伤寒沙门菌Ⅵ型分泌系统对SopE表达影响的初步研究

Preliminary study on the effect of Salmonella typhi typeⅥsecretion system on the expression of SopE

为探索伤寒沙门菌Ⅵ型分泌系统(type 6 secretion system,T6SS)对SopE表达的影响,阐明其中的分子机制,本研究利用自杀质粒同源重组法构建携带sopE∷3×Flag的伤寒沙门菌野生株(WT-pBAD33)、T6SS功能分子缺陷株(ΔsciG-pBAD33)和回补株(C-ΔsciG),用构建成功的菌株感染巨噬细胞THP-1并在模拟巨噬细胞内环境(LPM培养基、微需氧)下培养,通过实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)与蛋白质印迹法(Western blot,WB)试验探索T6SS对SopE表达的影响,采用β-半乳糖苷酶活性检测来进一步验证sopE基因启动子的转录水平,Ni柱纯化T6SS功能蛋白SciG,电泳迁移率变动分析(electrophoretic mobility shift assay,EMSA)研究SciG蛋白对sopE基因启动子的调控作用。结果显示:ΔsciG-pBAD33中SopE表达水平较于WT-pBAD33明显降低;SciG蛋白不能直接与sopE基因启动子区域结合。本研究结果表明,伤寒沙门菌T6SS影响了SopE的表达,但是其功能蛋白SciG不能直接调控sopE基因的表达。

In order to explore the effect of Salmonella typhi type 6 secretion system(T6SS)on SopE expression and clarify the molecular mechanism,the wild Salmonella typhi strain(WT-pBAD33),T6SS functional molecule-deficient strain(ΔsciG-pBAD33)and complementing strain(C-ΔsciG)carrying sopE∷3×Flag were constructed by suicide plasmid homologous recombination method.The strains were constructed to infect macrophage THP-1 and cultured in simulated macrophage environment(LPM medium,microaerobic).The effect of T6SS on the expression of SopE was investigated by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot experiments.Theβ-galactosidase activity test further verified the transcription level of sopE gene promoter,and the T6SS functional protein SciG was purified by Ni column.The regulation effect of SciG protein on the sopE gene promoter was analyzed by electrophoretic mobility shift assay(EMSA).The results demonstrated that the expression level of SopE inΔsciG-pBAD33 was significantly lower than that in WT-pBAD33;SciG protein could not directly bind to the sopE gene promoter region.It was concluded that Salmonella typhi T6SS affects the expression of SopE,but its functional protein SciG can not directly regulate the expression of sopE gene.