CFA/I菌毛在大肠埃希氏菌（O78:H11）对肠道上皮细胞感染中的分子机制

During the infection process, gram-negative pathogenic bacteria produce a variety of adhesive pili for attachment to specific host organ, tissue or cell. Thus, adhesive pili is a new type of anti-biotic drug target and effective antigen for vaccine development, the structural and functional studies of pili are very critical. In gram-negative pathogenic bacteria, the major mechanism for pili assembly is the chaperone-usher pathway. Due to the complexity and diversity of chaperone-usher pili assembly mechanisms and subtypes, the in-depth research and investigation of specific pili are urgently required for its clinical application. This project will focus on CFA/I pili from gram-negative bacteria enterotoxigenic Escherichia coli (ETEC，O78:H11), we have cloned and expressed the pili assembly subunits, solved the protein and protein-protein complex crystal structures. Combining with the molecular biology, cell biology and biochemistry assay, we will keep investigating the details of their assembly mechanism and molecular mechanism on host infection, and find out its host receptor and related signal pathway. The results would provide hints and basic theory for better understanding of pathogenesis mechanism, establishing diverse anti-pathogenic bacteria strategies and developing novel specific anti-bacteria drugs. At meantime, this knowledge would also expand our understanding on host cell infection especially human immune system and signal pathway.

革兰氏阴性病原菌在感染宿主的过程中能产生不同的粘性菌毛，实现对宿主器官,组织或细胞的特异性附着。菌毛作为新的抗菌药物设计靶点以及有效的疫苗抗原，其结构与功能的研究非常重要。革兰氏阴性病原菌中最主要的菌毛组装途径为分子伴侣-引导蛋白通路, 鉴于此类菌毛亚型和组装机制的复杂多样性，对其临床应用和研发都迫切需要研究者针对特异的菌毛进行深入的研究与探索。本课题以大肠埃希氏菌（O78:H11）表面CFA/I菌毛为研究对象，已经克隆并表达了它们各自组装的亚基蛋白,解析各亚基及其复合物的晶体结构。在进一步结合分子生物学,细胞生物学和生物化学的方法的同时，深入探索它们的组装机制与宿主感染的分子机制，寻找其在宿主细胞中的受体分子和相关信号通路。该研究结果将对了解病原生物的致病机制,建立灵活的防治措施和开发特异的新药提供重要的理论基础，同时也将加深人们对宿主细胞特别是人类细胞自身免疫系统信号转导机制的了解。

革兰氏阴性病原菌在感染宿主的过程中能产生不同的粘性菌毛,它对宿主器官、组织或细胞能特异性附着。本课题的研究对象是大肠埃希氏菌(O78:H11)表面CFA/I 菌毛，探究了CFA/I 菌毛的不同组成CfaA,CfaB及CfaE的作用。在本课题中，通过分子克隆构建，表达纯化了CFA/I 菌毛的重要组成部分CfaA-CfaB和CfaA-CfaE复合物，并且基于X-射线衍射，得到了该两个复合物的晶体结构。我们利用荧光分光光度计检测了CfaA-CfaB和CfaA-CfaE的亲和性，同时通过对这两个复合物的晶体结构进行分析，构建了可能对CFA/I 菌毛组装有重要影响的突变体，并通过pull-down,全菌-ELISA，负染色电子显微镜和甘露糖抗性血细胞凝集实验等证实了该类突变体的作用。基于前期和现有的实验结果，模拟了分子伴侣-粘附蛋白/引导蛋白对CFA/I菌毛的组装，为之后探索研究关于针对CFA / I菌毛的新型药物提供了一定的理论基础。

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