Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response

doi: 10.3389/fcimb.2024.1377077

Figure Lengend Snippet: CXCL-8 secretion varied with glycan alterations. (A) Experimental workflow for assessing pro-inflammatory cytokine secretion of glycosylation mutants compared to wildtype H. pylori . Image was created using BioRender. (B) The glycoprotein mutant with elaborated LPS structures, Δ579, induced increased CXCL-8 secretion from gastric epithelial cells relative to wildtype. (C) The LPS mutant, Δwzk, induced decreased CXCL-8 secretion from gastric epithelial cells relative to wildtype. (D–I) Variable impact on CXCL-8 secretion was observed with glycoprotein mutants Δ580 and Δ1179, and LPS mutant, ΔwaaL, compared to wildtype depending on the coculture experiment. Error bars reflect technical replicates. Tukey’s multiple comparison test one-way ANOVA was used. (**P< 0.01, ***P< 0.001, ****P< 0.0001, ns, not significant). Data are representative of independent replicate experiments (n > 3) that exhibited the same findings.

Article Snippet: Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- α IL-6, or IL-1 β conditioned media samples.

Techniques: Glycoproteomics, Mutagenesis, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response

doi: 10.3389/fcimb.2024.1377077

Figure Lengend Snippet: Immature dendritic cell cytokine secretion changed when challenged by H. pylori glycosylation mutants. (A) Experimental workflow used to assess TNF-α, IL-6, IL-1β, and IL-10 secretion from iDCs following challenge for 24-hours with glycosylation mutants or wildtype bacteria. Image was created using BioRender. Datasets from two different iDC challenge experiments are shown, with (B–E) collected in one experiment and (F–I) collected in a second experiment. Δ580.1 and Δ 580.2, as well as Δ1179.1 and Δ1179.2, represent different freeze lots of the same strain. Error bars reflect technical replicates. Tukey’s multiple comparison test one-way ANOVA was used. (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, ns, not significant).

Article Snippet: Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- α IL-6, or IL-1 β conditioned media samples.

Techniques: Glycoproteomics, Bacteria, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response

doi: 10.3389/fcimb.2024.1377077

Figure Lengend Snippet: The glycocalyx was probed to assess how Lewis Y expression and LPS elaboration correlate to cytokine secretion by immune cells. (A) Western blot analysis reveals robust Lewis Y expression in Δ579 and Δ580.2. The strain samples used in these experiments correspond to those used in iDC challenge experiments for the dataset shown in Figure 5 . (B, C) Analysis of LPS by gel electrophoresis reveals that Δwzk and ΔwaaL synthesize only truncated, low molecular weight LPS relative to wildtype H. pylori , whereas Δ579 appears to synthesize LPS with somewhat more prominent bands at higher molecular weights (e.g., >29kDa) than wildtype H. pylori . (B) The strain samples used in these experiments correspond to those used in dendritic cell challenge experiments for the dataset shown in Figure 5 . Δ580.1 and Δ 580.2 represent different freeze lots of the same strain. (C) Analysis of LPS by gel electrophoresis for strain samples used in dendritic cell challenge experiments for the dataset shown in Supplementary Figure S3 . Δ1179.1 and Δ1179.2 represent different freeze lots of the same strain and appear to have comparable LPS fingerprints.

Article Snippet: Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- α IL-6, or IL-1 β conditioned media samples.

Techniques: Expressing, Western Blot, Nucleic Acid Electrophoresis, Molecular Weight