DNA DAMAGE AND REPAIR IN SALMONELLA PATHOGENESIS

描述（由申请人提供）：吞噬细胞通过产生由NADPH吞噬细胞氧化酶（phox）和诱导型一氧化氮合酶（iNOS）产生的遗传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的杀伤并在小鼠中引起致死性感染。本项目的具体目标是：A）确定ROS/RNS对DNA修复缺陷沙门氏菌的体外作用; B）表征沙门氏菌体内感染过程中的DNA损伤和必要的DNA修复机制; C）鉴定RNS/ROS靶向的特异性DNA结合锌金属蛋白。初步观察表明，DNA复制的抑制是感染过程中DNA损伤的最终共同途径。复制停滞可以由多种机制引起，包括阻断损伤、链断裂、核苷酸耗尽或抑制重启折叠的复制叉所需的启动体装置。在没有RecBC修复蛋白的情况下，复制停滞可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结合锌金属蛋白抑制DNA复制。为了验证这一假设的核心，S。将构建切除修复、同源重组或翻译DNA合成缺陷的鼠伤寒沙门氏菌，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。野生型和同源phox/iNOS敲除巨噬细胞和小鼠将用于鉴定沙门氏菌毒力所需的修复机制，并表征宿主-病原体体内相互作用期间负责DNA损伤和复制停滞的宿主衍生介质。生化策略和定点突变将被用来确定锌金属蛋白修饰的RNS。这些研究将为先天宿主防御通过靶向DNA合成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。

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.