Analysis of regulatory networks in Salmonella pathogenesis.

项目总结： 在健康人中，非伤寒沙门氏菌感染经常与腹泻有关。一些人 像鼠伤寒杆菌这样的血清型也是艾滋病毒感染者菌血症的常见原因，而生命- 中性粒细胞缺陷的免疫受损个体可能出现播散性并发症， 巨噬细胞或CD4T细胞。双组分中的感受器蛋白及其同源反应调节因子 在沙门氏菌和许多其他致病细菌中，系统协调了许多毒力程序。在 双组分系统的典型激活，传感器激酶被磷酸化以响应提示 在哺乳动物宿主的定居和感染过程中遇到。磷酰基的转移 从传感器激酶到其同源反应调节器的接收域，启动毒力程序 对细菌致病是必不可少的。我们有一个意想不到的发现，即双组分反应 调节子由以前未知的与硫氧还蛋白的变构相互作用控制。我们的研究已经 研究表明，硫氧还蛋白翻译后控制着几种反应调节因子，如OmpR、Phop和 SSRB，所有这些都控制着沙门氏菌致病的关键方面。令人惊讶的是，后翻译控制 硫氧还蛋白对双组分信号的作用不依赖于普遍保守的硫醇二硫键 氧化还原酶这一祖先蛋白的酶活性，但取决于迄今尚未确定的 在细菌中硫氧还蛋白的整个进化过程中一直保存的疏水界面表面， 古生菌和真核生物。我们的调查表明，硫氧还蛋白对沙门氏菌的大部分贡献 其发病机制与其氧化还原酶活性无关，但由新发现的 界面表面。这项拟议的研究将检验硫氧还蛋白利用结合的假设 用于建立具有多种响应的蛋白质-蛋白质相互作用的保守疏水斑块的属性 调节器，从而对双组分信号施加广泛的翻译后控制。具体来说，我们将 确定介导硫氧还蛋白氧化还原酶非依赖性功能的界面残基，并将 量化新的硫氧还蛋白结合面使反应调节因子能够激活沙门氏菌的程度 毒力项目。我们的研究将阐明之前未被认识到的调节两个因素的因素- 成分信号转导，并将确定史无前例的氧化还原酶不依赖硫氧还蛋白的功能。 关于硫氧还蛋白新功能的知识不仅将有助于阐明 沙门氏菌的发病机制，但最终可能扩大我们对沙门氏菌原始功能的理解 普遍表达的硫氧还蛋白。我们的工作还将为监管提供深远的见解 二组分系统，这代表了细菌中的主要信号通路。药物特别是 抑制硫氧还蛋白和反应调节剂之间的相互作用可能有助于 下一代抗生素。

PROJECT SUMMARY: Nontyphoidal Salmonella infections are frequently associated with diarrhea in healthy people. Some serovars such as Typhimurium are also common causes of bacteremia in HIV-infected people, and life- threatening disseminated complications in immunocompromised individuals with defects in neutrophils, macrophages or CD4 T cells. Sensor kinases and their cognate response regulators in two-component systems orchestrate many virulence programs in Salmonella and many other pathogenic bacteria. In the canonical activation of two-component systems, the sensor kinase is phosphorylated in response to cues encountered during colonization and infection of the mammalian host. The transfer of the phosphoryl group from the sensor kinase to the receiver domain of its cognate response regulator turns on virulence programs essential for bacterial pathogenesis. We have made the unexpected discovery that two-component response regulators are controlled by previously unknown allosteric interactions with thioredoxin. Our research has shown that thioredoxin post-translationally controls several response regulators such as OmpR, PhoP and SsrB, all of which govern key aspects of Salmonella pathogenesis. Strikingly, the post-translational control exerted by thioredoxin on two-component signaling does not rely on the universally conserved thiol-disulfide oxidoreductase enzymatic activity of this ancestral protein, but is contingent upon a hitherto uncharacterized hydrophobic interfacial surface that has been preserved throughout the evolution of thioredoxin in bacteria, archaea and eukaryotes. Our investigations indicate that most contributions of thioredoxin to Salmonella pathogenesis are independent of its oxidoreductase activity but are carried out by this newly discovered interfacial surface. The proposed research will test the hypothesis that thioredoxin leverages the binding attributes of a conserved hydrophobic patch to establish protein-protein interactions with multiple response regulators, thereby exerting broad post-translational control of two-component signaling. Specifically, we will identify the interfacial residues that mediate oxidoreductase-independent functions of thioredoxin, and will quantify the extent that the novel thioredoxin-binding face enables response regulators to activate Salmonella virulence programs. Our research will elucidate previously unappreciated elements in the regulation of two- component signaling, and will ascertain unprecedented, oxidoreductase-independent functions of thioredoxin. The knowledge gained on the novel function of thioredoxin will not only shed light on key aspects of Salmonella pathogenesis, but may ultimately broaden our understanding of a primordial function of universally-expressed thioredoxin proteins. Our work will also provide far reaching insight into the regulation of two-component systems, which represent a dominant signaling pathway in bacteria. Drugs that specifically inhibit interactions between thioredoxin and response regulators may inform the rational development of the next generation of antibiotics.