二元信号调控系统PhoP/PhoQ对鼠伤寒沙门氏菌蛋白磷酸化的调节

Salmonella typhimurium, a bacterium capable of chronically hiding inside host cells, relies on two-component regulatory systems such as PhoP/PhoQ to sense host microenvironment and to regulate bacterial responses. Through systematic analysis of S. typhimurium phosphoproteome, we discovered that the phosphorylation sites identified from a PhoP-constitutive strain is about twice as much as those identified from a wild-type strain (83:49), suggesting that the downstream protein phosphorylation activities are more robust when PhoP/PhoQ is active. Subsequently, preliminary evidences to support such a claim were found through analysis of phosphoproteins from a PhoP-null strain. Since little is known regarding the role of protein phosphorylation in bacterial physiological and biochemical processes, and there is no report linking protein phosphorylation with PhoP/PhoQ regulation, we propose in this grant to systematically investigate PhoP/PhoQ-regulated protein phosphorylation events in S. typhimurium, and to study whether there are any common features in this regulation. We will also conduct in-depth studies on several functionally important phosphoproteins to investigate the roles of protein phosphorylation in microbial pathogenesis. This study not only will advance our understanding about PhoP/PhoQ regulatory network, but it will also shine lights on a potentially new concept in microbial pathogenesis.

可以长期藏身于宿主细胞中的鼠伤寒沙门氏菌依靠以PhoP/PhoQ为代表的二元信号系统去感应宿主微环境，并调控各种应答反应。通过系统分析鼠伤寒沙门氏菌磷酸化蛋白组，我们发现从PhoP-持续激活型菌株中可鉴定到的蛋白磷酸化位点数几乎是野生型菌株的两倍（83:49），推断PhoP/PhoQ激活后其下游的蛋白磷酸化反应更加活跃，并进而通过分析PhoP-缺陷型菌株初步验证了这个推断。由于蛋白质磷酸化在细菌生理生化活动中的作用目前尚不清楚，也从未见报道将蛋白磷酸化与细菌二元调控系统相连接，因此本项目拟通过定量磷酸化蛋白组学方法系统地研究有哪些鼠伤寒沙门氏菌蛋白磷酸化位点受PhoP/PhoQ调节，以及受调节的磷酸化蛋白的共性和规律。我们还将选择部分有重要生物学功能的受调控蛋白，研究磷酸化反应对细菌致病性的影响。本研究不仅将有助于对PhoP/PhoQ调控网络的认识，也将为细菌致病性机理提供一个新理念。

可以长期藏身于宿主细胞中的鼠伤寒沙门氏菌依靠以PhoP/PhoQ为代表的二元信号系统去感应宿主微环境，并调控各种应答反应。在本项目研究中，我们系统地分析了鼠伤寒沙门氏菌蛋白磷酸化位点，发现了分布于在224个蛋白中的337个磷酸化位点并探索了鼠伤寒沙门氏菌蛋白磷酸化是否与双组份系统PhoP/PhoQ调控相关联。我们发现PhoP和H-NS这两个重要的鼠伤寒沙门氏菌调控蛋白的磷酸化修饰对其生物学功能具有影响并深入探讨了分子机制。与此同时，我们发展了一种基于质谱技术的靶向蛋白相对定量技术用于研究细菌蛋白动态变化，并采用稳定同位素标记技术开展了鼠伤寒沙门氏菌蛋白在宿主细胞中的动态变化研究。