副溶血弧菌Ⅲ型分泌系统1内杆状蛋白基因vscI的功能分析

Functional analysis of the inner rod protein gene vscI in Vibrio parahaemolyticus TypeⅢsecretion system 1

[目的]Ⅲ型分泌系统(TypeⅢsecretion systems,T3SS)在副溶血弧菌(Vibrio parahemolyticus)致病过程中发挥重要作用。本试验旨在探讨T3SS1内杆状蛋白基因vscI的功能。[方法]以副溶血弧菌POR-1菌株为研究对象,构建vscI缺失株ΔvscI与回补株CΔvscI,检测野生株、缺失株与回补株的细胞毒性、细胞黏附等生物学特性,分析vscI缺失对副溶血弧菌感染细胞能力的影响;通过Western blot与ELISA检测HeLa细胞内效应蛋白的易位量。[结果]与POR-1菌株相比,缺失菌株ΔvscI在生长速度上并无显著差异,但对HeLa细胞的黏附能力下降85%。细胞毒性检测结果显示:与POR-1菌株相比,缺失株ΔvscI感染细胞组释放LDH量显著下降60%。同时与野生株POR-1感染HeLa细胞组相比,ΔvscI感染HeLa细胞组发生凋亡的细胞数量显著下降了85%。腺苷酸环化酶报告系统研究结果显示:与POR-1菌株感染组相比,ΔvscI菌株感染HeLa细胞内的cAMP含量显著下降,CyaA融合蛋白的易位量显著下降,而在回补株CΔvscI中上述生物性状均恢复。[结论]vscI提高了副溶血弧菌对细胞的黏附能力和毒力,促进细胞凋亡,同时对于T3SS1效应子的易位是必需的。

[Objectives]The structural components and the biological functions of Vibrio parahemolyticus T3SS1 have not been fully described.In this study we investigated the role of gene vscI.[Methods]To do some research about the vscI,we constructed the deletion strainΔvscI and complementation strain CΔvscI to analyze the cytotoxicity,adhesion and other biological characteristics of wild strain(POR-1),ΔvscI and CΔvscI when infected HeLa.Further,Western blot analysis and ELISA kits were used to research the translocation of effector protein in Hela cell.[Results]Compared with POR-1 infection group,the results showed that the number of apoptosis decreased significantly by about 80%(P<0.01),the adhesion decreased significantly by 85%(P<0.01),and the cytotoxicity decreased significantly by 60%(P<0.01)inΔvscI infection group.In addition,Western blot and ELISA data indicated that the intracellular cAMP levels of theΔvscI group was significantly lower than those of the POR-1,CΔvscI.[Conclusions]vscI improves the adhesion ability of V.parahaemolyticus to cells,and produces cytotoxicity,promotes cell apoptosis,and it is necessary for the translocation of T3SS1 effector.