Outer Membrane Vesicles (OMVs) are a component of Heicobacter pylori biofilms. The accumulation of eDNA on the surfaces of OMVs and microscopic evidence of aggregation suggests eDNA may play a structural role in “bridging” OMV-OMV and OMV-cell interactions. The aim of this study was to demonstrate the role of eDNA-OMVs interactions in biofilm development. H.pylori ATCC43629 biofilm formation was analyzed at 2, 6 and 10 days of incubation using live/dead staining and Confocal Laser Scanning Microscopy (CLSM) followed by COMSTAT image analysis. The OMV production from biofilm (bOMVs) and planktonic (pOMVs) phenotypes was measured using PicoGreen and PKH staining followed by flow cytometry. The amount of eDNA associated with the isolated vesicles was evaluated using Quant-iTTMPicoGreendsDNA. COMSTAT showed no significant changes in thickness, biomass and roughness suggesting the biofilm had reached maturity after 2 days. The biofilm had a well-developed 3D-structure with an equal amount of live and dead (membrane compromised) cells. Flow cytometry data demonstrated an increase of bOMVs associated with eDNA over time, while the pOMVs associated with eDNA peaked at 6 days. The number of vesicles (obtained by volumetric count-FACSVerse) containing eDNA was significantly higher for the biofilm, compared to the planktonic phenotype. eDNA associated with the isolated bOMVs increased over time confirming the flow cytometry data. Further, the association appeared to protect eDNA from enzymatic digestion from DNaseI. Our results demonstrate a key role of eDNA vesicles associated in H.pylori biofilm formation over time and highlight the structural role of the nucleic acid bOMV association.
Role of the extracellular DNA (eDNA) associated with the Outer Membrane Vesicles (OMVs) in Helicobacter pylori biofilm development
MINCIONE, Gabriella;LANUTI, PAOLA;DI MARCANTONIO, Maria Carmela;SIMEONE, PASQUALE;SAVINO, LUCA;ERCOLINO, EVA;BOLOGNA, GIUSEPPINA;MURARO, Raffaella;PAGOTTO, SARA;GRANDE, ROSSELLA
2016-01-01
Abstract
Outer Membrane Vesicles (OMVs) are a component of Heicobacter pylori biofilms. The accumulation of eDNA on the surfaces of OMVs and microscopic evidence of aggregation suggests eDNA may play a structural role in “bridging” OMV-OMV and OMV-cell interactions. The aim of this study was to demonstrate the role of eDNA-OMVs interactions in biofilm development. H.pylori ATCC43629 biofilm formation was analyzed at 2, 6 and 10 days of incubation using live/dead staining and Confocal Laser Scanning Microscopy (CLSM) followed by COMSTAT image analysis. The OMV production from biofilm (bOMVs) and planktonic (pOMVs) phenotypes was measured using PicoGreen and PKH staining followed by flow cytometry. The amount of eDNA associated with the isolated vesicles was evaluated using Quant-iTTMPicoGreendsDNA. COMSTAT showed no significant changes in thickness, biomass and roughness suggesting the biofilm had reached maturity after 2 days. The biofilm had a well-developed 3D-structure with an equal amount of live and dead (membrane compromised) cells. Flow cytometry data demonstrated an increase of bOMVs associated with eDNA over time, while the pOMVs associated with eDNA peaked at 6 days. The number of vesicles (obtained by volumetric count-FACSVerse) containing eDNA was significantly higher for the biofilm, compared to the planktonic phenotype. eDNA associated with the isolated bOMVs increased over time confirming the flow cytometry data. Further, the association appeared to protect eDNA from enzymatic digestion from DNaseI. Our results demonstrate a key role of eDNA vesicles associated in H.pylori biofilm formation over time and highlight the structural role of the nucleic acid bOMV association.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
