甲基化修饰调控沙门菌双组份系统PhoP-PhoQ的机制研究

The two-component regulatory system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. When PhoQ senses the environmental signals such as low concentration of magnesium ion or acidic pH produced by neutrophils and macrophages, it is activated by autophosphorylation. Subsequently, the phosphoryl of PhoQ is transferred to the conserved glutamic acid of PhoP (D52), and then the phosphorylated PhoP activates the transcription of phoP itself and PhoP-regulated genes. Recently, our mass spectrometry analysis showed the existence of methylation on PhoP. The further results showed that the transcription of phoP and PhoP-regulated genes decreased dramatically in the chromosome mutant E8A, in which glutamic acid residue 8 was substituted by alanine under low magnesium condition. Moreover, the mutant strain also was attenuated in the growth and cell viability. Therefore, we speculate that methylation may be involved in the regulation of PhoP activities. To test this speculation, we will 1) identify the methyltransferase(s) involved in PhoP methylation; 2) explore the mechanism behind PhoP activities regulated by methylation; 3) uncover the relationship between PhoP methylation and the Salmonella virulence. The information derived from this application will provide a new perspective for understanding the pathogenesis of Salmonella.

原核生物保守的双组份调控系统PhoP-PhoQ对病原菌的毒力至关重要，其转录调节因子PhoP的第52位天冬氨酸(D52)的磷酸化为PhoP激活关键步骤。最近，我们利用质谱技术发现PhoP还存在谷氨酸甲基化修饰。在低浓度镁离子环境下，甲基化修饰位点的突变株的phoP以及受PhoP调控基因的转录显著下调。同时低浓度镁离子环境下突变株的生长速度和存活率也出现明显下降，因此我们推测甲基化修饰可调控PhoP活性。为此，本课题拟采用细菌染色体基因敲除和敲入、同位素标记、巨噬细胞以及小鼠动物模型等手段，发现PhoP对应的甲基转移酶，研究PhoP甲基化修饰变化规律和调控PhoP活性的机制，揭示甲基化修饰与沙门菌毒力的关系。本申请将有助于深入理解PhoP活性调控机理，为认识沙门菌致病机制提供新的思路。

双组份调控系统PhoP-PhoQ对鼠伤寒沙门氏菌的毒力至关重要。其转录调节因子PhoP的第52位天冬氨酸(D52)的磷酸化为PhoP激活关键步骤。本项目中，我们发现PhoP存在甲基化修饰，PhoP的第八位谷氨酸和第112位精氨酸可以被甲基化修饰，这两个氨基酸位点对于PhoP的磷酸化，二聚体结构以及DNA的结合非常的关键，突变以后会显著降低沙门菌在巨噬细胞内的存活能力以及感染小鼠的能力。在沙门菌遭遇低浓度镁离子环境、低pH环境或者被巨噬细胞吞噬以后，E8和R112的甲基化水平会迅速下降。此外我们还鉴定出参与参与调控细菌趋化性的甲基转移酶CheR可以对R112进行甲基化修饰，过表达cheR可以抑制PhoP的降解从而导致PhoP蛋白积累。总的来说，本项目发现.甲基化在调节PhoP活性以响应环境变化方面起着重要作用，并且可以调节沙门氏菌的毒性。