四膜虫介导的嗜水气单胞菌毒力进化及其分子基础

The pathogen virulence is not only associated with co-evolve in reciprocal selection with its host organism, but also with ecological factors,especially with the selection by protozoa predation. The project is performed to explore the role of Tetrahymena in the virulence evolution of Aeromonas hydrophila. Firstly, we'll examine the resistance of various A. hydrophila strains to phagocytosis (predation) and digestion by Tetrahymena, and then determine the role of Tetrahymena in selection for strain virulence. Secondly, we'll measure the proportion of viable bacterial cells contained in expelled Tetrahymena vesicles, the levels of invasion and adhesion of entrapped bacteria to cultured cells, their growth levels in macrophages and virulence to animals.It is expected to investigate the role of protozoa in up- or down- regulation of bacterial virulence. Thirdly,to decipher the molecular mechanism of virulence regulation, the transcriptomics and Real-Time PCR techniques will be further used to search for the differentiated expressed genes of A. hydrophila in vesicles released by Tetrahymena and as free cells remaining uningested, and the functions of genes will be demonstrated by deleting the interesting genes followed by analyzing the virulence traits. Finally, we'll investigate the extent of conjugation transfer event between the bacterial strains within the digestive vacuoles and examine the virulence traits of recombinant bacteria.It is aimed to access the role of Tetrahymena in mediating bacterial gene transfer. This research project will not only have an important scientific significance in deciphering A. hydrophila virulence evolution, but also provide a meaningful revelation for the emerging bacteria.

病原菌的毒力进化不仅取决于病原与宿主的共进化，还与生态因素特别是原生动物的捕食选择有关。为探讨原生动物四膜虫在嗜水气单胞菌毒力进化上的作用，本项目拟从生态学角度出发，在调查虫菌互作关系的基础上，研究该菌不同毒力菌株对四膜虫的抗吞噬（捕食）消化能力，分析四膜虫对菌株毒力的选择作用；检查四膜虫吞噬细菌后，其所释放的排泄泡中细菌的活力，从细胞和动物水平分析四膜虫对吞噬菌毒力的影响；利用转录组学和Real-Time PCR技术从全基因组表达水平分析四膜虫吞噬菌与未吞噬菌的差异表达基因，并通过构建基因缺失株进行功能验证，从而解析四膜虫对细菌毒力调控的分子基础；研究排泄泡内细菌间毒力基因的接合转移，分析转移重组菌的致病特性，评估四膜虫在细菌毒力基因水平转移上的媒介作用。研究结果具有重要的科学意义，不仅丰富了对嗜水气单胞菌毒力进化机制的认识，同时可为探讨新病原菌或新毒力菌株的发生机理提供有意义的启示。

嗜水气单胞菌广泛分布于淡水环境中，与其生活在同一生境的原生动物主要是四膜虫（Tetrahymena）。为调查四膜虫对嗜水气单胞菌毒力和环境适应性的影响，课题以GFP标记的嗜水气单胞菌不同毒力菌株和四膜虫为研究对象，探讨不同菌株对四膜虫的抗吞噬消化能力以及菌株毒力进化的分子基础。结果表明，强毒株具有明显的抗四膜虫捕食消化能力；强毒株NJ-35 与四膜虫共培养并连续传代后，其生物被膜形成能力、对HEp-2细胞的黏附能力以及对大肠杆菌的竞争能力均显著增强，但运动能力及对噬菌体的敏感性显著降低，说明NJ-35在四膜虫的选择压力下，其抗捕食的防御能力及其在环境中的适应能力相应增强。外膜蛋白蛋白质组学分析结果显示，在与四膜虫共培养后，NJ-35中共检测到36个蛋白表达出现差异，对3个上调表达蛋白SurA、Slp、LpoB的基因进行缺失，特性分析证实这3个基因与细菌黏附、生物被膜形成能力及抗氧化应激能力有关。利用选择性捕获转录序列(SCOTS)和dot-blot技术研究嗜水气单胞菌在四膜虫细胞内存活相关基因的转录情况，结果筛选获得119个差异转录基因，其中3个基因msrA、msrB和norV被证实与嗜水气单胞菌的环境适应及毒力相关。另外，重点研究了虫菌互作后表达明显上调的T6SS 主要效应蛋白Hcp，对3个hcp基因功能分析证实Hcp蛋白在嗜水气单胞菌抵抗四膜虫吞噬上发挥重要作用。总之，本项目调查了四膜虫对嗜水气单胞菌毒力和环境适应性的影响，并解析了调控的分子基础，丰富了对嗜水气单胞菌毒力进化机制的认识。项目执行期间，发表SCI论文7篇，中文核心期刊论文2篇；获江苏省海洋与渔业科技创新一等奖1项；邀请美国专家合作交流1次；与美国奥本大学Mark教授联合培养博士生计划获国家留学基金委批准。