c-di-GMP信号系统调控钩端螺旋体毒力及介导免疫逃避的分子机制研究

Leptospirosis is a serious worldwide zoonotic disease caused by infection with Leptospira spp., but pathogenecity of the spirochete is poorly understood. Recently, we found that the expression of leptospiral outer membrane protein antigens was significantly down-regulated and the expression of leptospiral virulence factors such as adhesins was significantly up-regulated during pathogenic L. interrogans strain Lai infecting host cells. However, the exact regulatory mechanism remain to be discovered. Cyclic diguanylate acid (c-di-GMP) has been shown to widely involve in the regulation of bacterial virulence and membrane protein expression. The c-di-GMP system is composed of ① diguanylate cyclases (DGC), phosphodiesterases (PDE), the two key componets responsible for synthesis or hydrolyzation of c-di-GMP, and ② c-di-GMP. Although L. interrogans strain Lai posesses all the elements of c-di-GMP system, the function of them still unkown. We found that the expression levels of partial antigens and virulence factors were significantly changed after blockage of either DGC or PDE, but its regulation mechanism yet to be illustrated. In this project, we intend to generate the knockout and complemented mutants of leptospiral DGC or PDE encoding gene, and use leptospire-infected cell models to determine the c-di-GMP levels regulated by DGC and PDE proteins in c-di-GMP signalling systems after sensing infection environment, as well as the expression diversity of leptospiral outer membrane protein antigens, adhesins, invasive enzymes and hemolysins regulated by c-di-GMP through trigger or terminate the transcription of mRNA. Simultaneity, we plan to use leptospire-infected animal models to determine the functions of different c-di-GMP signalling systems in vivo such as decrease of the antigen expression to realize immune escape, and increase of the virulent factor expression to enhance virulence for survival and proliferation in hosts.

钩端螺旋体引起的钩体病是全球性人兽共患病，对其致病机制了解甚少。我们发现钩体感染细胞时外膜蛋白抗原表达显著下调、黏附素等毒力因子表达显著上调，但其机制不明。环二鸟苷酸（c-di-GMP）信号系统广泛参与细菌毒力与膜蛋白表达调控，钩体有该系统所有元件，我们阻断二鸟苷合成酶DGC或降解酶PDE后部分抗原和毒力因子表达水平改变，但调控机制有待阐明。本项目中，我们拟构建钩体DGC和PDE基因敲除及补回突变株，采用细胞感染模型探讨环二鸟苷系统中DGC和PDE感受感染环境信号并调控c-di-GMP水平，c-di-GMP通过启动或终止mRNA转录调控外膜蛋白抗原、黏附素、侵袭性酶及溶血毒素表达水平的作用及差异，另采用动物感染模型证实体内环二鸟苷系统功能，以期阐明感染宿主时钩体减少抗原表达以利于免疫逃避、提高毒力因子表达以增强毒力，从而实现在宿主体内生存并繁殖的分子机制，具有较高创新性和医学价值。

钩端螺旋体引起的钩体病是全球性人兽共患病，我们发现钩体感染细胞时外膜蛋白抗原表达显著下调、黏附素等毒力因子表达显著上调，环二鸟苷酸（c-di-GMP）信号系统广泛参与细菌毒力与膜蛋白表达调控，阻断二鸟苷合成酶DGC或降解酶PDE后部分抗原和毒力因子表达水平改变，但调控机制有待阐明。在国家自然科学基金资助下，我们采用细胞感染模型研究了不同环境条件下环二鸟苷系统中DGC和PDE功能蛋白的mRNA水平以及c-di-GMP水平改变情况；构建了钩体中推测具有DGC或PDE功能的GGDEF、EAL/HD-GYP结构域蛋白的原核表达系统并成功表达纯化了各目的蛋白；采用荧光方法和HPLC方法测定了各目的蛋白的酶学特性；采用目的基因重组入c-di-GMP相关基因敲除的枯草芽孢杆菌，检测了各基因的生物学功能及可能的下游靶基因，另采用结构域敲除等技术研究了环二鸟苷系统的调控，以期阐明感染宿主时钩体减少抗原表达以利于免疫逃避、提高毒力因子表达以增强毒力，从而实现在宿主体内生存并繁殖的分子机制，具有较高创新性和医学价值。.本研究已发表SCI论文1篇、另有5篇正在投稿中，国家核心期刊论文4篇、另有一篇在审稿中，获得国家发明专利1项，参编了高等教育出版社出版的普通高等教育十二五国家规划教材《医学微生物学》第三版，在本课题资助下参加两届国际钩体病学术会议做墙报展示并获得优秀学生研究奖，培养了博士后1名、博士生2名、硕士生2名。对照预定的完成本项目主要考核指标，我们基本完成了本项目研究任务。