Intimin及其受体Tir在致病性大肠杆菌致细胞线粒体功能障碍中的作用

目的探讨致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)外膜蛋白Intimin及其受体Tir在EPEC致HeLa细胞线粒体功能障碍中的作用。方法将EPEC外膜蛋白Intimin及其受体Tir删除株、相应质粒互补株或染色体互补株感染HeLa细胞,用线粒体膜电位(Mitochondria membrane potential,MMP)检测试剂JC-1染色细胞线粒体,通过多功能酶标仪检测MMP水平,Western blot检测Intimin的表达及Tir的转位。结果与野生型菌株相比,Eae删除株和Tir删除株感染细胞的MMP功能显著减弱(P<0.05),Eae删除株功能能被质粒表达相应蛋白所互补,Tir删除株不能被质粒表达Tir互补,但可被染色质表达野生型Tir或TirY474S互补,而染色质TirS434A突变株不能引起明显的MMP下降。结论 Intimin和Tir是参与线粒体功能障碍的重要分子;TirS434在线粒体功能障碍中起重要作用。

Roles of Intimin and Its Receptor Tir in Mitochondrial Dysfunction of Cells Infected with Enteropathogenic E. coli

Objective To investigate the role of outer membrane protein Intimin of enteropathogenic E.coli(EPEC),as well as its receptor Tir,in the mitochondrial dysfunction of HeLa cells infected with EPEC.Methods HeLa cells were infected with EPEC mutants with Intimin deleted and with Tir deleted,as well as the mutants complemented with the corresponding plasmid or with the corresponding chromosome,in which the mitochondria were stained with JC-1,a reagent for detection of mitochondria membrane potential(MMP).MMP was determined by multifunctional ELISA reader,and the expression of Intimin and transposition of Tir by Western blot.Results The MMPs of cells infected with the mutants with Eae deleted and with Tir deleted decreased significantly as compared with those of wild EPEC strain(P < 0.05).The function of mutant with Eae deleted was complemented by the plasmid expressing the corresponding protein.However,the function of mutant with Tir deleted was not complemented by the plasmid expressing Tir,while was complemented by the wild Tir or TirY474S inserted into the chromosome.TirS434A mutant caused no significant decrease of MMP.Conclusions Intimin and Tir are important molecules involving in mitochondrial dysfunction.Meanwhile,TirS434 plays an important role in mitochondrial dysfunction.