Helicobacter pylori Outer Membrane Vesicles (OMVs) as DNA shuttles and structural components of biofilm EPS

Extracellular DNA (eDNA) is a component of the H.pylori biofilm but the DNaseI ineffectiveness to disrupt the biofilm supports the hypothesis that eDNA might be protected by other EPS components and OMVs, which bleb from the cell wall during growth. The aim of this study was to both identify the eDNA presence on OMVs segregated from H.pylori biofilm (bOMVs) and its planktonic (pOMVs) phase and physiochemically characterize bOMVs and pOMVs. The eDNA was visualized using a DNaseI-gold complex and Transmission Electron Microscope analysis (TEM). Moreover,pOMVs and bOMVs were further isolated by ultracentrifugation and physiochemically characterized using the dynamic light scattering (DLS) analysis. eDNA was detected and quantified using the Quant-iTTMPicoGreendsDNA assay and NanoDrop2000cUV-VIS spectrophotometer, while its extraction was performed through QIAampDNAMiniKit. The eDNA was mainly identified on the OMVs membrane surface and the PicoGreen staining showed a 4-fold increase of dsDNA in bOMVs compared to pOMVs.The eDNA-OMVs association was also demonstrated by gel electrophoresis. The resultant data agreed those performed through TEM and PicoGreendsDNAassay. The DLS analysis demonstrated that H.pylori generate vesicles with sizes in the nanometer scales and a broad size distribution. The bOMVs appeared to “bridge” bacterial cells suggesting a possible structural role, however, the association with vesicles membrane may protect eDNA from the enzymatic activity of DNaseI in breaking up the biofilm. We further hypothesise that OMVs, generated from H.pylori, particularly in its biofilm phenotype, may represent a shuttle for eDNA transport, as well as possibly playing a structural role by preventing the nucleic acid degradation by nucleases.