CdaR-DAC/PDE信号系统调控钩端螺旋体侵袭相关毒力因子表达的作用及机制

We recently found that the expression levels of invasiveness-associated virulence factors of Leptospira were significantly up-regulated during infection of host cells, but the expression up-regulation mechanism remains unknown. Recent literatures reported that diadenylyl cyclase (DAC) and phosphodiesterase (PDE) compose a signaling system that can sense environmental signals and regulate c-di-AMP second messenger level to control target gene expression through KdpDE two-component system (TCS). Our preliminary study results showed that leptospiral genome contains DAC or PDE domain-containing protein and KdpDE encoding genes, and the c-di-AMP levels in leptospires during infection of host cells or under a low pH environment were increased. In this project, we will use LC-MS/MS, gene knockout, target protein phosphorylation determination and fluorescent probe-based laser confocal microscopy to determine that the product of LA3303, LA3304 or LA2999 gene of L. interrogans strain Lai acts as DAC-activated protein, DAC or PDE which composing a CdaR-DAC/PDE signaling system. Subsequently, we will determine the role and mechanism of the CdaR-DAC/PDE signaling system to sense the microenvironmental signals such as low pH and high ROS level during leptospiral infection, and up-regulate the expression levels of adhesins, invasion, collagenase and metalloproteases through c-di-AMP and its downstream KdpDE-TCS. The results of this study will present novel findings in pathogenicity of Leptospira with high medical significance.

我们发现钩端螺旋体（简称钩体）感染细胞时侵袭相关毒力因子表达较培养基中显著上调，其机制不明。新近报道细菌二腺苷环化酶（DAC）与磷酸二酯酶（PDE）组成的信号系统能感知环境信号、调节第二信使环二腺苷（c-di-AMP）水平并通过KdpDE双组分系统（TCS）调控靶基因表达。我们前期实验结果显示钩体基因组中有DAC、PDE结构域蛋白及KdpDE基因，感染细胞或低pH时钩体c-di-AMP显著升高。本项目拟采用LC-MS/MS、基因敲除、靶蛋白磷酸化测定、荧光探针激光共聚焦等技术，确定问号钩体赖株LA3303、LA3304和LA2999基因产物分别为DAC活化蛋白、DAC和PDE并组成CdaR-DAC/PDE信号系统，阐明该系统感知钩体感染时低pH或高ROS微环境信号并通过c-di-AMP及下游KdpDE-TCS上调黏附素、侵袭素、胶原酶和金属蛋白酶表达的作用及机制，具有较高创新性和医学意义。

钩体病是全球流行的自然疫源性人兽共患传染病，但其致病机制至今未完全明了。在国家自然科学基金资助下，我们研究证实了问号钩体LA3303、LA3304和LA2999基因产物分别为DAC活化蛋白、DAC和PDE且三者共同组成CdaR-DAC/PDE信号传导系统，该信号系统可感知问号钩体感染不同宿主细胞时的低pH或高渗透压等感染微环境信号，通过c-di-AMP第二信使及其下游KdpDE-TCS调控问号钩体黏附素、侵袭素、胶原酶和金属蛋白酶等侵袭力相关毒力基因表达。本研究共发表SCI论文4篇（其中1篇IF>5），国家核心期刊论文2篇，参与国内学术会议报告2次，国际学术报告1次，协助培养博士后1名，博士3名。对照项目计划书，我们已完成项目的研究任务并达到了考核要求。