肠炎沙门氏菌毒力蛋白与宿主细胞蛋白相互作用的筛选和功能研究

发布时间：2018-01-02 20:23

本文关键词：肠炎沙门氏菌毒力蛋白与宿主细胞蛋白相互作用的筛选和功能研究　出处：《武汉大学》2012年博士论文　论文类型：学位论文

【摘要】：肠炎沙门氏菌(Salmonella enterica)是一种典型的革兰氏阴性致病菌。该菌主要引起畜禽的胃肠炎以及人类肠炎和食物中毒,严重威胁着人畜健康。肠炎沙门氏菌表达两个Ⅲ型分泌系统(type Ⅲ secretion systems,T3SS),由毒力岛1和毒力岛2(pathogenicity islands1and2, SPI-1and SPI-2)分别编码。细菌依赖T3SS将毒力蛋白运输至宿主细胞内发挥作用。通常认为,毒力岛1编码的毒力蛋白在细菌入侵的过程中起到作用,而毒力岛2编码的蛋白则负责促进细菌在细胞中的生存,复制以及免疫逃逸等的调节。目前已经鉴定出了30几个T3SS毒力蛋白,但是它们中的绝大多数其生理功能尚未知晓或了解很少。为探讨T3SS两个毒力岛的分泌蛋白的潜在生物学功能,并分析它们是怎样帮助细菌从入侵上皮细胞到在细胞内生存、复制的,鉴定这些毒力蛋白与宿主细胞蛋白的相互作用关系成为了必不可少的途径。本文从以下两个方面对T3SS分泌的毒力蛋白与宿主细胞蛋白的相互作用进行了系统分析,分别是： 1.通过酵母双杂交技术严格系统地筛选20个T3SS毒力蛋白(SsPH2, Ssel、 SpiC、SseF、SseG、SlrP、SseJ、SopD2、SifB、SipC、SipB、SipA、SopD、SipD、 SptP、SopA、SopE2、SrfB、SrfA、SrfJ)与宿主细胞的相互作用蛋白,并最终得到了超过300对的阳性相互作用结果。我们对其中部分结果进行了免疫共沉淀验证并通过文献进行了初步的功能分类分析,并从蛋白质组学的角度构建了相互作用网络草图。这些蛋白功能分布广泛,涵盖了能量代谢、细胞骨架和细胞运动、物质运输以及细胞内多种信号通路的调节。结合文献,我们获得的大量数据为揭示细菌侵染细胞及之后的生物学功能和细菌对宿主细胞各种生物学通路系统的影响提供了理论基础和事实依据。 2.选取SPI-2的重要毒力蛋白SsPH2为研究对象,探讨其对宿主内源泛素通路的影响作用。SsPH2作为一个最近才知晓的细菌编码的E3泛素连接酶,通过影响宿主内源泛素通路来促进细菌在宿主内的生存、复制以及调节宿主免疫反应。我们系统地鉴定了酵母双杂交得到的SsPH2作用对象,并着重研究了SsPH2与细胞蛋白LM04的相互作用。我们首先分别用不同方式验证了二者于体内、体外的相互作用,并探讨了其作用区域。接着我们通过体内、体外实验证实了SsPH2可以催化LM04蛋白的泛素化修饰。后面的进一步研究阐释了SsPH2对LM04的表达调控作用,我们发现SsPH2在体内稳定表达具有提高LM04蛋白水平的作用,通过深入研究这种调控的分子机制,我们发现SsPH2增强LM04蛋白水平是通过其对LM04的泛素化修饰来实现的。SsPH2抑制了LM04在细胞中的降解途径,从而延长了LM04蛋臼的半衰期。考虑到LM04与白细胞介素6(IL-6)途径的重要关系,我们猜测这种依赖于细菌SsPH2的LM04蛋白水平调节作用很可能影响了宿主IL-6信号通路,并调节了由IL-6介导的Stat3转录活性,从而影响宿主的早期炎症反应。以上发现揭示了SsPH2作为细菌编码的E3泛素连接酶通过特异性识别并泛素化修饰宿主蛋白LM04来实现细菌对宿主细胞生理功能的调节作用。综上所述,通过本课题的研究我们鉴定出了数量庞大的肠炎沙门氏菌T3SS毒力蛋白在宿主细胞中的相互作用对象,为后续的进一步功能分析打下了坚实的理论基础。此外我们着重研究了毒力蛋白SsPH2与宿主蛋白LM04的相互作用机制,并发现了SsPH2对LM04的泛素化修饰以及调节作用。我们的研究工作为肠炎沙门氏菌乃至整个革兰氏阴性菌界毒力蛋白与宿主蛋白相互关系的思考提供很多新的研究思路和方向。
[Abstract]:Salmonella enteritidis (Salmonella enterica) is a kind of typical gram negative bacteria. The bacteria are mainly caused by livestock gastroenteritis and human enteritis and food poisoning, a serious threat to human and animal health. Salmonella enteritidis expression of two type III secretion system (type secretion systems, T3SS), and 1 by pathogenicity island pathogenicity island 2 (pathogenicity islands1and2, SPI-1and SPI-2) respectively. T3SS will depend on the encoding of bacterial virulence protein transport to host cells play a role. Generally, the pathogenicity island encoding virulence protein 1 plays a role in the process of the invasion of bacteria, and 2 pathogenicity island encoding protein is responsible for promoting the survival of bacteria in the cells. Replication and immune escape regulation. At present have identified 30 T3SS virulence proteins, but most of them in their physiological function are currently unknown or poorly understood.
As a potential biological function of secretory protein two T3SS pathogenicity island, and analyze how they help the bacteria from invading epithelial cells to survive and replicate inside cells, the interaction between the identification of these virulence proteins with host cells has become an indispensable way. This interaction on the secretion of T3SS from the following two aspects the virulence protein and host cell proteins were analyzed, respectively:
1. through the yeast two hybrid system to strictly screened 20 T3SS virulence proteins (SsPH2, Ssel, SpiC, SseF, SseG, SlrP, SseJ, SopD2, SifB, SipC, SipB, SipA, SopD, SipD, SptP, SopA, SopE2, SrfB, SrfA, SrfJ) protein interaction with host cells and finally, got more than 300 of the positive interaction. We some of these results were verified by immunoprecipitation and analyzed preliminary functional classification through the literature, from the perspective of proteomics constructed the interaction network sketch. These proteins function are widely distributed, covering energy metabolism, cytoskeleton and cell movement, substance transport and regulation of multiple signaling pathways in cells. Combining with literature, a large amount of data we obtained provides to reveal the biological function and the bacterial cells and bacterial infection after the host cell biological pathway system On the basis of the foundation and the facts.
The most important virulence protein SsPH2 2. SPI-2 is selected as the research object, to explore the role of endogenous ubiquitin pathway of host.SsPH2 as a recently known bacteria encoding E3 ubiquitin ligase, to promote the survival of bacteria in the host by affecting the host endogenous ubiquitin proteasome pathway, replication and regulation of host immune response. We systematically identified SsPH2 object yeast two hybrid, and focuses on the interaction between SsPH2 and cell protein LM04. We used different ways to verify the two in vivo interaction in vitro, and to explore the role of regional. Then we through in vivo, in vitro experiments confirmed that SsPH2 can catalyze LM04 ubiquitination protein. Further study behind the expression regulation of SsPH2 on LM04, we found that SsPH2 expression is stable in vivo with increased LM04 protein levels for With the further study of molecular mechanism, through this regulation, we found that SsPH2 increased LM04 protein level is achieved through the degradation pathway of LM04 ubiquitination of.SsPH2 inhibited LM04 in the cells, thereby prolonging the half-life of LM04 egg mortar. Considering LM04 and interleukin 6 (IL-6) important relationship way, we guess this depends on the level of LM04 protein in bacterial SsPH2 regulation is likely to affect the host IL-6 signaling pathway, and regulates the transcriptional activity of Stat3 mediated by IL-6, thus affecting the early inflammatory response of the host. The above findings reveal SsPH2 as bacteria encoding E3 ubiquitin ligase by specific recognition and ubiquitination modification of the host protein LM04 to achieve the regulatory role of bacteria on physiological function of host cells.
In summary, through this study we identified the interaction between a large number of Salmonella enteritidis T3SS virulence proteins in host cells, and lay a solid theoretical foundation for the further function of subsequent analysis. In addition we studied the interaction mechanism of virulence protein SsPH2 and host protein LM04, and found SsPH2 of LM04 ubiquitination and regulation. Our research provides many new research ideas and directions for the thinking of the relationship between Salmonella enteritidis and the gram negative bacteria community virulence proteins and host proteins.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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