Analysis of regulatory networks in Salmonella pathogenesis.

项目概要： 非伤寒沙门氏菌感染通常与健康人的腹泻有关。一些 血清型如鼠伤寒也是HIV感染者菌血症的常见原因， 在具有中性粒细胞缺陷的免疫功能低下个体中威胁播散性并发症， 巨噬细胞或CD 4 T细胞。传感器激酶及其同源反应调节剂在双组分 系统协调沙门氏菌和许多其它病原菌中的许多毒力程序。在 双组分系统的典型激活，传感器激酶响应于线索而磷酸化 在哺乳动物宿主的定殖和感染期间遇到的。磷酰基的转移 从感受器激酶到其同源反应调节因子的受体结构域， 对细菌致病至关重要。我们意外地发现双组分反应 调节剂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 表明硫氧还蛋白在免疫后控制几种反应调节因子，如OmpR，PhoP和 SsrB，所有这些都控制沙门氏菌致病的关键方面。引人注目的是， 硫氧还蛋白对双组分信号传导的作用不依赖于普遍保守的巯基-二硫键 氧化还原酶的酶活性，这一祖先的蛋白质，但取决于迄今未表征的 疏水性界面表面在细菌中硫氧还蛋白的整个进化过程中一直被保留， 古生菌和真核生物。我们的研究表明，硫氧还蛋白对沙门氏菌的大部分贡献 发病机制是独立的氧化还原酶活性，但进行了这一新发现的 界面表面这项拟议中的研究将测试硫氧还蛋白利用结合的假设， 保守的疏水斑块的属性，以建立具有多重响应的蛋白质-蛋白质相互作用 调节因子，从而对双组分信号传导进行广泛的翻译后控制。具体来说，我们将 鉴定介导硫氧还蛋白的氧化还原酶非依赖性功能的界面残基，并将 量化新型硫氧还蛋白结合面使反应调节剂能够激活沙门氏菌的程度 毒力计划我们的研究将阐明以前不受重视的因素，在调节两个- 组分信号，并将确定前所未有的，氧化还原酶独立的功能硫氧还蛋白。 关于硫氧还蛋白新功能的知识不仅将阐明 沙门氏菌的致病机制，但最终可能会扩大我们的理解的原始功能， 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 双组分系统，代表了细菌中的主导信号通路。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会为合理开发 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.