鼠伤寒沙门菌STM1344调控鞭毛合成及免疫逃逸的分子机制研究

Salmonella typhimurium is the main pathogen of foodborne illness. Its flagellin can be recognized by host immune system and cause a serious immune response. After invasion, Salmonella shut down its flagellum synthesis and achieve immune escape. However, the regulatory mechanism of this process remains unclear. At the top of flagellum synthesis pathway is FlhDC operon who controls the expression of all other flagellar proteins by recruiting RNA polymerase to their DNA. In Salmonella, there are two proteins, STM1344 and STM1697, regulate flagellum synthesis through interaction with FlhD, however, with different mechanisms: STM1697 inhibits RNA polymerase recruitment of FlhDC but does not affect its DNA-binding activity, while STM1344 directly destroys the DNA binding ability of FlhDC. In this project, we will try to determine the crystal structure of STM1344-FlhD complex, combining with biochemical experiments, such as EMSA and SEC, we will answer how STM1344 inhibits the DNA-binding activity of FlhDC. Further in vitro and in vivo studies will reveal the important role of STM1344-mediated flagellum control in the process of Salmonella infection. Our research will expound the molecular mechanism by which STM1344 regulates flagellum synthesis to achieve immune escape and provide a theoretical basis for pathogenetic mechanism of Salmonella infection.

鼠伤寒沙门菌是引起食物中毒的重要病原菌。其鞭毛蛋白是宿主免疫识别的重要抗原，入侵后沙门菌迅速关闭其合成实现免疫逃逸，但该过程的调控机制还不明确。鞭毛合成通路最上游的FlhDC蛋白是鞭毛调控的最关键靶点，沙门菌中存在STM1344和STM1697两种蛋白均通过与其D亚基互作调控鞭毛合成。课题组前期研究表明两者的作用机制差异显著：STM1697抑制FlhDC招募RNA聚合酶但不影响其与DNA结合，而STM1344却直接破坏FlhDC的DNA结合能力。本项目拟在前期获得STM1697-FlhD结构的基础上解析STM1344-FlhD结构，结合EMSA、SEC等生化实验解答STM1344如何影响FlhDC与DNA结合，并利用体外、细胞和动物实验探讨STM1344介导的鞭毛调控在致病过程的作用。研究结果将系统完整地阐明STM1344调控鞭毛合成、介导免疫逃逸的分子机制，为其致病机制研究提供理论基础。

鼠伤寒沙门菌是引起人和动物食物中毒的重要病原菌，它依靠周身长有的鞭毛，通过运动来趋利避害。其鞭毛还与致病能力密切相关。研究发现，进入宿主细胞后野生沙门菌可迅速关闭鞭毛合成实现免疫逃逸，而持续表达鞭毛的沙门菌会迅速被宿主识别并杀灭。因此，沙门菌的鞭毛合成调控在其致病过程中发挥重要的作用。. flhDC操纵子编码的FlhD4C2复合体是最关键的鞭毛合成调控位点。沙门菌中STM1344和STM1697蛋白同属EAL家族，均与FlhD亚基相互作用负调控鞭毛合成。在基因组容量有限的情况下，沙门菌进化出STM1344和STM1697两种蛋白对同一位点进行调控，预示着该调控过程具有重要意义。本课题组之前报道了沙门菌STM1697通过影响RNA聚合酶招募来抑制下游鞭毛基因的转录从而实现负调控鞭毛合成。本研究结果则详细揭示了STM1344在沙门菌鞭毛合成调控中的作用机制。. 沙门菌入侵宿主细胞后，迅速启动关闭鞭毛合成通路，STM1344基因转录水平迅速升高，6h后可达几百倍甚至上千倍，并且STM1344发挥了对入侵后鞭毛蛋白基因转录的抑制作用。蛋白组学结果也验证了STM1344对鞭毛合成的调控最终导致了各级鞭毛蛋白的减少。与STM1697不同，STM1344对鞭毛通路的调控存在浓度效应，在低摩尔浓度时（STM1344：FlhD4C2≤2：1）与STM1697作用相仿，通过抑制FlhD4C2招募RNA聚合酶暂时关闭下游鞭毛基因的转录和鞭毛蛋白的合成；而高摩尔浓度下（STM1344：FlhD4C2＞2：1）可直接破坏FlhD4C2六元环结构，导致其丧失与目的DNA结合的能力，造成下游鞭毛基因转录停滞，从而持久地关闭鞭毛合成途径，实现免疫逃逸。本课题的研究结果为沙门菌基于FlhD4C2蛋白的复杂的鞭毛调控机制提供了更全面更深刻的认识和理解。