本文關(guān)鍵詞： 沙門(mén)氏菌 效應(yīng)蛋白 SipA 腸炎　出處：《復(fù)旦大學(xué)》2011年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：沙門(mén)氏菌是革蘭氏陰性菌,它可以引起自限性腸炎、傷寒等疾病。沙門(mén)氏菌的致病性主要與染色體上成簇分布的編碼致病相關(guān)基因的特定區(qū)域-致病島(Salmonella pathogenicity island, SPI)相關(guān)。目前,已在沙門(mén)氏菌中發(fā)現(xiàn)了5個(gè)致病島,即SPI-1-SPI-5。其中SPI1和SPI2各自編碼不同的Ⅲ型分泌系統(tǒng),TTSS1(type Ⅲ secretion system1)和TTSS2(type Ⅲ secretion system2)。其中SPI1-TTSS與細(xì)菌入侵宿主細(xì)胞相關(guān),而SPI2-TTSS在細(xì)菌的存活和復(fù)制過(guò)程中發(fā)揮重要作用。 SipA是SPI1編碼Ⅲ型分泌系統(tǒng)分泌的效應(yīng)蛋白之一,它可以與actin相互作用。促進(jìn)G-actin聚合成為F-actin;結(jié)合F-actin,導(dǎo)致細(xì)胞骨架的重排從而促進(jìn)沙門(mén)氏菌入侵宿主細(xì)胞。同時(shí)也有研究發(fā)現(xiàn)在動(dòng)物感染模型中,SipA促進(jìn)腸炎的發(fā)生。但是,對(duì)于SipA導(dǎo)致腸炎的分子機(jī)制還不清楚。因此,本課題首先找到SipA與actin相互作用的具體位點(diǎn),構(gòu)建SipA的突變菌株使其失去與actin的作用,然后利用動(dòng)物感染模型探討突變菌株對(duì)腸炎的影響,從而闡明SipA與actin相互作用的結(jié)構(gòu)域是否與其導(dǎo)致腸炎的功能相關(guān)。 已有研究結(jié)果表明SipA497-669是與actin相互作用的最小結(jié)構(gòu)域。在此基礎(chǔ)上,本研究首先構(gòu)建了一系列SipA的截短突變體,利用共沉的實(shí)驗(yàn)方法驗(yàn)證這些截短突變體的功能,從而找到SipA與actin目互作用的最小結(jié)構(gòu)域。利用點(diǎn)突變?cè)噭┖?構(gòu)建SipA的點(diǎn)突變體,利用共沉的實(shí)驗(yàn)方法驗(yàn)證這些點(diǎn)突變體的功能,從而找到SipA與actin相互作用的位點(diǎn)。研究結(jié)果表明SipA514-651是與actin相互作用的最小結(jié)構(gòu)域；SipA635位和637位氨基酸是與actin相互作用的氨基酸位點(diǎn)。同時(shí)利用pyrene-actin實(shí)驗(yàn)來(lái)研究SipA514-651,SipA635,637對(duì)actin聚合臨界濃度的影響,發(fā)現(xiàn)SipA514-651, SiPA635,637均可以降低G-actin聚合的臨界濃度,從而促進(jìn)G-actin的聚合。 為了進(jìn)一步研究SipA的功能,利用同源重組方法構(gòu)建了沙門(mén)氏菌染色體sipA點(diǎn)突變的突變菌株：包括sipAK551A S.Typhimurium, sipAK635A S.Typhimurium, S.Typhimurium,sipAK635AE637W S.Typhimurium, sipA1580SK581AS. Typhimurium。在細(xì)胞水平,通過(guò)感染HeLa細(xì)胞研究這些突變菌株侵襲宿主細(xì)胞的能力是否發(fā)生改變。在動(dòng)物水平,利用小鼠的感染模型研究這些突變菌株是否可以導(dǎo)致嚴(yán)重的腸炎。研究結(jié)果表明,sipAK635AE637WS. Typhimurium的侵襲力明顯低于野生株；但是,S. Typhimurium引起腸炎程度與野生株相似,也就是說(shuō)把SipA與actin相互作用的氨基酸位點(diǎn)突變后并不影響其促進(jìn)腸炎的發(fā)生。以上研究豐富了SPI1-TTSS效應(yīng)蛋白SipA的認(rèn)識(shí)及其與宿主相互作用機(jī)制的探討。該研究將促進(jìn)理解微生物與宿主相互作用的機(jī)制,為細(xì)胞微生物學(xué)的研究提供新思路。
[Abstract]:Salmonella is a gram-negative bacteria, it can cause self limiting enteritis, typhoid and other diseases. The pathogenic Salmonella specific area - and clusters on chromosomes encoding pathogenicity related genes (Salmonella pathogenicity pathogenicity island island, SPI). At present, 5 have been found in Salmonella pathogenicity island bacteria, which is SPI-1-SPI-5. type III SPI1 and SPI2 respectively encoding different secretion system, TTSS1 (type III secretion system1) and TTSS2 (type secretion system2 SPI1-TTSS. 3) associated with bacterial invasion of host cells, and SPI2-TTSS play an important role in bacterial survival and replication process.
SipA is SPI1 encoding type III secretion of secreted effector proteins, which can interact with actin. To promote the polymerization of G-actin into F-actin; with F-actin, leading to cytoskeleton rearrangement and promote Salmonella invasion of host cells. There was also a study found in the animal model of infection, promote SipA enteritis. However, for the molecular mechanisms leading to SipA enteritis is not clear. Therefore, this paper firstly find the specific sites of SipA interaction with actin, construction of mutant strain SipA lose its interaction with actin, and then use the animal strains to explore the mutation effect of enteritis model of infection, so as to clarify the domain of SipA interacts with actin and the function of enteritis.
The research results show that SipA497-669 is the smallest domain interaction with actin. Based on this, we first constructed a series of truncated mutant SipA, verify these truncated mutant function using the experimental method of co precipitation, the smallest domain in order to find the SipA and actin interaction. Using point mutation point kit. Construction of mutant SipA, the validation of these point mutants function using the experimental method of co precipitation, in order to find the SipA and actin interaction sites. The results of the study show that SipA514-651 is the smallest domain interaction between actin and SipA635; and 637 amino acids are amino acid sites of interaction with actin. At the same time using the pyrene-actin experiment to study SipA514-651 effect of SipA635637, the critical concentration of actin polymerization of SipA514-651, SiPA635637 can reduce the critical concentration of G-actin in polymerization. Promote the polymerization of G-actin.
In order to further study the function of SipA by homologous recombination method to construct the Salmonella chromosome sipA mutation mutation strains including sipAK551A S.Typhimurium, sipAK635A S.Typhimurium, S.Typhimurium, sipAK635AE637W S.Typhimurium, sipA1580SK581AS. Typhimurium. at the cellular level, the changes on these mutant host cell invasion ability to infect HeLa cells. At the animal level, research whether these mutant strains can cause severe enteritis by infected mouse model. The results show that the sipAK635AE637WS. Typhimurium invasion were significantly lower than that of wild strain; however, S. Typhimurium caused by enteritis extent similar to wild strains, that does not affect the occurrence of the amino acid sites to promote enteritis SipA interaction with actin mutation. Study on the effect of SPI1-TTSS rich protein SipA The understanding and mechanism of interaction with host will promote the understanding of the mechanism of interaction between microorganisms and host, and provide new ideas for the research of cell microbiology.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R378.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Victor Tunje Jeza;;Salmonella Typhi:from a Human Pathogen to a Vaccine Vector[J];Cellular & Molecular Immunology;2008年02期

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本文編號(hào)：1518385