【摘要】 目的:探讨致病性大肠杆菌(EPEC)效应分子在Hela细胞线粒体功能障碍中的作用.方法:用EPEC效应分子删除株、质粒互补株或染色质互补株感染Hela细胞,用线粒体膜电位(MMP)检测试剂盒(JC-1)染色细胞线粒体,通过多功能酶标仪检测MMP,蛋白印迹法检测效应分子的转位,免疫荧光法检测效应分子的定位,以此判断效应分子在EPEC致MMP下降中的作用.结果:与野生型组相比较,Δmap,ΔespF,Δeae,Δtir感染组单删除株降低细胞MMP功能显著减弱(P<0.05),但ΔespZ感染组删除株的功能却增强(P<0.05).与Δmap,ΔespF感染组单删除株相比较,ΔmapespF感染组双删除株功能进一步减弱(P<0.05).Δmap,ΔespF,Δeae感染组删除株的功能可被质粒表达相应蛋白所互补,EspFL16E定位于细胞质,也能互补ΔespF的功能.Δtir不能被质粒表达转位受体(Tir)互补,可以被染色质表达野生型Tir或突变TirY474S互补,但不能被突变TirS434A互补.结论:除Map,EspF外,EspZ,外膜蛋白intimin和其受体Tir也是参与细胞MMP下降的重要效应分子,TirS434在Tir引起MMP下降中起重要作用.更多还原

【Abstract】 AIM:To explore the roles of enteropathogenic Escherichia Coli(EPEC) effectors in mitochondrial dysfunction in Hela cells infected by EPEC.METHODS:Hela cells were infected with EPEC strains missing one or two gene encoding effectors and strains complement with relative missing effectors expressed by plasmids or chromosome.The mitochondrial membrane potential(MMP) was detected with multifunctional microplate reader after staining the mitochondria with MMP Detection Kit JC-1,and the translocation and distribution of effectors were detected with Western blot and immunofluorescence assays respectively.RESULTS:Compared with that of wild type EPEC,the ability to decrease MMP reduced significantly in the individual gene missing strains(Δmap,ΔespF,Δeae and Δtir)(P<0.05),while the ability increased significantly in the espZ missing strain(ΔespZ)(P<0.05).Compared with that of Δmap or ΔespF,the ability of the double gene missing strain(Δmap espF) to decrease MMP reduced significantly(P<0.05).But plasmids expressing Map,EspF and intimin restored the defect associated with strain missing these effectors.EspF carrying the L16E substitution that abolished targeting to mitochondria retained its ability to function like the native EspF protein.Plasmids expressing Tir did not restore the function of Δtir,but it was restored by native Tir and substitution of the 474th tyrosine of Tir with serine(TirY474S) by re-introducing genes into chromosome.Substitution of the 434th serine of Tir with alanine(TirS434A) abrogated the ability of Tir to restore the function of Δtir.CONCLUSION:In addition to Map and EspF,outer membrane protein intimin and its receptor Tir are important molecules involved in decreasing MMP of Hela cells and 434th serine residue of Tir plays an important role in the decrease of MMP.更多还原