DNA DAMAGE AND REPAIR IN SALMONELLA PATHOGENESIS

沙门氏菌发病机制中的 DNA 损伤和修复

• 批准号: 6574877
• 负责人: Ferric C Fang
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6574877
• 关键词: DNA binding protein; DNA damage; DNA repair; DNA replication; NAD(P)H dehydrogenase; SDS polyacrylamide gel electrophoresis; Salmonella infections; bacterial DNA; host organism interaction; immunoprecipitation; laboratory mouse; matrix assisted laser desorption ionization; metalloproteins; nitric oxide synthase; polymerase chain reaction; site directed mutagenesis

DESCRIPTION (provided by applicant): Phagocytic cells inhibit microbes through production of genotoxic reactive oxygen and nitrogen species (ROS/RNS) produced by the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS). Salmonella typhimurium must repair DNA damage to resist killing by phagocyte-derived ROS/RNS and cause lethal infection in mice. The specific aims of this project are to: A) Determine effects of ROS/RNS on DNA repair-deficient Salmonella in vitro; B) Characterize DNA damage and essential DNA repair mechanisms during Salmonella infection in vivo; C) Identify specific DNA-binding zinc metalloproteins targeted by RNS/ROS. Preliminary observations suggest the hypothesis that inhibition of DNA replication is the final common pathway of DNA damage during infection. Replication arrest can be caused by multiple mechanisms including blocking lesions, strand breaks, nucleotide depletion or inhibition of the primosome apparatus required to restart collapsed replication forks. In the absence of the RecBC repair proteins, replication arrest can result in lethal double-strand breaks. Mobilization of zinc by RNS strongly correlates with cytostasis in vitro, suggesting that RNS inhibit DNA replication by targeting DNA-binding zinc metalloproteins. To test the central hypothesis of this proposal, strains of S. typhimurium deficient in excision repair, homologous recombination, or translation DNA synthesis will be constructed and examined for susceptibility to ROS/RNS. Measurement of DNA synthesis, strand breaks, and mutagenesis will clarify mechanisms of ROS/RNS-mediated DNA damage. Wild-type and congenic phox/iNOS knock-out macrophages and mice will be used to identify repair mechanisms required for Salmonella virulence and characterize host-derived mediators responsible for DNA damage and replication arrest during host-pathogen interactions in vivo. Biochemical strategies and site-specific mutagenesis will be utilized to identify zinc metalloproteins modified by RNS. These studies will provide novel insights into mechanisms by which innate host defenses limit microbial replication by targeting DNA synthesis and establish critical mechanisms of microbial resistance to ROS/RNS-related DNA damage.

描述（由申请人提供）：吞噬细胞通过 NADPH 吞噬细胞氧化酶 (phox) 和诱导型一氧化氮合酶 (iNOS) 产生基因毒性活性氧和氮物质 (ROS/RNS) 来抑制微生物。鼠伤寒沙门氏菌必须修复 DNA 损伤，以抵抗吞噬细胞衍生的 ROS/RNS 的杀伤，并导致小鼠致命感染。该项目的具体目标是： A) 体外确定 ROS/RNS 对 DNA 修复缺陷沙门氏菌的影响； B) 表征体内沙门氏菌感染期间的 DNA 损伤和必需的 DNA 修复机制； C) 鉴定 RNS/ROS 靶向的特定 DNA 结合锌金属蛋白。初步观察表明，抑制 DNA 复制是感染过程中 DNA 损伤的最终共同途径。复制停滞可由多种机制引起，包括阻断损伤、链断裂、核苷酸耗尽或重新启动折叠的复制叉所需的引发体装置的抑制。在缺乏 RecBC 修复蛋白的情况下，复制停滞可能导致致命的双链断裂。 RNS 动员锌与体外细胞停滞密切相关，表明 RNS 通过靶向 DNA 结合锌金属蛋白来抑制 DNA 复制。为了检验该提案的中心假设，将构建缺乏切除修复、同源重组或翻译 DNA 合成的鼠伤寒沙门氏菌菌株，并检查其对 ROS/RNS 的敏感性。 DNA 合成、链断裂和诱变的测量将阐明 ROS/RNS 介导的 DNA 损伤的机制。野生型和同类 phox/iNOS 敲除巨噬细胞和小鼠将用于识别沙门氏菌毒力所需的修复机制，并表征在体内宿主与病原体相互作用期间负责 DNA 损伤和复制停滞的宿主衍生介质。将利用生化策略和位点特异性诱变来鉴定经 RNS 修饰的锌金属蛋白。这些研究将为先天宿主防御通过靶向 DNA 合成来限制微生物复制的机制提供新的见解，并建立微生物抵抗 ROS/RNS 相关 DNA 损伤的关键机制。