Grande Rossella, Di Marcantonio Maria C, Robuffo Iole, Pompilio Arianna, Celia Christian, Di Marzio Luisa, Paolino Donatella, Codagnone Marilina, Muraro Raffaella, Stoodley Paul, Hall-Stoodley Luanne, Mincione Gabriella
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.
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.
Front Microbiol. 2015 Dec 16;6:1369. doi: 10.3389/fmicb.2015.01369. eCollection 2015.
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.
幽门螺杆菌的持续存在与其形成生物膜以应对不断变化的环境条件和压力的能力有关。细胞外DNA（eDNA）是幽门螺杆菌生物膜基质的一个组成部分，但缺乏DNase I活性支持了这样一种假说，即eDNA可能受到其他细胞外聚合物（EPS）和/或外膜囊泡（OMV）的保护，这些物质在生长过程中从细菌表面出芽。本研究的目的是确定从幽门螺杆菌ATCC 43629/NCTC 11639生物膜（bOMV）及其浮游期（pOMV）分离出的OMV上eDNA的存在情况，并表征OMV的物理化学性质。首先使用DNase I-金复合物标记和透射电子显微镜分析（TEM）来检测bOMV和pOMV中eDNA的存在。进一步分离bOMV和pOMV，并使用动态光散射（DLS）分析进行物理化学表征。使用PicoGreen分光光度计测定法检测和定量与OMV相关的eDNA，同时用DNA试剂盒进行提取。TEM图像显示eDNA主要与OMV膜表面相关；而PicoGreen染色显示bOMV中的双链DNA（dsDNA）比pOMV增加了四倍。使用凝胶电泳观察从OMV中提取的eDNA。DLS分析表明，浮游型和生物膜型幽门螺杆菌表型均产生囊泡，其大小在纳米尺度上分布广泛。DLS聚集试验表明，eDNA可能在生物膜表型中OMV的聚集中起作用。此外，与囊泡膜相关的eDNA可能会阻碍DNase I对幽门螺杆菌生物膜的活性。这些结果表明，源自幽门螺杆菌生物膜表型的OMV可能通过防止核酸酶降解eDNA而发挥结构作用，在OMV表面的eDNA链之间提供桥接功能并促进聚集。
