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

DksA 作为沙门氏菌发病机制核心智能开关的分子研究

• 批准号: 8716509
• 负责人: Matthew Anthony Crawford
• 金额: $ 5.43万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716509
• 关键词: Address; Affect; Americas; Area; Bacteria; Bacterial RNA; Biochemical; Biological; Biophysics; Cancer Biology; Cause of Death; Cessation of life; Clinical; Communicable Diseases; Cysteine; DNA Sequence Rearrangement; DNA-Directed RNA Polymerase; Data; Development; Diarrhea; Disease; Economics; Eubacterium; Evaluation; Event; Fingers; Foundations; Gastroenteritis; Gram-Negative Bacteria; Guanine; Health; Host Defense; Immune response; Infection; Investigation; Laboratories; Mediating; Metabolic; Metabolism; Modeling; Modification; Molecular; Morbidity - disease rate; Multi-Drug Resistance; Mus; NADP; Natural Immunity; Nitrogen; Nutrient; Nutritional; Output; Oxidases; Oxidation-Reduction; Oxidative Stress; Oxygen; Pathogenesis; Pathogenicity; Phagocytes; Play; Process; Proteins; Research; Role; Salmonella; Salmonella infections; Sensory; Signal Transduction; Source; Stress; Sulfhydryl Compounds; Testing; Transcriptional Regulation; Typhoid Fever; Vaccine Design; Virulence; base; cost; disulfide bond; experience; genetic regulatory protein; global health; human NOS2A protein; innovation; insight; macrophage; mortality; multidisciplinary; nitrosative stress; novel; pathogen; pathogenic bacteria; programs; public health relevance; response; sensor; therapeutic target

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.

描述（由申请人提供）：沙门氏菌是一种革兰氏阴性致病菌，在世界范围内造成显着的发病率、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，近 400 万人死亡。此外，由于多重耐药性，沙门氏菌感染的治疗变得越来越复杂。 为了引起疾病，病原菌（特别是沙门氏菌）必须感知、响应和限制先天宿主防御所施加的相当大的应激，包括伴随宿主对感染反应的氧化和亚硝化应激。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。我们假设 DksA 的 Zn 指形成一种新型氧化还原传感器，能够区分特定宿主来源的活性氧和氮物种并对其做出差异响应，从而赋予沙门氏菌快速且可逆的机制来影响转录响应。这种“智能开关”概念将使 DksA 将营养、氧化和亚硝化信号整合到协调的调节输出中，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期间遇到的动态和敌对微环境。本文提出的研究将确定 DksA 用于感知活性氧和氮物种的分子机制，以及该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定义响应活性物质的二硫键形成，以及该键在 DksA 结构重排和调节功能变化中所起的作用。目标 2 将确定 DksA 促进抗氧化和抗亚硝化防御的分子机制； DksA 介导的氧化还原感应和响应在沙门氏菌发病机制中的作用也将使用巨噬细胞和沙门氏菌病小鼠模型进行检查。 总的来说，所提出的研究将描述 DksA 以前未知的感觉作用，并极大地扩展我们对生物氧化还原传感器的理解，这些传感器在不同的原核和真核过程中介导多效性作用。这些研究还将对沙门氏菌发病机制和传染病的分子机制产生新的见解。革兰氏阴性细菌对 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。对细菌发病机制以及细菌感知、响应和限制宿主防御的过程的机械理解将为识别独特的广谱治疗靶点提供重要基础，并有助于制定有效治疗由致病性、潜在多重耐药性细菌引起的感染的创新策略。