乙酰化修饰调节沙门菌双组份系统PhoP磷酸化的机制及意义

The two-component system PhoP/PhoQ is critical to bacterial resistance to stresses and pathogenicity. In a previous study, we found that the important two-component regulatory system transcriptional regulatory protein PhoP can be acetylated in Salmonella enterica serovar Typhimurium. This modification is mediated by acetyltransferase Pat. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP can be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. Recently, we found that PhoP can also be acetylated non-enzymatically at K102 by metabolic intermediate, acetyl phosphate (AcP), and its transcriptional activity changed accordingly. Notably, the acetylation of K102 may inhibit PhoP phosphorylation. Therefore, we propose that the crosstalk between acetylation of PhoP K102 and PhoP phosphorylation ensures Salmonella promptly respond to different stresses in host cells. To address this key question, this proposal is aimed to identify the law of dynamic change of K102 acetylation and reveal the molecular regulatory mechanism of acetylation on PhoP phosphorylation, and interaction between acetylation and phosphorylation on PhoP. The information derived from this application will provide a new perspective for understanding the pathogenesis of Salmonella.

双组份系统PhoP/PhoQ在细菌应对环境压力以及对宿主致病方面非常关键。我们报道了鼠伤寒沙门菌双组份系统转录调控因子PhoP的DNA结合域的赖氨酸残基存在乙酰基转移酶控制的乙酰化修饰，且乙酰化修饰抑制PhoP的DNA结合能力并减弱沙门菌的毒力。最近我们发现PhoP存在依赖细菌代谢中间产物乙酰磷酸(AcP)的赖氨酸乙酰化修饰。该修饰可抑制PhoP的激活关键步骤—磷酸化。因此，本项目拟采用体内和体外研究策略，研究不同条件(包括不同碳源培养基、小鼠/人巨噬细胞以及小鼠感染模型)下PhoP乙酰化修饰响应外界环境变化的规律，探索AcP对PhoP乙酰化修饰调节机制，研究乙酰化修饰与磷酸化修饰之间互作并调控PhoP活性的机制。本申请将有助于深入理解PhoP磷酸化调控机理，发现初级代谢调节致病性的内在原因，为认识沙门菌致病机制提供新的思路。

越来越多的证据表明代谢在细菌毒力中起着重要作用。乙酰磷酸（AcP）是磷酸转乙酰酶-乙酸激酶途径的中间代谢物，是大肠杆菌中主要的乙酰基供体。双组份系统PhoP/Q是一种重要的转录调控系统，对细菌毒力和应对外界应激压力至关重要。我们的研究发现在鼠伤寒沙门菌中显示PhoP可以被AcP以非酶学的方式乙酰化修饰，AcP修饰影响其转录活性。进一步研究证明第102位赖氨酸（K102）的乙酰化依赖于细胞内AcP。在PhoP激活条件下，包括低镁、酸胁迫或巨噬细胞吞噬后，K102的乙酰化水平降低。另外体外实验表明K102乙酰化影响PhoP磷酸化并抑制其转录活性。最后细胞和小鼠模型均显示K102对沙门菌毒力至关重要。综上所述我们的研究证实了代谢在细菌毒力中的重要性，并表明AcP可能是一个关键的分子。