Biological Characterization of Helicobacter pylori Outer Membrane Vesicles isolated by the Biofilm and Planktonic Phenotypes

Helicobacter pylori generates outer membrane vesicles (OMVs) during its growth in both planktonic and biofilm phenotypes. It has been demonstrated that OMVs are a component of H. pylori biofilm and that their interactions with associated extracellular DNA (eDNA) has a role in “bridging” OMV-OMV and OMV-cell interactions. The aim of the present study was the biological characterization of OMVs isolated by the planktonic (pOMVs) and biofilm (bOMVs) phenotypes at different time points, to elucidate the role of the OMVs in H. pylori growth and pathogenesis as a developmental process. H. pylori ATCC 43629 biofilm formation was evaluated at 2, 6 and 10 days of incubation using confocal microscopy and live/dead staining and quantified image analysis. Enumeration of bOMVs and pOMVs associated with eDNA was performed using PicoGreen and PKH staining followed by flow cytometry. The bOMVs and pOMVs collected were subsequently analysed by nanoLC-MS/MS to determine the exoproteome. COMSTAT analysis showed no significant changes in thickness, biomass and roughness for each day, suggesting that biofilm reaches maturity after 2 days. Flow cytometry data demonstrated an increase of bOMVs and pOMVs over time and most of the detected vesicles (>60%) contained eDNA suggesting a possible key role of OMVs in eDNA delivery as well as in the biofilm formation. Proteomic analysis revealed a time and phenotype dependent modulation of many virulence related proteins including the Vacuolating cytotoxin transporter which was 5 times more abundant in the biofilm, linking OMVs to virulence via biofilm formation.