Roles of effectors in mitochondrial dysfunction of cells infected by enteropathogenic Escherichia Coli
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 (△map, △espF, △eae and △tir) (P ＜ 0.05), while the ability increased significantly in the espZ missing strain(△espZ) (P ＜ 0.05). Compared with that of △map or △espF, the ability of the double gene missing strain (△map 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 △tir, 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 △tir. 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.
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万方数据
维普
