沙眼衣原体隐秘质粒致病作用及机理研究

Chlamydia trachomatis is a medically important bacterial pathogen that causes diseases of eye, uroreproductive tract, and respiratory tract in humans. It has been noticed that sexually transmitted chlamydial infection increased recently in China. Majority of C. trachomatis strains have a small highly conserved cryptic plasmid. This plasmid has been linked to immuno-pathological damage in up-reproductive organs induced by C. trachomatis infection. The mechanism underlying the impairment remains unclear. Toll-like receptor 2 (TLR2) signaling pathway is essential in response to C. trachomatis infection and the activation relies upon chlamydial cryptic plasmid. We hypothesize that gene product encoded by chlamydial plasmid is involved in the activation of TLR2 signaling, thus, plays a key role in the pathogenesis of C. trachomatis. This project seeks to use new C. trachomatis shuttle plasmid transformation and targeted-gene mutation techniques to analyze the pathogenic role of plasmid. This proposed work will systematically and carefully establish the relationship of virulence phenotypes and pathogenesis by the mutation of individual C. trachomatis plasmid gene for the first time in this unique obligate intracellular pathogen in situ. Elucidation of pathogenic role of the plasmid is significant as this will advance our understanding of C. trachomatis pathogenicity in order to aid in the development of new effective and safe therapies against C. trachomatis infection.

沙眼衣原体是人类眼、生殖道和呼吸道等多种疾病的重要致病菌。近年来我国性活跃人群中生殖道衣原体感染不容忽视。衣原体生殖道感染可导致盆腔炎性疾病、宫外孕、不孕不育，经围生期转播，引起婴儿肺炎等疾病，故严重危害妇女、其性伙伴和子女的健康。至今，衣原体致病机制了解甚少。研究发现，衣原体菌株隐秘质粒的存在可能与衣原体诱发的免疫组织损伤有关。TLR2信号途径是宿主对衣原体感染的重要应答信号通路之一，其激活依赖于衣原体隐秘质粒的存在。我们推测，衣原体质粒编码的特定产物参与TLR2信号通路激活，从而在其致病机制中起重要作用。本项目拟采用新的衣原体穿梭质粒载体转化、定向基因突变和互补等分子遗传方法，率先阐明质粒基因产物对衣原体致病性和对宿主TLR2信号通路激活的影响。阐明衣原体质粒的致病、免疫修饰功能和作用机制，对深入了解衣原体的高致病性有重要意义，为发展新且有效安全的衣原体疾病治疗剂提供重要靶标。

衣原体质粒在其致病性中扮演重要角色，本项目主要探究质粒基因产物对衣原体致病性和对宿主 TLR2 信号通路激活的影响。首先，我们完善了衣原体转化方法学，构建了质粒基因突变体并转化衣原体并获得突变菌株。通过生长曲线、激活TRL2/4 和NF-κB信号通路、宿主细胞糖原累积分析，发现衣原体质粒表达蛋白Pgp3和Pgp4与衣原体的胞内寄生相关且可能与宿主糖原累积能力有关；Pgp3和Pgp4诱发的宿主固有免疫反应可能与致病性相关。由于原拟定的技术路线，衣原体和大肠杆菌穿梭质粒pGFP::SW2对高表达衣原体pgp4有明显不足，我们改造了穿梭质粒，使用pBOMB为基础，构建新的可控蛋白表达兼报告质粒载体，这个困难使我们后续研究工作推进有一定的延迟。后续工作中我们建立并完善了沙眼衣原体主要外膜蛋白基因ompA启动子（P3）驱动的GFP报告基因系统并确定了ompA 启动子的关键原件。此系统提供了经遗传学强荧光标记衣原体的工具，为深入实时研究活细胞内衣原体基因表达、生理、和病理特性提供了一个有用的分子平台。另外，首次使用定量GFP报告基因法，评估抗微生物药物的疗效。项目资助发表论文5篇，其中3篇SCI，2篇CSCD核心。培养硕士研究生2名。项目投入经费70万，支出22.8926万元。剩余经费 47.1074 万元，剩余经费计划用于本项目研究后续支出。