The Study Of Effectors Involved In Mitochondrial Dysfunction And Attaching And Effacing Lesion In Cells Infected By Enteropathogenic Escherichia Coli

OBJECTIVE:1,To investigate the tanslocation and location of EspH protein of enteropathogenic Escheichia Coli(EPEC) and the roles of EspH in attaching and effacing lesion (A/E lesion) of host cells infected with EPEC strains.2,To investigate the roles of EPEC effectors in mitochondrial dysfunction in Hela cells infected with EPEC strains.3,To investigate the effectors of EPEC involved in A/E lesion after infection TC-7 cells with EPEC strains.METHODS:1. PCR was employed to amplify the close neighbor upstream DNA fragment (about 500 bp) of espH gene, the kanamycin open reading frame (ORF) and the close neighbor downstream DNA fragment (about 500 bp) of espH, and the three PCR products were cloned into T vector pSC-A in tandem, subsequently the long DNA fragment (about 2200 bp) was subcloned into suicide vector pCVD442 to construct pCVD442-ΔespH::kana. TheΔespH mutant was made after conjugation, allelic exchange and selection with kanamycin and nalidixic acid and identified with PCR and sequencing. The plasmid pTrc99a-EspH:HA expressing EspH labeled with HA tag was constructed and transformed intoΔespH mutant to getΔespH /pTrc99a-EspH:HA strain, the translocation and distribution of EspH:HA was investigated with Western Blot and immunofluorescence microscopy after infection Hela cells withΔespH /pTrc99a-EspH:HA strain. The vector pEspH-EGFP which expressed the fusion protein of EspH and Enhanced Green Fluorescence Protein (EGFP) was constructed and the distribution of fusion protein was detected with fluorescence microscopy after transfection Hela cells with pEspH-EGFP. The roles of EspH in A/E lesion were investigated with fluorescence microscopy, scanning electron microscopy (SEM) and transepithelial electrical resistance (TER) measurement after infection Hela cells, caco-2 cells and TC-7 cells withΔespH mutant respectively.2. TheΔespF,Δmap andΔmap espF mutants were got after conjugation, allelic exchange and resistance selection and identified with PCR and Western Blot. The mitochondria of Hela cells were stained with JC-1 and mitochondrial membrane potential (MMP) was detected with the fluorescence reader after Hela cells were infected with EPEC effectors defective mutants, mutants complemented with relative effectors expressed by plasmids or chromosome, the translocation and location of effectors were detected with Western Blot and immunofluorescence microscopy. 3. SEM was employed to detect which effectors contributed to the A/E lesion after infection TC-7 cells with EPEC wide type, T3SS defective strainΔcfm-14, BFP defective mutantΔbfpA, other effectors defective mutants and defective mutants complemented with relative effectors expressed by plasmids.RESULTS:1. The suicide vector pCVD442-ΔespH::kana was constructed successfully after identification with PCR, digestion and sequencing. The resulting mutantΔespH was analysed with PCR and sequencing and indicated that espH gene was replaced with kanamycin ORF andΔespH was resistant to kanamycin. After infection Hela cells withΔespH /pTrc99a -EspH:HA strain, EspH was found to be translocated to host cells dependant on T3SS and distribute in Tritonx-100 soluble fraction by Western Blot and on the membrane, the position of pedeatal formation by immuno- fluorescence microscopy. The fusion protein of EspH and EGFP was detected on the membrane of Hela cells and the cells transfected with the plasmid turned round. Infection assays indicated that EspH mutant formed pedestal in Hela cells, decreased tight junction of Caco-2 cells and effaced the microvilli of TC-7 cells resembled with EPEC wild type.2. The resulting mutants were analyzed with PCR and the results showed that map and espF gene were defective inΔmap,ΔespF andΔmap espF accordingly, furthermoreΔespF andΔmap espF didn't secrete EspF protein. Compared with EPEC wide type,Δmap orΔespF mutant lose part of the function to induce mitochondrial dysfunction in Hela cells (P< 0.05) andΔmap espF double mutant lose more (P <0 .05), but the function ofΔmap espF is stronger than the T3SS defective mutant (Δcfm-14) (P< 0.05), and they were complemented with the relative protein expressed by plasmids, furthermore EspFL16E complemented the function ofΔespF though it located in the cytoplasm. Compared with EPEC wide type, Outer membrane protein intimin defective mutant (Δeae) and its receptor Tir defective mutant (Δtir) lose the function to induce mitochondrial dysfunction to the level ofΔcfm-14 (P < 0.05), andΔeae was complemented with intimin expressed by plasmid, butΔtir wasn't complemented with Tir expressed by plasmid, yet it was complemented with the wide type Tir and mutation TirY474S inserted into chromosome, and not by mutation TirS434A in the chromosome.ΔespG andΔespH mutants induced mitochondrial dysfunction comparable to EPEC wide type andΔespZ mutant adjusted the phosphorylation of Tir and induced mitochondrial dysfunction more severe than wide type (P < 0.05).3. Many bacteria first localized adhesion and subsequently adhered intimately to TC-7 cells and microvilli of TC-7 cells were effaced severely when infection with EPEC wide type, yet only a few bacteria attached to TC-7 cells when infection withΔbfpA mutant. When infection withΔcfm-14, many bacteria localized adhesion to host cells, but, there were no effacement just the disturbance of microvilli. When infection withΔeae andΔtir mutants, many bacteria localized adhesion to host cells, but the effacement of microvilli was more severe than wide type, but when complementedΔeae andΔtir mutants with the relative protein expressed by the plasmids, the function of mutants were rescued to the level of EPEC wide type.ΔespF andΔespF map double mutant adhered to host cells comparable to wide type and some bacteria sink into microvilli, but microvilli effacement was slight and the EspF protein expressed by plasmids complemented the function ofΔespF to the similar phenotype of wide type.CONCLUSION:1. EspH is a translocated effector of EPEC dependant on T3SS and EspH protein distributes on the membrane after translocation, but EspH doesn't play an important role in the pedestal formation, tight junction disruption and microvilli effacement.2. Map and EspF induce mitochondrial dysfunction individually and synergistically and EspF functions independant on its mitochondria location. Intimin and Tir are the important molecules in mitochondrial dysfunction induced by EPEC and TirS434 plays an important role in the function of Tir. EspG and EspH don't play a role in mitochondrial dysfunction but EspZ adjusts the phosphorylation of Tir and is implicated in the function.3. BFP is a key adhesin of EPEC, T3SS is an important apparatus for the intimate adherence and mocrovilli effacement of EPEC, intimin and Tir are crucial molecules for intimate adherence and involved in microvilli effacement and EspF is a crucial molecule of EPEC to efface the microvilli of host cells.