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

描述（由申请方提供）：沙门氏菌是一种革兰氏阴性致病菌，在全球范围内导致严重的发病率、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，每年造成14亿人患病，近400万人死亡。此外，沙门氏菌感染的治疗由于多药耐药性而日益复杂。 为了引起疾病，一般的病原菌和沙门氏菌特别是必须感测、响应和限制由先天宿主防御施加的相当大的压力，包括伴随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鉴定了细菌RNA聚合酶调节蛋白DksA作为氧化和亚硝化应激的直接传感器。我们假设，锌指DksA形成一种新的氧化还原传感器，能够区分，并差异响应，特定的主机衍生的活性氧和氮物种，从而赋予沙门氏菌的快速和可逆的机制，影响转录反应。这种“智能开关”的概念将允许DksA将营养，氧化和亚硝化信号整合到一个协调的调节输出中，能够有效地定制细菌代谢和防御程序，以最好地解决沙门氏菌在感染过程中遇到的动态和敌对的微环境。本文提出的研究将确定DksA用于检测活性氧和氮的分子机制，以及这种调节蛋白在沙门氏菌致病性中的作用。在目标1中，生化分析和转录评估将定义二硫键形成反应的物种，这种键合在DksA结构重排和调节功能的变化中发挥的作用。目的2将确定DksA促进抗氧化和抗亚硝化防御的分子机制; DksA介导的氧化还原传感和反应在沙门氏菌发病机制中的作用也将使用巨噬细胞和鼠沙门氏菌病模型进行研究。 总的来说，拟议的调查将表征一个以前未知的感觉作用DksA和大大扩展了我们的理解，生物氧化还原传感器介导多效性的作用，在不同的亲和真核过程。这些研究也将产生新的见解沙门氏菌的发病机制和感染性疾病的分子机制。革兰氏阴性菌对dksA的保护表明，拟议的研究可能与一系列医学上重要的细菌相关。对细菌发病机制和细菌用于感知、响应和限制宿主防御的过程的机械理解将为鉴定独特的广谱治疗靶标提供重要基础，并有助于开发有效治疗致病性、潜在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.