Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA).
Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA).
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DOI：
10.3389/fmicb.2015.01369
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发表时间：
2015
影响因子：
5.2
通讯作者：
Mincione G
中科院分区：
文献类型：
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作者：
Grande R;Di Marcantonio MC;Robuffo I;Pompilio A;Celia C;Di Marzio L;Paolino D;Codagnone M;Muraro R;Stoodley P;Hall-Stoodley L;Mincione G
幽门螺杆菌的持久性与其形成生物膜的能力有关，生物膜是对不断变化的环境条件和压力的反应。细胞外DNA（extracellular DNA，eDNA）是H. pylori生物膜基质，但缺乏DNA酶I活性支持eDNA可能受到其他细胞外聚合物（EPS）和/或外膜囊泡（OMV）保护的假设，这些物质在生长过程中从细菌表面起泡。本研究的目的是鉴定与H. pylori ATCC 43629/NCTC 11639生物膜（bOMV）和其增殖相（pOMV），并表征OMV的物理-化学性质。bOMV和pOMV中eDNA的存在最初使用DNase I-金络合物标记和透射电子显微镜分析（TEM）进行。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，同时使用DNA试剂盒进行其提取。TEM图像显示，eDNA主要与OMV膜表面相关;而PicoGreen染色显示，与pOMV相比，bOMV中的dsDNA增加了4倍。使用凝胶电泳观察从OMV提取的eDNA。DLS分析结果表明，无论是厌氧消化还是生物膜H. pylori表型产生囊泡，其尺寸在纳米尺度上广泛分布。DLS聚集试验表明，eDNA可能在生物膜表型中的OMV聚集中起作用。此外，与囊泡膜结合的eDNA可能会阻碍DNase I对H.幽门生物膜。这些结果表明，OMV来源于H。pylori生物膜表型可能通过阻止核酸酶降解eDNA，在OMV表面的eDNA链之间提供桥接功能并促进聚集而发挥结构作用。
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.