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

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.

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。

钩端螺旋体（简称钩体）病是一种全球分布、洪涝时重点监控的人畜共患传染病，是我国重点防疫和监控传染病之一，然而钩体致病机制至今所知甚少。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。本研究结果发现：问号钩体LA2103/2104/2558/2559/1953是问号钩体ClpS/A/X/P1/P2的相应编码基因，生物信息学分析结果显示其表达产物均为亲水性蛋白、半衰期长、不可外分泌，故均在细菌胞内发挥作用。qRT-PCR结果显示，感染巨噬细胞后问号钩体clpS/A/X/P1-mRNAs水平显著升高，其中clpS/clpA-mRNAs水平提高可达近7倍。体外实验表明钩体ClpX/P1/P2以ClpX-ClpP1-ClpP2的组合形式发挥蛋白降解作用，可降解含钩体SsrA标签的蛋白。因基因敲除实验未能获得目标基因敲除突变株，我们推测clpS/A/X/P1基因可能是问号钩体赖株的重要基因，对其生存可能具有重要作用，随后的文献调研结果显示细菌ClpP对细菌生存具有重要意义，是抗菌类物质研发的理想靶标之一。采用GST-Pull down实验筛选并获得了14个可能的问号钩体ClpXP1P2天然蛋白底物和156个可能的肺炎克雷伯菌ClpXP天然蛋白底物，这些蛋白涉及DNA修复、细菌分裂、核糖体蛋白、tRNA连接、转录和离子转运等多种重要生理过程。总之，本项目结果显示ClpP相关蛋白酶复合物在问号钩体感染过程中发挥重要作用，研究结果可为问号钩体对宿主致病机制提供新的研究热点，也可为其他病原菌致病机制研究提供线索和依据，具有重要医学意义。此外，该项目资助期内，国内外爆发了新冠疫情，在本项目及其他项目联合资助下，我们还完成了基于LAMP的新冠核酸快速检测技术研发、申请了2项专利等工作，具有一定的应用价值。