弗氏枸橼酸杆菌CF74的FliS蛋白功能研究

目的 研究弗氏枸橼酸杆菌CF74的FliS蛋白功能。方法 构建弗氏枸橼酸杆菌CF74 fliS的精确缺失突变株(CF74ΔfliS)及其互补菌株(CF74pfliS),并进行细菌游动性实验,检测它们对THP1细胞的粘附性和细胞毒性的作用。并用Western blotting方法检测FliC蛋白在培养上清中的分泌。结果 弗氏枸橼酸杆菌CF74ΔfliS突变株没有游动性,而回补株CF74pfliS回补了游动性。而且,CF74ΔfliS突变株降低对THP1细胞的粘附和细胞毒作用,其回补株恢复了粘附性和细胞毒性。此外,CF74ΔfliS突变株降低FliC蛋白在培养上清中的分泌。结论 FliS蛋白影响弗氏枸橼酸杆菌CF74的游动性,并参与其对宿主细胞的粘附和细胞毒作用。

FliS function in C.freundii strain CF74

FliS as an export chaperone contributes to stabilization of flagellin subunit interactions during polymerisation. The fliS mutant leads to most of the FliC protein being accumulated in inclusion bodies formed inside the cell cytoplasm. C. freundii strain CF74 has a complete flagellar system. To analyze FliS function in CF74, we constructed deletion mutant of fliS and complementation in CF74 and analysed its effects on the swimming motility, adherence and cytotoxicity. CF74ΔfliS mutant was found to be no motile, and motility was restored by CF74pfliS. Moreover, CF74ΔfliS mutant was defective in adhesion and cytotoxicity to THP1 cells and restored upon complementation. Significant differences between wild type and CF74△fliS mutant were shown with P<0.01. The band of FliC in CF74ΔfliS mutant was weak by immunoblot analysis of FliC protein in the culture supernatant prepared from CF74 and CF74ΔfliS mutant grown in LB. These results suggested that the FliS in CF74 enhances motility, involves in the adherence to host cells, and induces cytotoxicity to host cells.