Molecular study of DksA as a smart switch central to Salmonella pathogenesis

说明(申请人提供)：沙门氏菌是一种革兰氏阴性致病菌，在世界范围内造成重大的发病率、死亡率和经济损失。沙门氏菌病是一种广泛的临床疾病，从胃肠炎到伤寒，每年导致14亿人患病和近400万人死亡。此外，沙门氏菌感染的治疗因多重耐药而日益复杂。为了引起疾病，病原菌，特别是沙门氏菌，必须感知、反应和限制由固有宿主防御施加的相当大的压力，包括伴随着宿主对感染的反应的氧化和亚硝化压力。本申请中提供的初步数据表明，细菌RNA聚合酶调节蛋白DksA是氧化和亚硝化应激的直接传感器。我们推测，Dks A的锌指形成了一种新型的氧化还原传感器，能够区分特定宿主衍生的活性氧和氮物种，并对其做出不同的反应，从而赋予沙门氏菌一种快速和可逆的机制来影响转录反应。这一“智能开关”概念将使Dks A能够将营养、氧化和亚硝化信号整合到一个协调的调节输出中，从而能够有效地定制细菌新陈代谢和防御计划，以最好地应对沙门氏菌在感染期间遇到的动态和有害的微环境。本文提出的研究将确定Dks A用来感知活性氧和氮物种的分子机制，以及这种调节蛋白在沙门氏菌致病性中所起的作用。在目标1中，生化分析和转录评估将确定反应活性物种的二硫键的形成，以及这种键在Dks A结构重排和调节功能变化中所起的作用。目的2将确定Dks A促进抗氧化和抗亚硝酸盐防御的分子机制；也将使用巨噬细胞和沙门氏菌病的小鼠模型来研究Dks A介导的氧化还原感觉和反应在沙门氏菌发病中的作用。总之，拟议的研究将描述Dks A以前未知的感觉角色，并极大地扩展我们对生物氧化还原传感器的理解，该传感器在不同的亲核和真核过程中介导多效性角色。这些研究还将对沙门氏菌的发病机制和传染病的分子机制产生新的见解。革兰氏阴性菌对dks A的保守表明，拟议的研究可能与一系列医学上重要的细菌有关。从机制上了解细菌的发病机制以及细菌感知、反应和限制宿主防御的过程将为确定独特的、广谱的治疗靶点提供重要基础，并有助于开发有效治疗由致病性、潜在的多重耐药细菌引起的感染的创新策略。

DESCRIPTION (provided by applicant): Salmonella is a Gram-negative pathogenic bacterium that causes significant morbidity, mortality, and economic loss worldwide. Salmonellosis represents a broad spectrum of clinical diseases, ranging from gastroenteritis to typhoid fever, and is responsible for 1.4 billion illnesses and nearly 4 million deaths annually. Additionally, th treatment of Salmonella infection is increasingly complicated by multidrug resistance. In order to cause disease, pathogenic bacteria in general and Salmonella in particular must sense, respond to, and limit the considerable stresses imposed by innate host defense, including oxidative and nitrosative stress that accompanies the host response to infection. Preliminary data presented in this application identifies the bacterial RNA polymerase regulatory protein DksA as a direct sensor of oxidative and nitrosative stress. We hypothesize that the Zn finger of DksA forms a novel redox sensor capable of distinguishing amongst, and differentially responding to, specific host-derived reactive oxygen and nitrogen species, thereby endowing Salmonella with a rapid and reversible mechanism to affect transcriptional responses. This "smart switch" concept would allow DksA to integrate nutritional, oxidative, and nitrosative signals into a coordinated regulatory output capable of effectively tailoring bacterial metabolism and defense programs to best address the dynamic and hostile microenvironments Salmonella encounters during infection. The investigations proposed herein will determine the molecular mechanism used by DksA to sense reactive oxygen and nitrogen species, and the role this regulatory protein plays in Salmonella pathogenicity. In Aim 1, biochemical analysis and transcriptional evaluation will define disulfide bond formation in response to reactive species, and the role this bonding plays in DksA structural rearrangement and changes in regulatory function. Aim 2 will determine the molecular mechanism by which DksA promotes antioxidative and antinitrosative defenses; the role of DksA-mediated redox sensing and response in Salmonella pathogenesis will also be examined using macrophages and a murine model of salmonellosis. Collectively, the proposed investigations will characterize a previously unknown sensory role for DksA and greatly expand our understanding of biological redox sensors that mediate pleiotropic roles in diverse pro- and eukaryotic processes. These studies will also yield novel insights into Salmonella pathogenesis and the molecular mechanisms of infectious diseases. Conservation of dksA by Gram-negative bacteria indicates the proposed investigations are likely relevant to a range of medically important bacteria. A mechanistic understanding of bacterial pathogenesis and the processes used by bacteria to sense, respond to, and limit host defense will provide an important foundation for the identification of unique, broad-spectrum therapeutic targets and aid in the development of innovative strategies for effectively treating infections caused by pathogenic, potentially multidrug-resistant bacteria.