鼠伤寒沙门菌CadC响应宿主酸信号启动STM1344表达的分子机制研究

Salmonella typhimurium is an important foodborne pathogen. STM1344 is the key factor for Salmonella to adapt to the intracellular environment, but its expression regulation mechanism has not been elucidated. We found that acid signal can significantly increase the expression of STM1344, and further confirmed that acid signal activated transcription factor CadC can directly interact with STM1344 promoter. When Salmonella was cultured in LB medium, the transcription of stm1344 gene was inhibited by another protein H-NS. Therefore, we speculate that Salmonella senses the host acid signal and then reverses the inhibition of H-NS by CadC to initiate the expression of STM1344. This project intends to analyze the structure of related protein-DNA complexes, identify key sites of action, establish a molecular mechanism model in which CadC reverses H-NS inhibition to initiate STM1344 expression, and elucidates the molecular mechanism by which CadC responds to environmental acid signals to rapidly initiate STM1344 expression. To construct the corresponding mutant strains and to explore the role of CadC-mediated STM1344 expression regulation in the intracellular survival and pathogenicity of Salmonella at the level of cell and animal infection. This topic will fill in the gap of STM1344 initiation mechanism and provide an important theoretical basis for the study of intracellular survival mechanism of Salmonella.

鼠伤寒沙门菌是重要的食源性病原菌，STM1344是沙门菌适应宿主胞内环境的关键因子，但其表达调控机制未被阐明。课题组前期发现酸信号可显著提高STM1344的表达，并进一步确定酸信号激活型转录因子CadC可与STM1344启动子直接作用。而在体外生存的沙门菌中，stm1344基因的转录被另一蛋白H-NS抑制。因此我们推测沙门菌感受宿主酸信号后通过CadC逆转H-NS的抑制作用来启动STM1344的表达。本项目拟解析相关蛋白-DNA的复合物结构，确定关键作用位点，建立CadC逆转H-NS抑制作用启动STM1344表达的分子机制模型，阐明CadC响应环境酸信号快速启动STM1344表达的分子机理。构建相应突变菌株，在细胞和动物感染水平探讨CadC介导的STM1344表达调控在沙门菌胞内生存和致病过程中的作用。本课题将填补STM1344启动机制研究的空白，为沙门菌胞内生存机制研究提供重要理论基础。

鼠伤寒沙门菌是重要的食源性病原菌。当其进入宿主细胞后，为避免被宿主免疫系统识别，沙门菌的鞭毛合成会迅速关闭，然而具体的调控机制还未被阐明。在本研究中，我们证明了环境中的酸信号可以抑制沙门菌的鞭毛合成及其运动性，并且这一调控发生在主鞭毛基因flhDC之后，并依赖于调控因子YdiV。酸处理后，沙门菌YdiV的表达被激活。进一步用YdiV启动子DNA和酸处理后的沙门菌总蛋白进行DNA-pull down实验，筛选得到了一个新的YdiV调控因子-CadC，CadC为一酸相关转录因子。CadC DNA结合区CadCc与ydiV启动子的33bp区域直接结合，KD值为0.2μM。此外，CadC能在较低浓度下使H-NS-ydiV启动子DNA复合体解离，形成CadC-DNA复合体。结构模拟和突变分析表明，CadC的H43和W106是与ydiV启动子结合所必需的。与WT菌株相比，CadC敲除株或YdiV敲除株在酸性条件下没有观察到鞭毛抑制表型，这表明在酸适应过程中鞭毛的调控依赖于CadC和YdiV。细胞实验结果表明，与WT菌株相比，CadC敲除株的胞内生存能力显著下降，且不能有效抑制鞭毛蛋白的表达。综上所述，我们的研究表明，酸胁迫是一个重要的宿主信号，能够通过CadC-YdiV-FlhDC这一途径实现沙门菌鞭毛调控，使沙门菌感知恶劣的环境，并在感染期间抑制鞭毛的合成。