本文關(guān)鍵詞：腸出血性大腸桿菌（EHEC O157：H7）VT2毒素致病機(jī)理研究　出處：《西北農(nóng)林科技大學(xué)》2010年博士論文　論文類型：學(xué)位論文

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【摘要】：腸出血性大腸桿菌(EHEC)是產(chǎn)VT毒素大腸桿菌(VTEC)的一個(gè)亞群。O157:H7血清型是EHEC中典型的致病菌株,是大規(guī)模O157感染爆發(fā)的主要病原菌。O157:H7是一種食源性致病菌,牛是其首要的天然寄主,主要是食物被牛糞污染,導(dǎo)致感染爆發(fā)。EHEC感染會(huì)引起輕度腹瀉到出血性腸炎、嚴(yán)重者會(huì)引起出血性尿毒綜合癥,甚至死亡。EHEC最關(guān)鍵的兩個(gè)致病因子是由噬菌體編碼的VT毒素和介導(dǎo)粘附作用的LEE毒力島(腸道上皮細(xì)胞損傷位點(diǎn))。LEE毒力島編碼的蛋白負(fù)責(zé)形成粘附抹平損傷,其典型特征是通過病原菌和寄主腸道表面的緊密粘附,在寄主細(xì)胞表面細(xì)菌粘附位點(diǎn)肌動(dòng)蛋白富集,形成杯狀基座和細(xì)胞表面邊緣刷狀微絨毛損傷。LEE毒力島由LEE1到LEE5主要5個(gè)操縱子組成,編碼的Ⅲ型分泌系統(tǒng)涉及到移位因子的分泌、效應(yīng)蛋白和外膜表面緊密粘附素Intinmin與其受體蛋白Tir(由細(xì)菌編碼轉(zhuǎn)運(yùn)到宿主細(xì)胞上的粘附素受體)。粘附素Intinmin除了和其主要受體蛋白Tir結(jié)合之外,還會(huì)和寄主細(xì)胞表面的粘附素受體(HIR)結(jié)合,如Intinmin與腸道細(xì)胞表面的β1整合素(β1-integrin)和核仁素(nucleolin)結(jié)合。有研究認(rèn)為在形成穩(wěn)定的intimin-Tir結(jié)構(gòu)之前,粘附素intimin首先和寄主細(xì)胞的粘附素受體HIRs結(jié)合,使得細(xì)菌和寄主細(xì)胞足夠接近從而完成Tir和其他效應(yīng)因子從細(xì)菌到寄主上皮細(xì)胞表面的注射轉(zhuǎn)運(yùn)過程。 EHEC O157:H7產(chǎn)生的VT毒素是其重要的致病因素之一,本研究關(guān)注腸出血性大腸桿菌EHEC86-24感染豬空腸細(xì)胞系(IPEC-J2)、人結(jié)腸細(xì)胞系(CaCo-2)和人喉癌細(xì)胞系(HEp-2)過程中,產(chǎn)生的VT毒素對(duì)其粘附定植過程的影響,以及在細(xì)菌感染IPEC-J2細(xì)胞過程中,VT毒素對(duì)其相關(guān)致病因子的基因表達(dá)水平影響,包括對(duì)致病粘附因子β1整合素(β1-integrin)和核仁素( nucleolin)的基因表達(dá)影響,對(duì)VT毒素受體Gb3合成表達(dá)的影響,從而深入分析VT毒素在EHEC致病過程中的作用機(jī)理。試驗(yàn)利用λ-Red同源重組技術(shù)構(gòu)建了vt2基因敲除突變菌株86-24-vt2S,同時(shí)產(chǎn)生了編碼VT2毒素的噬菌體缺失突變菌株86-24△933W、以及突變菌株的vt2基因補(bǔ)充菌株86-24-vt2S(pVT2)和86-24-933W(pVT2)。將野生型菌株EHEC 86-24和不同vt2基因突變菌株與真核細(xì)胞系進(jìn)行粘附試驗(yàn),并對(duì)感染過程真核細(xì)胞IPEC-J2的相關(guān)粘附因子基因進(jìn)行實(shí)時(shí)定量PCR檢測。試驗(yàn)結(jié)果顯示:與其親本野生型EHEC菌株和vt2基因補(bǔ)充菌株比較,vt2基因敲除突變株對(duì)3株真核細(xì)胞系的粘附能力均顯著降低;野生型EHEC菌株在感染IPEC-J2細(xì)胞系過程中,伴隨宿主細(xì)胞相關(guān)粘附因子β1-integrin、nucleolin和Gb3合成基因的表達(dá)增加;IPEC-J2細(xì)胞系與野生型EHEC O157:H7或vt2基因補(bǔ)充突變株相互作用時(shí),β1整合素的表達(dá)水平高于IPEC-J2與vt2基因敲除突變菌株或無細(xì)菌粘附時(shí)的表達(dá)水平;IPEC-J2與vt2基因敲除突變菌株粘附感染時(shí),核仁素的表達(dá)水平降低,但是其基因補(bǔ)充菌株沒有修復(fù)核仁素的表達(dá)到野生型水平。試驗(yàn)數(shù)據(jù)揭示VT2毒素在EHEC O157:H7致病菌對(duì)腸道上皮細(xì)胞的粘附過程中有促進(jìn)作用,這種促進(jìn)作用從基因水平上是通過提高宿主細(xì)胞表面受體因子β1-integrin和nucleolin的表達(dá)水平,從而提高了細(xì)菌的粘附功能。 核仁素(Nucleolin)是一種可以表達(dá)在多種類型真核細(xì)胞表面的多功能核蛋白,作為一些病毒和粘附素的受體。核仁素是與廣泛的細(xì)胞和細(xì)胞之間相互作用相關(guān)的一個(gè)受體大家族。腸致病性大腸桿菌桿(EPEC)的粘附素與β1整合素特異結(jié)合。在豬和牛的組織感染試驗(yàn)中,免疫染色發(fā)現(xiàn)β1整合素聚集在細(xì)菌粘附位點(diǎn),說明整合素在EHEC字O157:H7感染過程中是一個(gè)潛在的受體蛋白。VT毒素是一種典型的AB_5結(jié)構(gòu)蛋白,能抑制真核細(xì)胞蛋白合成。VT毒素在EHEC感染過程中導(dǎo)致出血性腸炎(HC)、溶血性尿毒綜合癥(HUS)等嚴(yán)重的臨床癥狀。VT毒素的B亞基和目標(biāo)細(xì)胞表面的糖脂類受體(Gb3)結(jié)合,通過受體介導(dǎo)毒素內(nèi)化。最近研究報(bào)道VT2毒素通過刺激真核細(xì)胞表達(dá)核仁素,從而促進(jìn)EHEC在腸道上的定植過程。然而,關(guān)于VT毒素促進(jìn)細(xì)菌粘附定植的理論依然具有爭議。 本實(shí)驗(yàn)室前期研究工作中構(gòu)建了萘啶酮酸抗性EHEC菌株的vt2基因插入突變菌株,實(shí)驗(yàn)結(jié)果顯示vt2基因突變導(dǎo)致EHEC 86-24對(duì)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菌株基礎(chǔ)上,產(chǎn)生了vt2基因敲除突變菌株和編碼VT2噬菌體缺失突變菌株,評(píng)估了兩株EHEC O157:H7突變菌株對(duì)IPEC-J2細(xì)胞系的粘附特征,并和細(xì)胞系HEp-2和CaCo-2進(jìn)行了比較。實(shí)驗(yàn)室前期用小豬做了動(dòng)物腸道感染體內(nèi)實(shí)驗(yàn),在此基礎(chǔ)上本研究建立新的豬結(jié)腸IPEC-J2細(xì)胞體外感染模型,研究了EHEC O157:H7 86-24及其等位基因vt2-突變菌株對(duì)IPEC-J2細(xì)胞的粘附受體nucleolin、β1-intergrin和Gb3合成的表達(dá)影響,為進(jìn)一步準(zhǔn)確分析EHEC O157:H7的感染機(jī)理提供理論依據(jù)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 邱素君;呂春華;楊冬梅;;產(chǎn)腸毒素性大腸桿菌的研究進(jìn)展[J];草食家畜;2009年03期

2 楊琴;閻有功;;腸出血性大腸桿菌Ⅲ型分泌系統(tǒng)及轉(zhuǎn)位效應(yīng)器蛋白的研究進(jìn)展[J];華南國防醫(yī)學(xué)雜志;2008年02期

3 羅霞;徐建國;;產(chǎn)志賀毒素型大腸桿菌的研究進(jìn)展[J];疾病監(jiān)測;2006年04期

4 劉雪連;孫振鈞;王沖;崔東良;廖燕;;大腸桿菌O157:H7的疫病生態(tài)學(xué)研究進(jìn)展及其控制對(duì)策[J];家畜生態(tài)學(xué)報(bào);2008年04期

5 孫洋,岳瑛,劉軍,馮書章,姜力;腸出血性大腸桿菌志賀毒素的研究進(jìn)展[J];吉林醫(yī)學(xué);2002年05期

6 樊蕊,劉謙民;腸出血性大腸桿菌O157∶H7研究進(jìn)展[J];臨床醫(yī)藥實(shí)踐;2003年08期

7 周勇;萬成松;;大腸桿菌O157:H7的毒力島與毒力因子的研究進(jìn)展[J];微生物學(xué)免疫學(xué)進(jìn)展;2006年02期

8 鄒全明;腸出血性大腸桿菌O157感染防治研究進(jìn)展[J];微生物學(xué)雜志;2004年05期

9 嚴(yán)亞賢,華修國;大腸桿菌O157:H7的毒力因子的研究進(jìn)展[J];畜牧與獸醫(yī);2003年04期

10 馮翔宇;腸出血性大腸桿菌毒力因子研究進(jìn)展[J];醫(yī)學(xué)綜述;2002年01期

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