肠出血性大肠杆菌（EHEC O157：H7）VT2毒素致病机理研究

发布时间：2018-01-11 22:33

本文关键词：肠出血性大肠杆菌（EHEC O157：H7）VT2毒素致病机理研究　出处：《西北农林科技大学》2010年博士论文　论文类型：学位论文

【摘要】：肠出血性大肠杆菌(EHEC)是产VT毒素大肠杆菌(VTEC)的一个亚群。O157:H7血清型是EHEC中典型的致病菌株,是大规模O157感染爆发的主要病原菌。O157:H7是一种食源性致病菌,牛是其首要的天然寄主,主要是食物被牛粪污染,导致感染爆发。EHEC感染会引起轻度腹泻到出血性肠炎、严重者会引起出血性尿毒综合症,甚至死亡。EHEC最关键的两个致病因子是由噬菌体编码的VT毒素和介导粘附作用的LEE毒力岛(肠道上皮细胞损伤位点)。LEE毒力岛编码的蛋白负责形成粘附抹平损伤,其典型特征是通过病原菌和寄主肠道表面的紧密粘附,在寄主细胞表面细菌粘附位点肌动蛋白富集,形成杯状基座和细胞表面边缘刷状微绒毛损伤。LEE毒力岛由LEE1到LEE5主要5个操纵子组成,编码的Ⅲ型分泌系统涉及到移位因子的分泌、效应蛋白和外膜表面紧密粘附素Intinmin与其受体蛋白Tir(由细菌编码转运到宿主细胞上的粘附素受体)。粘附素Intinmin除了和其主要受体蛋白Tir结合之外,还会和寄主细胞表面的粘附素受体(HIR)结合,如Intinmin与肠道细胞表面的β1整合素(β1-integrin)和核仁素(nucleolin)结合。有研究认为在形成稳定的intimin-Tir结构之前,粘附素intimin首先和寄主细胞的粘附素受体HIRs结合,使得细菌和寄主细胞足够接近从而完成Tir和其他效应因子从细菌到寄主上皮细胞表面的注射转运过程。 EHEC O157:H7产生的VT毒素是其重要的致病因素之一,本研究关注肠出血性大肠杆菌EHEC86-24感染猪空肠细胞系(IPEC-J2)、人结肠细胞系(CaCo-2)和人喉癌细胞系(HEp-2)过程中,产生的VT毒素对其粘附定植过程的影响,以及在细菌感染IPEC-J2细胞过程中,VT毒素对其相关致病因子的基因表达水平影响,包括对致病粘附因子β1整合素(β1-integrin)和核仁素( nucleolin)的基因表达影响,对VT毒素受体Gb3合成表达的影响,从而深入分析VT毒素在EHEC致病过程中的作用机理。试验利用λ-Red同源重组技术构建了vt2基因敲除突变菌株86-24-vt2S,同时产生了编码VT2毒素的噬菌体缺失突变菌株86-24△933W、以及突变菌株的vt2基因补充菌株86-24-vt2S(pVT2)和86-24-933W(pVT2)。将野生型菌株EHEC 86-24和不同vt2基因突变菌株与真核细胞系进行粘附试验,并对感染过程真核细胞IPEC-J2的相关粘附因子基因进行实时定量PCR检测。试验结果显示:与其亲本野生型EHEC菌株和vt2基因补充菌株比较,vt2基因敲除突变株对3株真核细胞系的粘附能力均显著降低;野生型EHEC菌株在感染IPEC-J2细胞系过程中,伴随宿主细胞相关粘附因子β1-integrin、nucleolin和Gb3合成基因的表达增加;IPEC-J2细胞系与野生型EHEC O157:H7或vt2基因补充突变株相互作用时,β1整合素的表达水平高于IPEC-J2与vt2基因敲除突变菌株或无细菌粘附时的表达水平;IPEC-J2与vt2基因敲除突变菌株粘附感染时,核仁素的表达水平降低,但是其基因补充菌株没有修复核仁素的表达到野生型水平。试验数据揭示VT2毒素在EHEC O157:H7致病菌对肠道上皮细胞的粘附过程中有促进作用,这种促进作用从基因水平上是通过提高宿主细胞表面受体因子β1-integrin和nucleolin的表达水平,从而提高了细菌的粘附功能。核仁素(Nucleolin)是一种可以表达在多种类型真核细胞表面的多功能核蛋白,作为一些病毒和粘附素的受体。核仁素是与广泛的细胞和细胞之间相互作用相关的一个受体大家族。肠致病性大肠杆菌杆(EPEC)的粘附素与β1整合素特异结合。在猪和牛的组织感染试验中,免疫染色发现β1整合素聚集在细菌粘附位点,说明整合素在EHEC字O157:H7感染过程中是一个潜在的受体蛋白。VT毒素是一种典型的AB_5结构蛋白,能抑制真核细胞蛋白合成。VT毒素在EHEC感染过程中导致出血性肠炎(HC)、溶血性尿毒综合症(HUS)等严重的临床症状。VT毒素的B亚基和目标细胞表面的糖脂类受体(Gb3)结合,通过受体介导毒素内化。最近研究报道VT2毒素通过刺激真核细胞表达核仁素,从而促进EHEC在肠道上的定植过程。然而,关于VT毒素促进细菌粘附定植的理论依然具有争议。本实验室前期研究工作中构建了萘啶酮酸抗性EHEC菌株的vt2基因插入突变菌株,实验结果显示vt2基因突变导致EHEC 86-24对IPEC-J2和HEp-2的粘附能力降低。本研究中重新在萘啶酮酸敏感的野生型EHEC 86-24菌株基础上,产生了vt2基因敲除突变菌株和编码VT2噬菌体缺失突变菌株,评估了两株EHEC O157:H7突变菌株对IPEC-J2细胞系的粘附特征,并和细胞系HEp-2和CaCo-2进行了比较。实验室前期用小猪做了动物肠道感染体内实验,在此基础上本研究建立新的猪结肠IPEC-J2细胞体外感染模型,研究了EHEC O157:H7 86-24及其等位基因vt2-突变菌株对IPEC-J2细胞的粘附受体nucleolin、β1-intergrin和Gb3合成的表达影响,为进一步准确分析EHEC O157:H7的感染机理提供理论依据。
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) is VT toxin producing Escherichia coli (VTEC) a subgroup of.O157:H7 serotypes of pathogenic strains in a typical EHEC,.O157:H7 is the main pathogenic bacteria infection outbreak of large-scale O157 is a kind of food borne pathogens, cattle is the natural host of the first, is the main food contaminated with feces the outbreak of.EHEC infection, the infection can cause mild diarrhea to hemorrhagic enteritis, severe cases can cause hemolytic uremic syndrome, even death.EHEC two pathogenic factor is the most critical adhesion by phage encoding and VT toxin mediated LEE pathogenicity island (intestinal epithelial cell injury site).LEE pathogenicity island encoding protein responsible for the formation of intimin damage, which is characterized by tight adhesion of pathogenic bacteria and host intestinal surface, bacteria in the host cell surface adhesion sites of actin enriched, forming a cup-shaped base and cell surface The edge brush damage microvilli.LEE pathogenicity island from LEE1 to LEE5 5 operon, encoding the type III secretion system involves the secretion of the shift factor, the effect of protein and the membrane surface of the tight adhesion of Intinmin and its receptor protein Tir (encoding by adhesion bacteria translocated into the host cell receptors on the adhesion of Intinmin and in addition). The main receptor binding protein Tir, and also the host cell surface adhesion receptor (HIR) binding, such as Intinmin and intestinal cell surface integrin beta 1 (beta 1-integrin) and nucleolin (nucleolin). Combined with the research that intimin-Tir before the formation of a stable structure, adhesion and adhesion with the first intimin host cell receptor HIRs, the bacteria and the host cell close enough to complete the Tir and other effect factors from bacteria to host epithelial cell surface transport process of injection.
VT toxin EHEC produced by O157:H7 is one of the important pathogenic factors, this study focuses on enterohemorrhagic Escherichia coli EHEC86-24 infection of porcine jejunum cell line (IPEC-J2), human colon carcinoma cell line (CaCo-2) and human laryngeal cancer cell line (HEp-2) process, effects of VT toxin produced by the adhesion and colonization process. In bacterial infection of IPEC-J2 cells, VT toxin on the related pathogenic factors affecting gene expression, including pathogenic adhesion factor beta 1 integrin (beta 1-integrin) and nucleolin (nucleolin) expression of gene effects on VT toxin receptor Gb3 expression, so as to further analysis of the mechanism of VT toxin in EHEC disease in the process of the test. The use of lambda -Red homologous recombination technique to construct vt2 gene knockout mutant strain 86-24-vt2S, while producing VT2 toxin encoding phage mutant strain 86-24 933W, and the mutant strain vt2 Gene of strain 86-24-vt2S (pVT2) and 86-24-933W (pVT2). The wild type strain EHEC 86-24 and different vt2 gene mutant strain and eukaryotic cell lines were adhesion test and related adhesion factor on the infection process of eukaryotic cells IPEC-J2 real-time quantitative PCR was performed. The test results showed that the wild type EHEC strain and its parent on comparison of strains and vt2 gene, vt2 gene knockout mutant of 3 strains of eukaryotic cell line adhesion ability decreased significantly; wild type strain EHEC infection in IPEC-J2 cell line in the process, along with the associated host cell adhesion factor beta 1-integrin, increased expression of nucleolin and Gb3 genes; and wild type EHEC O157:H7 or vt2 the gene of mutant interactions of IPEC-J2 cell lines, the expression of integrin beta 1 was higher than that of IPEC-J2 and the expression level of vt2 gene knockout mutant strains with or without bacterial adhesion of IPEC-J2 and vt2; Gene knockout mutant strains of adhesion of infection, reduce the level of the expression of nucleolin, but the gene strain did not repair the nucleolin expression to the level of the wild type. The adhesion process test data revealed that the VT2 toxin in EHEC O157:H7 pathogenic bacteria of intestinal epithelial cells in the role, this role in gene level by to improve the expression level of the host cell surface receptor factor beta 1-integrin and nucleolin, so as to improve the adhesion of bacteria.
Nucleolin (Nucleolin) is a multifunctional nuclear protein can be expressed in many types of eukaryotic cell surface, as some viruses and adhesion receptors. Nucleolin is widespread and between cells and cell interactions related to a large family of receptors. Enteropathogenic Escherichia coli adhesin (EPEC) rod combined with the specific integrin beta 1. In the experimental infection of porcine and bovine tissues, immunostaining showed that beta 1 integrin aggregation in bacterial adhesion sites that integrin in EHEC words in the course of O157:H7 infection is a potential receptor protein of.VT toxin is a kind of typical structural protein AB_5 can inhibit the synthesis of.VT protein in eukaryotic cells the toxin in the process of EHEC infection cause hemorrhagic enteritis (HC), hemolytic uremic syndrome (HUS) and the clinical severity of the.VT toxin B subunit and target cell surface glycolipid receptor (Gb3) binding, receptor mediated through drug guide Recently, studies have reported that VT2 toxin promotes the colonization process of EHEC on the intestine by stimulating eukaryotic cells to express nucleolus. However, the theory of VT toxin promoting bacterial adhesion and colonization is still controversial.
Our previous research work in the construction of vt2 gene of nalidixic acid resistant strain EHEC mutant strains, the results show that vt2 gene mutation leads to the decrease of adhesion ability of EHEC 86-24 on IPEC-J2 and HEp-2. In this study, again in the wild type EHEC nalidixic acid sensitive strain 86-24 on the basis of the vt2 gene knockout mutant strain and VT2 mutant strain of phage encoding, evaluation of two strains of EHEC O157:H7 mutant strain adhesion characteristic of IPEC-J2 cell line, and cell lines HEp-2 and CaCo-2 were compared. The laboratory with pig made in vivo animal intestinal infection, this study on the basis of the establishment of porcine colon IPEC-J2 cells in vitro infected model. Study on EHEC O157:H7 86-24 and allele vt2- of nucleolin on IPEC-J2 cell adhesion receptor expression strain, beta 1-intergrin and Gb3 synthesis effect, as in It provides a theoretical basis for the accurate analysis of the infection mechanism of EHEC O157:H7.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378

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