ExsA上游调节因子的乙酰化修饰对溶藻弧菌T3SS致病性的影响机制

ExsA是病原菌III型分泌系统（T3SS）的直接诱导因子，可受到调节因子的调控，进而影响T3SS的致病性。目前，调节因子通过乙酰化介导T3SS的致病机制在人类病原菌中已有研究，但在鱼类病原菌中尚不明确。本项目拟比较溶藻弧菌T3SS诱导（HY9901:pexsA）和非诱导（HY9901:pexsD）菌株的蛋白质乙酰化修饰组学图谱，系统鉴定ExsA的上游调节因子，并验证其乙酰化修饰位点；构建各调节因子缺失株、互补株、乙酰化修饰位点突变株，并评价其毒力、细胞毒性、胞外蛋白酶活性；通过qRT-PCR、引物延伸、LacZ报告基因融合试验确定调节因子修饰位点对ExsA的调控关系；免疫印迹、EMSA及Dnase I足迹试验鉴定调节因子修饰位点与ExsA的互作关系。研究结果有望揭示ExsA调节因子的乙酰化修饰对T3SS致病性的影响机制，为更好地预防和控制弧菌病提供靶点。

ExsA is a direct inducer of type III secretion system (T3SS), which can be regulated by regulators, and has influence on pathogenicity of T3SS. Although there are reports about acetylation of regulator mediating pathogenicity of human pathogenic bacteria, it is poorly known whether such mechanism is in fish pathogens. In this study, we intend to further study the effect of acetylation of ExsA upstream regulators on T3SS pathogenicity of V. alginolyticus. The proposal is as following: 1) comparing the proteomic patterns of T3SS induced (HY9901:pexsA) and non-induced (HY9901:pexsD) strains of V. alginolyticus, screening the regulators of ExsA and verifying the acetylation modification of upstream regulators; 2) constructing the regulator deletion strains and acetylation modification site mutant strains, then measuring their toxicity, cytotoxicity and extracellular protease activity; 3) verifying the specific regulatory relationships between regulator and ExsA with qRT-PCR , the primer extension and LacZ reporter gene fusion methods; 4) identifying the interaction between regulator and ExsA with Western blotting, EMSA and Dnase I footprint. The results will reveal the mechanism of the acetylation modification of ExsA regulator on the pathogenicity of T3SS and provide targets for better prevention and control of Vibriosis.