沙眼衣原体分泌蛋白HtrA作用分子的鉴定及其致病机理研究

Chlamydia trachomatis was the pathogen of human trachoma and sexual transmitted diseases of genitourinary tract. Recently, the infection caused by C.trachomatis has exceeded that of neisseriagonorrhoeae and Treponema pallidum, become the first reason of genitourinary tract and threatened to human seriously. HtrA, existing widely in bacteria, animals and plants, was a highly conserved serine proteinase with the property of Heat Shock Protein. HtrA works as a molecular chaperone at a low temperature while as a serine proteinase at a high temperature, participating in cell resisting heat shock, closely related with bacteria pathogenesis. In our previous study, we discovered that cHtrA was secreted to inclusion body of Chlamydia and cytoplasm of host cells through a sec-dependent Type II Secretion System, and its cytotoxicity correlates with Toll-like receptor 4; we have screened many positive molecules interacting with host cells by yeast two hybrid technique successufully. To further illuminate the relationship with pathogenic mechanism, we will elucidate the relationship between its cytotoxity and signal pathway; further identify interaction molecules with host pathogenic mechanism and clarify its pathogenic mechanism.It will lay the rationale foundation for the prevelence and new drug development of C.trachomatis.

沙眼衣原体是引起人体沙眼及泌尿生殖道性传播疾病的病原体,近年来，沙眼衣原体的感染率已经超过淋球菌和梅毒，成为泌尿生殖道感染性疾病最常见的致病菌，严重危害人们的健康。HtrA,是一种具有热休克蛋白性质的高保守丝氨酸蛋白酶，广泛存在于各种微生物及动、植物体内，低温时作为一个分子伴侣，高温时作为丝氨酸蛋白酶,参与细胞抵御热休克，与细菌的致病关系非常密切。在前期的研究中，我们首次报道沙眼衣原体（cHtrA）以一种Sec依赖的Ⅱ型分泌途径分泌到衣原体包涵体和宿主细胞的细胞质中，且发现其细胞毒性作用与Toll样受体4缺失呈相关性；并通过酵母双杂交技术筛选出一系列与宿主细胞相互作用的阳性分子。为了进一步阐明该蛋白与致病机理的关系，我们拟进一步鉴定cHtrA与宿主的相互作用分子；并阐明其细胞毒性机理与信号传导通路的关系。

沙眼衣原体是引起人体沙眼及泌尿生殖道性传播疾病的病原体,近年来，沙眼衣原体的感染率已经超过淋球菌和梅毒，成为泌尿生殖道感染性疾病最常见的致病菌，严重危害人们的健康。HtrA,是一种具有热休克蛋白性质的高保守丝氨酸蛋白酶，广泛存在于各种微生物及动、植物体内，低温时作为一个分子伴侣，高温时作为丝氨酸蛋白酶,参与细胞抵御热休克，与细菌的致病关系非常密切。本研究旨在采用酵母双杂交、PULL down,免疫共沉淀、基因敲除、染色质免疫沉淀等技术系统地研究沙眼衣原体分泌蛋白HtrA与宿主的相互作用分子，阐明cHtrA与宿主的相互关系和致病机理、阐明其生物学功能。主要计划完成以下研究内容：1.成功构建pGBKT7-CtHtrA载体，构建了2.成功构建HeLa细胞的全长cDNA文库；3. 筛选了与CtHtrA相互作用的宿主分子的筛选， 酵母双杂交及回交实验，4.相互作用分子生物信息学分析、gene ontology（GO注释）；5.对发现16个与CtHtrA相互作用的宿主分子进行了相互作用的功能验证。6.由于技术原因，cHtrA基因敲除及宿主分子的RNAi工作，正在进行，大约在2018年9月份完成。