细菌性软腐病菌Cpx双组分信号转导系统的功能及其调控机制

The Cpx signal transduction system is one prevalent two-component systems (TCSs) in gram-negative bacteria which consists of the membrane integrated sensor histidine kinase CpxA, the cytoplasmic response regulator CpxR and the periplasmic auxiliary CpxP protein. Cpx two-component system is involved in the process of bacterial adherence, invasion, motility and cell division. Previous results showed that S. typhimurium deleted cpxP gene is more invasive and explained first molecular mechanism of the structural basis for the Cpx-signalling-cascade inhibition by CpxP in E. Coli. In this project, the correlation between cpxP gene of Pectobacterium carotovorum subsp. carotovorum (PC1)　and its pathogenicity will be clarified by gene knockout; CpxP protein structure will be obtained by using gene reconstitution, protein expression, purification, crystallization and X-ray diffraction technique, and its function will be revealed by gene single-site-substitution and prediction basis on its structure; Cpx-signalling-cascade of PC1 will be studied by gene cloning, protein expression, purification and the interaction experiments between CpxRAP proteins in vivo and in vitro, which will provide a new sight of the study on invasion mechanism in gram-negative plant pathogens and provide a principle of effective prevention and remedy of these plant diseases.

Cpx信号转导系统是革兰氏阴性菌中普遍存在的双组分调控系统之一，由膜感应蛋白组氨酸激酶CpxA、胞质效应调节蛋白CpxR和周质辅助调节蛋白CpxP组成。Cpx双组分调控系统参与菌体的黏附、侵染、迁移和细胞分化等过程。申请人前期研究结果表明敲除掉cpxP基因的沙门氏菌更具有侵染性，并首次基于大肠杆菌CpxP蛋白空间结构诠释了其抑制Cpx途径信号级联的分子机制。本课题以植物病原菌细菌性软腐病菌为出发菌株，利用基因敲除方法明确cpxP基因与菌体致病性的关系；通过基因重组，蛋白表达、纯化、结晶和X射线衍射技术获得该菌CpxP蛋白空间结构，通过结构功能预测和原位替换等技术揭示CpxP蛋白的作用机理；利用基因克隆，蛋白表达纯化和CpxRAP蛋白体内体外相互作用等实验方法研究细菌性软腐病菌中的Cpx信号级联，从而为研究革兰氏阴性植物病原细菌的致病机制开辟新的途径，为该类植物病害的有效防治奠定理论基础。

细菌性软腐病菌（PC1）属于肠杆菌科家族成员，是一种会造成许多经济作物，如胡萝卜、番茄、马铃薯等获得细菌性软腐病的革兰氏阴性植物病原菌，目前仍找不到有效的方法防治此病。Cpx信号转导系统是革兰氏阴性菌中普遍存在的双组分调控系统之一，由膜感应蛋白组氨酸激酶CpxA、胞质效应调节蛋白CpxR和周质辅助调节蛋白CpxP组成。Cpx双组分调控系统参与菌体的黏附、侵染、迁移和细胞分化等过程。本课题以PC1为出发菌株，利用基因敲除方法发现了 cpxP 基因与菌体致病性的关系进而为研究革兰氏阴性植物病原细菌的致病机制开辟新的途径；通过体外基因重组技术获得高表达细菌性软腐病菌 CpxP 蛋白的重组子，并通过蛋白表达、纯化技术为其结晶和空间结构的解析建立了一种稳定制备高纯度该蛋白的方法，并获得了CpxP 蛋白的微晶体从而为研究细菌性软腐病菌 CpxP 的结构与功能及其参与的 Cpx 信号级联提供前期保障。