Characterization of Helicobacter pylori Outer Membrane Vesicles over time, in biofilm and planktonic phenotypes

Introduction Helicobacter pylori is known to be a major pathogen causing gastric diseases via its direct colonization of the gastric mucosa. H. pylori releases Outer Membrane Vesicles (OMVs) throughout the growth process both in planktonic and biofilm phenotypes. The number, size and content of H. pylori OMVs over time, especially in H. pylori biofilm, remain unclear. Methods In this study, we analyzed H. pylori biofilm at 2, 6, and 10 days as well as we extracted and characterized H. pylori pOMVs and bOMVs over time by transmission electron microscopy, nanoparticle tracking analysis, dynamic light scattering, electrophoretic light scattering and proteomic technology. Results Helicobacter pylori ATCC 43629 formed a multi-structured biofilm with large clusters characterized by mostly live cells and some fractures corresponding to water channels. Analysis of H. pylori OMVs reveals that the bacterial growth time and phenotype affect their number, size, and composition. Proteomic analysis revealed that in the early growth phase pOMVs are enriched with multiple virulence factors associated with host cell destruction whereas during later growth phases vesicles contain factors involved in the metabolic processing. The proteome of bOMVs was much more homogeneous and stable over time: in late growth stages, bOMVs proteomic analysis identified proteins involved in iron accumulation, protection against oxidative stress, immunosuppression in the gastric environment, and virulence promoting inflammation and tumorigenesis. Conclusion This study suggests that H. pylori induces pathogenicity at least partially by secreting bOMVs that could promote tissue destruction related to tumorigenesis; therefore, the development of gastric cancer could be associated not only with the microorganism itself, but also with OMVs that it produces.