title: Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA).
abstract: Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation.
authors.affiliation: Department of Pharmacy, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Center of Excellence on Aging, Ce.S.I., "G. d'Annunzio" University of Chieti-PescaraChieti, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Grande
authors.affiliation: Center of Excellence on Aging, Ce.S.I., "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Department of Medical, Oral, and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Di Marcantonio
authors.affiliation: Department of Biological Sciences, Institute of Molecular Genetics, National Research Council Chieti, Italy.
authors.forename: Iole
authors.initials: I
authors.lastname: Robuffo
authors.affiliation: Center of Excellence on Aging, Ce.S.I., "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Department of Medical, Oral, and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Pompilio
authors.affiliation: Department of Pharmacy, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Department of Nanomedicine, Houston Methodist Research InstituteHouston, TX, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Celia
authors.affiliation: Department of Pharmacy, "G. d'Annunzio" University of Chieti-Pescara Chieti, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Di Marzio
authors.affiliation: Department of Clinical and Experimental Medicine, University of Catanzaro "Magna Graecia" Catanzaro, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: Paolino
authors.affiliation: Center of Excellence on Aging, Ce.S.I., "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Department of Medical, Oral, and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy.
authors.forename: Marilina
authors.initials: M
authors.lastname: Codagnone
authors.affiliation: Center of Excellence on Aging, Ce.S.I., "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Department of Medical, Oral, and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Muraro
authors.affiliation: Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, The Ohio State UniversityColumbus, OH, USA; Department of Orthopaedics, The Ohio State UniversityColumbus, OH, USA; Faculty of Engineering and the Environment, University of SouthamptonSouthampton, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Stoodley
authors.affiliation: Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, The Ohio State UniversityColumbus, OH, USA; NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation TrustSouthampton, UK.
authors.forename: Luanne
authors.initials: L
authors.lastname: Hall-Stoodley
authors.affiliation: Center of Excellence on Aging, Ce.S.I., "G. d'Annunzio" University of Chieti-PescaraChieti, Italy; Department of Medical, Oral, and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-PescaraChieti, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Mincione
keywords: Helicobacter pylori; biofilm; eDNA; nanoparticles; outer membrane vesicles (OMVs)