【作者】 杨健；

【导师】 黄爱龙； Kenny Brendan；

【作者基本信息】 重庆医科大学 ， 内科学， 2009， 博士

【摘要】 目的:1、探讨致病性大肠杆菌(EPEC)感染宿主上皮细胞后,效应分子EspH的转位、分布以及在EPEC致宿主细胞粘附、脱落损伤(A/E)中的作用。2、探讨EPEC感染Hela细胞后,效应分子在EPEC致Hela细胞线粒体功能障碍中的作用。3、探讨EPEC感染TC-7细胞,参与细菌粘附及引起肠上皮细胞微绒毛脱落损伤的效应分子。方法:1、分别用PCR方法扩增espH基因紧邻上游DNA片段(约500 bp)、卡那霉素开放读码框(约1200 bp)和espH基因下游DNA片段(约500 bp),按顺序把三个DNA片段串联克隆到PCR克隆载体pSC-A中,再亚克隆该大片段(约2200bp)到自杀质粒pCVD442中得到自杀质粒pCVD442-ΔespH::kana,通过结合传递、等位交换,抗性筛选制备EPECΔespH删除株,并对删除株进行PCR鉴定和相应染色体DNA区段做测序鉴定;构建带HA标签的EspH表达载体pTrc99a-EspH:HA,电转化载体到ΔespH删除株制备ΔespH/pTrc99a-EspH:HA,并用其感染Hela细胞,Western Blot和免疫荧光技术检测EspH的转位以及转位后EspH的分布;构建EspH和增强型绿色荧光蛋白(EGFP)融合表达质粒pEspH-EGFP,转染Hela细胞,荧光显微镜观察融合蛋白的分布;并用ΔespH删除株感染Hela、Caco-2和TC-7细胞,分别用荧光显微镜技术,跨上皮细胞电阻(TER)检测和扫描电子显微镜(SEM)技术探讨EspH在基垫形成,屏障功能障碍和微绒毛脱落损伤中的作用。2、通过结合传递、等位交换,抗性筛选制备ΔespF、Δmap、Δmap espF等删除株,并用PCR和Western Blot对删除株进行鉴定;用制备的删除株和其他效应分子删除株、质粒表达相应效应分子互补株或染色体表达效应分子互补株感染经用线粒体膜电位试剂盒JC-1染色的Hela细胞,并用多功能酶标仪检测感染前后线粒体膜电位(MMP)的改变,Western Blot和免疫荧光技术检测效应分子的转位及分布,从而探索效应分子在EPEC致线粒体功能障碍中的作用及可能机制。3、分别用EPEC野生型、III型分泌系统(T3SS)删除株Δcfm-14、束状菌毛(BFP)删除株ΔbfpA以及其他效应分子删除株感染经极化培养的TC-7细胞,通过扫描电镜检测参与A/E损伤的效应分子,并用质粒表达相应蛋白互补删除株,证实效应分子在A/E损伤中的作用。结果:1、酶切、测序及PCR鉴定表明自杀质粒pCVD442-ΔespH::kana成功构建;PCR和测序鉴定表明:ΔespH删除株构建成功,卡那霉素基因取代espH基因,且ΔespH删除株获得卡那霉素抗性;感染Hela细胞以后,对细胞组分分离,Western Blot检测发现:EspH依赖于T3SS转位到宿主细胞,并在细胞Tritonx-100可溶性组分中检测到表达,免疫荧光检测发现EspH分布于细胞膜,基垫形成处;转染实验发现EspH与EGFP融合蛋白分布于细胞膜,且转染的细胞变成球形;感染实验发现:ΔespH删除株能有效诱导基垫形成,其降低Caco-2细胞屏障功能的能力和造成TC-7细胞微绒毛脱落损伤能力与EPEC野生型相似。2、PCR和Western Blot证实:Δmap、ΔespF和Δmap espF等删除株构建成功,MMP检测发现:与EPEC野生型相比,Δmap和ΔespF删除株降低线粒体膜电位功能减弱(P<0.05),Δmap espF双删除株功能较Δmap或ΔespF进一步减弱(P<0.05),但Δmap espF功能较T3SS删除株Δcfm-14强大(P<0.05),而且,删除株缺失功能能被质粒表达相应蛋白互补,并且,定位于细胞质的突变体EspFL16E也能互补删除株的功能。对Δeae和Δtir删除株的检测发现:其降低MMP功能较野生型显著减弱(P<0.05),Δeae删除株可以被质粒表达intimin互补,但是Δtir不能被质粒表达Tir所互补,但可以被染色体表达野生型Tir和突变TirY474S所互补,而不能被染色体表达突变TirS434A互补;而ΔespG和ΔespH降低MMP能力与野生型比较无显著性差异,但ΔespZ删除株具有比野生型更强大的功能(P<0.05),Western Blot检测发现:ΔespZ删除株转位的Tir蛋白具有不同的磷酸化修饰迁移条带。3、EPEC野生型感染TC-7细胞,最先细菌呈局限性粘附,菌落呈三维球状,随后可见有大量细菌紧密粘附在细胞表面,菌落致密平坦,并可见大量微绒毛脱落;BFP删除株ΔbfpA感染后只有个别细菌粘附,细胞微绒毛完好;而T3SS删除株Δcfm-14感染细胞以后,虽有大量细菌粘附,但细菌成局限性粘附,且只见微绒毛紊乱,未见微绒毛脱落损伤;外膜蛋白intimin删除株(Δeae)和其受体Tir删除株(Δtir)感染后,可见细菌皆呈局限性粘附,微绒毛大量脱落,但质粒互补表达相应效应分子以后,恢复删除株到野生型相似表型;而用ΔespF或Δmap espF双删除株感染后,有大量细菌粘附,细菌下陷入微绒毛丛,但是微绒毛脱落损伤轻微,质粒表达EspF互补ΔespF删除株后,恢复删除株到野生型相似表型。结论:1、EspH是EPEC毒力岛基因LEE编码的一个依耐T3SS转位的效应分子,转位以后,蛋白分布于细胞膜,基垫形成处,且对转染细胞有一定毒性,但未发现EspH在EPEC基垫形成、屏障功能障碍和微绒毛脱落方面具有重要作用。2、EPEC感染Hela细胞,效应分子Map和EspF可以单独并协同地引起线粒体功能障碍,且EspF作用不依耐其靶向线粒体;intimin、Tir是参与线粒体功能障碍的重要分子,TirS434在Tir功能发挥中起重要作用,EspZ调节Tir的磷酸化修饰,也参与线粒体功能障碍。3、BFP是EPEC感染细菌粘附的重要分子,T3SS是EPEC感染细菌紧密粘附和造成微绒毛脱落损伤的重要结构,intimin和Tir是细菌感染紧密粘附的重要分子,也参与了微绒毛脱落损伤,而EspF是造成微绒毛脱落损伤的主要分子。更多还原

【Abstract】 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.更多还原