The role of outer membrane vesicles of Helicobacter pylori in pathogenesis

Background: H. pylori continuously produces outer membrane vesicles (OMV) during active growth. These vesicles contain virulence factors and are cytotoxic, but the properties and contents of bacterial OMV can vary depending on the environmental conditions. Aim: The study aimed to characterise H. pylori OMV isolated from different environmental conditions in terms of their quantity, size, cytotoxicity, and protein contents. Methods: OMV were purified from H. pylori cultures in BHI broth with 0.2% β-cyclodextrin after 1 or 6 days or blood agar plates after 24 hours. OMV size and quantity were characterised by Zetaview. Their toxicity on human gastric epithelial (AGS) cells was determined by CellTiter, RealTime-Glo assays, IncuCyte and microscopy. Proteins were characterised and quantified by LC-MS/MS and SWATHMS, label-free, to determine changes in protein expression between growth conditions. A cagA mutagenesis plasmid was constructed in the 444A strain background. Results: H. pylori produced more OMV in broth than on agar plates with differences also observed in average OMV diameter between conditions. OMV isolated from late broth and plate cultures were significantly more toxic to AGS cells than those from early broth (p