溶藻弧菌T3SS效应蛋白的鉴定及其致鱼类细胞死亡机制的研究

The gram-negative bacterium Vibrio alginolyticus is one of marine Vibrio species that have been identified as pathogenic for marine animals. The type III secretion system (T3SS) is the main mechanisms to secret proteins, called effectors (T3SEs) which can trigger host-cell death. Although the gene structure and function of T3SS in Vibrio alginolyticus had been studied, the T3SEs and its mechanism were not clear. In our previous study, we had constructed △vscO and △vscC mutant strains of V.alginolyticus, and compared the proteins secreted from mutant strains and the wild strain, receiving 309 different expressive proteins. In this study, we will search T3SEs from different expressive proteins by bioinformatics and molecular biology techniques. Then we construct eukaryotic expression vector of T3SEs.The above recombinant plasmid was transferred into fish cells by liposome and then characterized the T3SEs induced fish cell death by some cell toxicity method,such as the activity of TUNEL、Caspase-3 and so on. Constructing the knockout mutant strain of T3SEs and the complementary strain at the same time. The wild strain, mutant strain and complementary strain were transferred into fish cells. Immuoprecipitation and mass spectrometry was used to identify the host proteins interaction with T3SEs. A rough drawing of fish cell-T3SEs protein interaction network will make. The study will provide an insight into the molecular machanism of T3SS causing fish cell death in Vibrio species, and help to find new targets for the control of Vibrio alginolyticus.

溶藻弧菌(Vibrio alginolyticus)是海水养殖动物弧菌病的重要致病菌之一。Ⅲ型分泌系统(T3SS)是弧菌的毒力装置，通过分泌效应蛋白(T3SEs)诱导宿主细胞死亡。虽然溶藻弧菌T3SS的装置蛋白得到了初步研究，但其效应蛋白及作用机制还未得到阐明。本项目在申请者已获得溶藻弧菌T3SS装置蛋白缺失株△vscO和△vscC与野生株的差异蛋白库的基础上，拟通过生物信息学、免疫印迹技术筛查潜在T3SEs；通过构建T3SEs真核重组表达载体并转染鱼类细胞，运用TUNEL、Caspase-3等细胞死亡的检测方法查明T3SEs的细胞毒性功能；将野生株、T3SEs基因缺失株和互补株转染鱼类细胞后，运用免疫共沉淀联合质谱测序方法初步研究T3SEs与鱼类细胞的作用机制，并绘制网络互作图谱。本研究结果将有助于揭示溶藻弧菌T3SS致鱼类细胞死亡的分子机理，也为寻找控制弧菌病的新分子靶标提供理论依据。

III型分泌系统(T3SS)是溶藻弧菌（Vibrio alginolyticus）保守存在的毒力装置，通过分泌效应蛋白（T3SEs）诱导宿主细胞死亡。本研究以溶藻弧菌T3SEs为研究对象，通过为期三年的研究，项目组完成了以下内容：①采用生物信息学筛选溶藻弧菌潜在 T3SEs，共筛选得到5个潜在T3SEs，分别为 Type III effector HopPmaJ、Va1686、TyeA、type3hy322、type III secretion protein。②对效应蛋白HopPmaJ、Va1686、type3hy322分别进行了克隆和生物信息学分析，构建了HopPmaJ、Va1686、type3hy322的原核表达载体和真核表达载体，对原核表达载体进行重组蛋白表达，进而制备HopPmaJ、Va1686、 type3hy322的特异性抗体，q-PCR验证了HopPmaJ、Va1686 、type3hy322均与T3SS相关。③分别构建溶藻弧菌 HopPmaJ、Va1686基因缺失突变株和互补株，分别转染鱼类细胞，通过激光扫描共聚焦显微镜观察到Va1686能导致细胞骨架网状结构降解，Caspase-3 活性检测、LDH 活力检验证明Va1686是引起鱼类细胞凋亡的关键蛋白。④运用免疫共沉淀联合质谱鉴定的方法初步发现Va1686与鱼类细胞作用的蛋白为Cdc42和Rac1蛋白，这两个蛋白均为Rho家族成员，功能是调节细胞骨架重组以及基因转录，该结果与显微镜观察到的Va1686能导致细胞骨架网状结构降解相吻合。通过整合以上研究成果，与弧菌致病机制的已有认识进行对比分析，揭示了溶藻弧菌T3SS致鱼类细胞死亡的分子机理，为溶藻弧菌的分子致病机理提供了新的理论和技术支撑。