-Forsythe and Welch ANOVA with post hoc Dunnett’s test; n.s. – not significant.

Article Snippet: Helicobacter pylori strains 26695 (ATCC ® 700392, cagA + , vacA s1/m1), 60190 (ATCC ® 49503, cagA + , vacA s1/m1) and Tx30a (ATCC ® 51932; cagA – , vacA s2/m2) were routinely cultured in Trypticase TM Soy Agar (TSA) supplemented with 5% Sheep Blood (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) and incubated in a sealed jar with a microaerophilic atmosphere (GENBox microaer; bioMérieux S.A., Marcy l’Etoile, France) at 37°C for 48 h. Bacteria were sub-cultured for a maximum of 12 passages.

Techniques: Negative Staining, Isolation, Staining, Generated

Journal: Frontiers in Microbiology

Article Title: Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

doi: 10.3389/fmicb.2021.654193

Figure Lengend Snippet: Protein profile and proteomic analysis of OMVs secreted by Helicobacter pylori grown in F12-cholesterol medium. (A) Protein profile of 10 11 OMVs isolated from 48, 64, and 72 h- H. pylori 26695 bacterial cultures, after staining a SDS-PAGE with BlueSafe, and corresponding (B) protein quantification. Data are shown as mean ± SEM of four biological replicates and statistical significance was evaluated using the one-way ANOVA with post hoc Tukey’s test; n.s. – not significant. Proteomic analysis of 48, 64, and 72 h-OMVs isolated from H. pylori 26695 F12-cholesterol liquid cultures by nanoLC-MS/MS and prediction of the (C) cellular localization, (D) biological process, and (E) molecular function of the identified proteins using the gene ontology UniProt database. Data are shown as the abundance of proteins in each group; for each bar, the number of proteins and respective abundance is indicated.

Article Snippet: Helicobacter pylori strains 26695 (ATCC ® 700392, cagA + , vacA s1/m1), 60190 (ATCC ® 49503, cagA + , vacA s1/m1) and Tx30a (ATCC ® 51932; cagA – , vacA s2/m2) were routinely cultured in Trypticase TM Soy Agar (TSA) supplemented with 5% Sheep Blood (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) and incubated in a sealed jar with a microaerophilic atmosphere (GENBox microaer; bioMérieux S.A., Marcy l’Etoile, France) at 37°C for 48 h. Bacteria were sub-cultured for a maximum of 12 passages.

Techniques: Isolation, Staining, SDS Page, Tandem Mass Spectroscopy