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. ? ?
