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

Journal: eLife

Article Title: PRR adjuvants restrain high stability peptides presentation on APCs

doi: 10.7554/eLife.99173

Figure Lengend Snippet: ( A ) Peptide locations across the Helicobacter pylori genome from MHC-II immunopeptidomes. ( B ) Rank plot of each protein abundance detected in the whole-proteome of bacterial ultrasonic supernatant antigens. Proteins identified in immunopeptidomes are annotated with their respective gene names. ( C ) MHC-II presentation potential of bacterial proteins. All reported H. pylori proteins were ranked according to the ratio between the number of peptides predicted to be presented by MHC-II alleles (rank ≤2) and the total number of 13- to 17-mer. Proteins identified in immunopeptidomes are annotated with their respective gene names. ( D ) Heatmap of exogenous MHC peptides from different adjuvant groups. The identified sequences are shown. ( E ) Numbers of MHC peptides derived from bacteria and hosts were compared among different adjuvant groups. n=3. **p<0.01, ****p<0.0001.

Article Snippet: Helicobacter pylori strain NCTC 11637 (ATCC) was cultured on brain–heart infusion plates with 10% rabbit blood (Sbjbio, Nanjing, China) at 37 °C under microaerophilic conditions.

Techniques: Quantitative Proteomics, Adjuvant, Derivative Assay, Bacteria