C-di-GMP激活效应蛋白YcgR调控细菌鞭毛运动的结构基础和分子机制研究

Bacterial biofilm (BBF) is the sessile state in bacterial lifestyle and have been reported to experience in increased tolerance to the severe environment, antibiotic challenge and immune phagocytosis. About 80% chronic microbial infection and many problems in industrial processes are due to the biofilms. Therefore, it is very critical to explore the mechanisms of bacterial biofilm formation and dispersal for preventing the problems. C-di-GMP, as one of the most common and important bacterial second messengers, can control the motile-sessile transition. The increased concentrations of c-di-GMP promote surface attachment and the formation of biofilms. In the initial attachment process, the PilZ domain-containing protein YcgR in Escherichia coli and its homologs in Salmonella enterica have been shown to bind to c-di-GMP to inhibit the flagellar motility with interacting with flagellar motor proteins directly. But there remains controversy about the target proteins which the c-di-GMP bound to YcgR interacts with, and the molecular mechanisms of how the binding of YcgR to c-di-GMP can regulate the flagellar motility are poorly understood. To data, we have solved the crystal structure of the complex of YcgR-c-di-GMP and the stator protein MotA have been determined as the target protein in vitro. On this basis, the complex between YcgR-c-di-GMP and MotA would be constructed and the atomic structure would be determined. The interaction details from the structure combined with the investigations of the interaction between YcgR-c-di-GMP and MotA in vivo and vitro would illustrate the molecular mechanisms of YcgR binding to c-di-GMP interferes with the cell motility in the beginning of biofilm formation. The new method of inhibiting the biofilm formation would be further explored based on this regulation mechanisms.

细菌生物被膜作为细菌的固着生存形态使可其对环境、抗生素、免疫吞噬等产生极强的耐受性，引起细菌慢性感染、工业污染等问题。深入研究细菌生物被膜的形成、降解机制对解决生物被膜带来的问题有非常重要的理论指导意义。在生物被膜形成和降解的过程中，细菌最普遍和最重要的第二信使c-di-GMP全程参与。其中，在起始粘附阶段，含有PilZ domain的蛋白YcgR结合c-di-GMP后可通过直接作用于鞭毛马达蛋白来抑制鞭毛运动，使细菌停留在表面上，但具体作用的马达蛋白和分子机制尚不明确。本项目拟在已解析YcgR结合c-di-GMP晶体结构和已确定作用马达蛋白为定子蛋白MotA基础上，构建YcgR-c-di-GMP与MotA的复合物，解析复合物的晶体结构，并结合体内和体外相互作用分析，阐明c-di-GMP激活YcgR后在生物被膜形成早期调控鞭毛运动的分子机制，为利用这一调控来抑制生物被膜的形成提供思路和理论基础。

细菌生物被膜作为细菌的固着生存形态可使其对环境、抗生素、免疫吞噬等产生极强耐受性，造成慢性感染、工业污染等问题。深入研究细菌生物被膜的形成、降解机制对防治生物被膜带来的问题有非常重要的理论指导意义。细菌中特有的第二信使分子c-di-GMP全程调控了细菌“游离”和“固着”的转换。其中在起始粘附阶段，含有PilZ domain的蛋白YcgR结合c-di-GMP后可通过直接作用于鞭毛马达蛋白来抑制鞭毛运动，使细菌停留在表面上，进而促进生物被膜的形成。本项目围绕c-di-GMP激活YcgR作用于鞭毛马达蛋白的分子机制这一科学问题开展研究。我们发现c-di-GMP激活YcgR后可按照1:4的比例稳定的结合在鞭毛定子蛋白MotA上，并利用结构生物学和相关功能实验阐明c-di-GMP的结合使YcgR的构象发生很大改变，使位于PilZ domain上的RxxxR motif和C-末端的α-helix形成MotA的结合区，且YcgR结合在MotA与转子蛋白FliG发生相互作用的区域。但与MotA的结合是不足以完成YcgR对细菌鞭毛运动的调控的，YcgR-N domain极有可能通过与转子蛋白的作用参与了这一调控。基于此，我们提出c-di-GMP的结合使YcgR通过其PilZ domian牢牢结合在定子蛋白MotA与转子蛋白FliG的作用面上，同时通过其YcgR-N domain作用在其他蛋白（FliG最有可能）上，从而实现对细菌鞭毛运动的调控。该机制的阐明可为利用这一调控来防治生物被膜的形成提供思路和理论基础，并为探究PilZ类蛋白结合c-di-GMP后行使不同生物学功能的分子机理提供新的线索。