乙酰化修饰调控氨同化的机制及对沙门氏菌致病性的影响

The metabolism of ammonia assimilation can provide the ammonia moiety for the amino acid synthesis, by balancing the level of glutamate and glutamine for the regulation of physiology and expression of the virulent proteins, and then having effect on the virulence of the Salmonella. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS) are the key metabolic enzymes responsible for this process, and the adenylylation modification is the well-known protein translational modification for the GS and further ammonia assimilation regulation. We firstly reported that the acetylation is a conserved and global regulation mechanism from prokaryotes to eukaryotes. In preliminary study, we found that the enzymatic activities of GDH and GS are regulated by acetylation. However, the mechanism of how the acetylation modulates the enzymatic activities of GDH and GS for the ammonia assimilation and then regulates Salmonella virulence is not clear. In this study, we will uncover the molecular mechanism how the acetylation coordinating the enzymatic activities of GDH and GS, and discover the crosstalk mechanism between the acetylation and the adenylylation, and then demonstrate the role of acetylation in regulating Salmonella ammonia assimilation for its physiology and virulence. If it is well done, we can expand the basic theory how acetylation in participating in regulation of microbial metabolism, which can provide the new concept of Salmonella virulence and the new targets for the Salmonella-mediated diseases in clinic.

氨同化为氨基酸合成代谢提供氨基供体，通过调节胞内谷氨酸和谷氨酰胺水平影响沙门氏菌的生理及病理。谷氨酸脱氢酶（Glutamate dehydrogenase, GDH）和谷氨酰胺合成酶（Glutamine synthetase, GS）是氨同化关键酶，腺苷酰化修饰调控GS酶活力。申请者在国际上首次报道蛋白的乙酰化修饰是一种保守和全局的代谢调控机制，且预实验结果表明乙酰化可以改变GDH和GS的酶活力，但乙酰化的GDH和GS如何参与氨同化，并进而调控沙门氏菌致病性及其相关机理尚不清楚。基于此，本研究拟解析乙酰化调控沙门氏菌GDH和GS酶活力的机制，揭示GS乙酰化修饰和腺苷酰化修饰的相互作用模式，并阐明乙酰化修饰调控沙门氏菌氨同化的生理和病理意义。项目研究成果有望进一步完善乙酰化参与微生物代谢调控的基础理论，为沙门氏菌的致病性提供新的作用机制，也将为沙门氏菌感染导致的临床相关疾病的干预提供新靶点。

本研究对乙酰化如何协同调控氨同化和碳代谢进行了研究。以氨同化关键酶GS和GDH为切入点，首先确定GS和GDH是被乙酰化修饰调控的，然后确定其乙酰化的上游信号，并对其乙酰化修饰酶进行确认；然后对乙酰化与GS上的腺苷酰化相互作用进行了深入研究，发现乙酰化可以激活被腺苷酰化失活的GS而不能激活没有被腺苷酰化失活的GS；通过分子模拟技术，在结构水平对乙酰化调控GS和GDH的分子机制进行了探讨，发现乙酰化可以使腺苷酰化的GS发生别构效应，从而使底物分子更容易进入到活性中心，进而激活被腺苷酰化失活的GS，相比较而言GDH的K128的乙酰化则封闭了底物分子进入活性中心的通道，从而抑制了GDH的活力。进一步从葡萄糖响应实验、生长表型互补实验和比活力实验三方面对乙酰化协同调控氨同化和碳代谢的生理意义进行了研究；并利用遗传敲除技术，构建乙酰化突变的菌株感染小鼠，评估小鼠粪便中的活菌数和小鼠的体重下降，研究乙酰化在沙门氏菌的致病过程中发挥重要的作用。