沙门氏菌Ⅲ型分泌系统效应蛋白SipA导致肠炎的分子机制

发布时间：2018-02-20 00:55

本文关键词： 沙门氏菌效应蛋白 SipA 肠炎　出处：《复旦大学》2011年博士论文　论文类型：学位论文

【摘要】：沙门氏菌是革兰氏阴性菌,它可以引起自限性肠炎、伤寒等疾病。沙门氏菌的致病性主要与染色体上成簇分布的编码致病相关基因的特定区域-致病岛(Salmonella pathogenicity island, SPI)相关。目前,已在沙门氏菌中发现了5个致病岛,即SPI-1-SPI-5。其中SPI1和SPI2各自编码不同的Ⅲ型分泌系统,TTSS1(type Ⅲ secretion system1)和TTSS2(type Ⅲ secretion system2)。其中SPI1-TTSS与细菌入侵宿主细胞相关,而SPI2-TTSS在细菌的存活和复制过程中发挥重要作用。 SipA是SPI1编码Ⅲ型分泌系统分泌的效应蛋白之一,它可以与actin相互作用。促进G-actin聚合成为F-actin;结合F-actin,导致细胞骨架的重排从而促进沙门氏菌入侵宿主细胞。同时也有研究发现在动物感染模型中,SipA促进肠炎的发生。但是,对于SipA导致肠炎的分子机制还不清楚。因此,本课题首先找到SipA与actin相互作用的具体位点,构建SipA的突变菌株使其失去与actin的作用,然后利用动物感染模型探讨突变菌株对肠炎的影响,从而阐明SipA与actin相互作用的结构域是否与其导致肠炎的功能相关。已有研究结果表明SipA497-669是与actin相互作用的最小结构域。在此基础上,本研究首先构建了一系列SipA的截短突变体,利用共沉的实验方法验证这些截短突变体的功能,从而找到SipA与actin目互作用的最小结构域。利用点突变试剂盒,构建SipA的点突变体,利用共沉的实验方法验证这些点突变体的功能,从而找到SipA与actin相互作用的位点。研究结果表明SipA514-651是与actin相互作用的最小结构域；SipA635位和637位氨基酸是与actin相互作用的氨基酸位点。同时利用pyrene-actin实验来研究SipA514-651,SipA635,637对actin聚合临界浓度的影响,发现SipA514-651, SiPA635,637均可以降低G-actin聚合的临界浓度,从而促进G-actin的聚合。为了进一步研究SipA的功能,利用同源重组方法构建了沙门氏菌染色体sipA点突变的突变菌株：包括sipAK551A S.Typhimurium, sipAK635A S.Typhimurium, S.Typhimurium,sipAK635AE637W S.Typhimurium, sipA1580SK581AS. Typhimurium。在细胞水平,通过感染HeLa细胞研究这些突变菌株侵袭宿主细胞的能力是否发生改变。在动物水平,利用小鼠的感染模型研究这些突变菌株是否可以导致严重的肠炎。研究结果表明,sipAK635AE637WS. Typhimurium的侵袭力明显低于野生株；但是,S. Typhimurium引起肠炎程度与野生株相似,也就是说把SipA与actin相互作用的氨基酸位点突变后并不影响其促进肠炎的发生。以上研究丰富了SPI1-TTSS效应蛋白SipA的认识及其与宿主相互作用机制的探讨。该研究将促进理解微生物与宿主相互作用的机制,为细胞微生物学的研究提供新思路。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378.22

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