副溶血性弧菌原噬菌体样基因簇介导的效应蛋白及其毒力效应的研究

Vibrio parahaemolyticus contamination in aquatic products is the major contributor to food safety issues caused by pathogenic microorganism in China. Recent research revealed that V. parahaemolyticus isolates lacking the primary virulence factors of thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH), which are required to be detected by government standards, also showed varying degrees of pathogenicity to human gastrointestinal cells, suggesting other virulence-associated factors exist in the bacterium. In this project, we will identify and characterize effector proteins (EFs) mediated by four unknown prophage gene clusters (designated as VpaProphage I-IV) present in V. parahaemolyticus CHN25 genome. The ∆VpaProphage I-IV mutants will be individually constructed using the unmarked in-frame gene deletion system. The virulence of the wild type and four mutant strains will be assessed against host human cells and prokaryotic bacteria. Secretomic patterns of the wild type and ∆VpaProphagestoxic mutant strains will be obtained using the two-dimensional gel electrophoresis analysis, and the EPs mediated by VpaProphagestoxic will be individually identified using the liquid chromatography tandem mass spectrometry (LC-MS/MS) technique. Each of the identified EPs will be confirmed for their virulence. The distribution of the genes encoding toxic EPs and their VpaProphagestoxic will be investigated in aquaculture products of shrimps and shellfish. The results of this project will lead to new insights into the molecular mechanisms underlying the pathogenicity of V. parahaemolyticus and suggest the future research directions. The outputs of this project will also provide technical support to the control of the leading food-borne disease in China.

副溶血性弧菌(Vibrio parahaemolyticus)污染引发的水产品安全事件在我国微生物性食品安全事件中位居首位。针对目前研究发现该菌不携带国标检测毒力基因的菌株也具有致病性的严峻问题，本课题以副溶血性弧菌CHN25基因组上四个原噬菌体样基因簇(VpaProphageI-IV)为研究对象，运用基因敲除技术与蛋白质组学方法，分别构建∆VpaProphageI-IV缺失突变株，比较野生型和四种突变型菌株对宿主真核细胞以及原核细菌的毒力效应；获取其中∆VpaProphagestoxic突变型和野生型菌株的分泌组图谱，发现、鉴定毒性VpaProphagestoxic各自介导的效应蛋白，验证其毒力效应；揭示携带毒性效应蛋白的副溶血性弧菌在虾、贝类水产品中的分布状况。本课题的开展将为副溶血性弧菌污染造成的食源性疾病及其防控的研究提供理论基础和技术支撑,进而提升我国在该领域的研究水平。

副溶血性弧菌(Vibrio parahaemolyticus)污染引发的水产品安全事件在我国微生物性食品安全事件中位居首位。摄食该菌污染的水产品可引起以发热、腹泻、呕吐等为主要症状的急性胃肠炎，严重者脱水、休克，甚至死亡。针对目前研究发现该菌不携带国标检测毒力基因的菌株也具有致病性的严峻问题，本项目基于项目组的前期研究发现，以水产动物源副溶血性弧菌CHN25基因组上四个原噬菌体样基因簇(VpaProphageI-IV)为研究对象，综合运用基因敲除技术、蛋白质组学、转录组学和细胞生物学等技术和方法，已敲除，鉴定，得到15种原噬菌体样基因簇中单基因的缺失突变型菌株，2种双基因的缺失突变型菌株，以及2种三基因的缺失突变型菌株；已构建，鉴定，得到15种回补突变型菌株；已测定野生型、15种基因缺失突变型及其回补突变型菌株响应环境应激的表型特征，发现原噬菌体样基因缺失显著影响副溶血性弧菌CHN25在不同环境条件下的生长力、泳动性和生物被膜形成；已测定，获得其中10种基因缺失突变型及其回补突变型，以及野生型菌株的分泌组图谱，发现、鉴定了多种差异分泌蛋白；已着重分析编码未知功能蛋白的原噬菌体样基因(如VpaChn25_0724、VpaChn25_0734等)对副溶血性弧菌CHN25–宿主人肠道上皮细胞相互作用，及其全局性水平基因转录的影响，证实了其中VpaChn25_0724基因贡献副溶血性弧菌CHN25细胞膜完整性的生物学功能；已分析携带潜在毒力效应蛋白编码基因的副溶血性弧菌在虾、贝类水产品中的分布状况，发现原噬菌体样基因(如VpaChn25_0724等)在水产动物源副溶血性弧菌种内，弧菌属种间，甚至非弧菌属的细菌属间散播。本项目的研究结果为我国水产品头号致病菌污染造成的食源性疾病及其防控的研究提供了理论基础和技术支撑，且为其它食源性疾病的研究提供了新思路。