钩端螺旋体ClpP相关蛋白酶复合物的变性蛋白降解作用及其分子致病机制研究

钩端螺旋体（简称钩体）病是全球性人兽共患传染病，至今其致病机制所知甚少。近年发现细菌ClpP相关蛋白酶复合物在细菌抵抗宿主固有抗感染免疫力、宿主体内生存与繁殖及致病过程中发挥重要作用。我们前期工作发现钩体含多个ClpP相关蛋白编码基因，感染巨噬细胞后钩体clpS、clpA、clpX和clpP基因表达显著上调，但其致病功能不明。本项目拟采用分子酶学技术确定钩体ClpS、ClpA/X和ClpP的功能及组合类型，采用细胞、动物感染模型比较靶基因敲除前后钩体抗杀伤能力、致病能力差异，采用iTRAQ LC-MS/MS技术检测ClpS/A/X特异识别的变性蛋白种类，以期揭示ClpP相关蛋白酶复合物ClpAP/APS/XP在钩体抗吞噬及致病过程中的实际作用和意义，从钩体抵抗力角度出发阐明钩体ClpAP/APS/XP致病相关分子机制，为其他病原菌致病机制研究提供线索和依据，具有较高创新性和医学意义。

Leptospirosis is a serious worldwide zoonotic disease, but the mechanisms of its pathogenicity remain poorly understood. In recent years, it has been found that the bacterial ClpP related protease complexes play a crucial role in resistance to host innate anti-infection immunity, the survival and proliferation in hosts and the bacterial pathogenic processes. We found that Leptospira possess several genes that encode ClpP related proteins and the expression of leptospiral clpS, clpA, clpX and clpP gene was significantly up-regulated during infection of macrophages, but its pathogenic features is unknown. In this project, we plan to determine the function of ClpS/A/X/P and their combination type by using techniques of molecular enzymology, and then observe differences of the resistibility against macrophages and the ability to infect hamster between before and after the deletion of target gene by using cell and animal infection models. Subsequently, we determine the diversity of denatured protein recognized by ClpS/A/X using iTRAQ LC-MS/MS techniques. Finally, we will uncover the practical role and significance of leptospiral ClpP related protease complexes ClpAP/APS/XP in anti-phagocytosis and pathogenic processes, and elucidate the molecular pathogenicity mechanism of ClpAP/APS/XP from the standpoint of resistance, which will provide clues and basis for the study of pathogenesis of other pathogens, and has high originality and medical significance.