小RNA对病原细菌中致病因子的表达调控研究

Bacterial infection tends to become more and severe recently. The pathogenicity of bacterial infection depends on the expression of virulent factors. Although some knowledge has become available regarding the major regulatory proteins of virulence factors, the role of small RNA molecules in virulent factor regulation is still poorly understood. In our previous work, we discovered that small RNA chaperon, Hfq, has regulatory roles in the expression of virulent factors. We also established a database for virulence factor-related small RNAs and their potential mRNA binding sites through high-throughput screening techniques in preliminary experiments. The present program will build on this database, verify the effect of small RNA on virulence factor expression, explore the molecular mechanisms underlying small RNA-based virulence factor regulation, and eventually validate the importance of the small RNA regulatory network on the expression of major virulence factors. The feasibility of our experimental design and approaches has been validated by our ability to show that a small RNA selected from the pre-established database exhibited regulatory effects on the expression of multiple virulence factors. Further work will involve knocking out and overexpressing small RNA genes in Enterohemorrhagic Escherichia coli (EHEC), followed by investigating the effects of these knockout and overexpression on the expression of Shiga toxin-Stx, H7 flagellum, Curli fimbriae, type 3 secretion system, and haemolysin using biochemical and molecular biological techniques. The outcome of our work will expand virulence factor regulation into a new territory and provide knowledge base and new targets for development of novel antimicrobials that suppress virulence factor expression.

细菌感染近年来有愈演愈烈的趋势，其致病力取决于致病因子的表达。虽然人们对其主要调控蛋白已经有了一定认识，但小RNA分子对细菌致病因子的表达调控研究尚不完善。申请人在前期工作中发现小RNA伴侣蛋白Hfq对细菌致病因子的表达有调控作用，并应用高通量技术筛选出与致病因子相关的小RNA及其在mRNA上可能的靶点。基于这个数据库，本项目将验证小RNA对细菌中致病因子表达的影响并研究其调控机制，阐明小RNA网络调控在细菌主要致病因子表达中的重要性。我们的预实验表明，选自该数据库中的小RNA对于细菌多个致病因子的表达有调控作用，证明相关实验设计合理可行。申请人拟通过在出血性大肠杆菌中敲除/过表达小RNA，应用生化/分子生物学等技术对细菌中志贺毒素、H7鞭毛、Curli菌毛、三型分泌系统和溶血素表达进行调查研究，拓展细菌毒力调控的新领域，为抑制细菌致病因子表达，筛选新型抗感染药物提供理论基础和新靶点。

细菌感染近年来有愈演愈烈的趋势，其致病力取决于致病因子的表达。虽然人们对其主要调控蛋白已经有了一定认识，但小 RNA 分子对细菌致病因子的表达调控研究尚不完善。我们发现小 RNA 伴侣蛋白 Hfq 对细菌致病因子的表达有调控作用，并应用高通量CRAC（cross-linking and analysis of cDNAs）技术筛选出与致病因子相关的小 RNA 及其在 mRNA 上可能的靶点。基于这个数据库，本项目筛选并验证了一系列小RNA以及相关应激条件对于细菌生长和致病因子表达的影响，并通过软件模拟和分子生物学手段验证了小 RNA在mRNA上的具体作用位点，确定其作用靶标，初步揭示其调控机制。我们通过一系列实验发现多个细菌小RNA及其相关应激条件（包膜刺激、营养条件、二甲亚砜）能改变细菌生长特性或特定致病因子（三型分泌系统、H7鞭毛、耐药性、Curli菌毛）表达水平；而且小RNA可以通过与三型分泌系统毒力岛上基因的mRNA靶点配对结合，对毒力基因的表达进行精细的瞬时调控。本项目中，我们获得了一系列应激条件下细菌的关键表型数据，并进一步通过小RNA缺失、突变或过表达，收集了其对于细菌毒力影响关键靶点数据。综上所述：本项目通过研究小RNA及其相关应激条件对于细菌毒力、生长相关基因表达调控的影响，使我们认识到，细菌的毒力或其他生理过程可以通过小RNA调控，使其表型发生改变，以应对快速变化的外界环境或应激条件；同时，该研究也探索了通过调节细菌外界环境，改变细菌毒力表达及其生存能力的可能性，为细菌感染的控制、预防提供了新策略、新思路。