Mechanisms of Nitric Oxide Cytotoxicity in Salmonellosis

Nitric oxide (NO) is a biological mediator with remarkably broad-spectrum antimicrobial activity NO can exist in different redox forms, each with distinctive stability, reactivity, and biological actions. Although NO has been investigated in numerous animal models of infection, the effects of specific redox forms of NO on microbes have not been extensively investigated at a molecular level. This proposal analyzes the molecular mechanism and biological relevance of NO antimicrobial activity in a murine salmonellosis model. Preliminary data are presented suggesting that S-nitrosothiols are important endogenous antimicrobial redox forms of NO, and that microbial homocysteine antagonizes the cytostatic effects of S-nitrosothiols. The specific aims of this proposal are the following: A) Identification of Salmonella genes which determine susceptibility to different redox forms of nitric oxide in vitro; B) Identification of nitric oxide-regulated Salmonella genes; C) Correlation of Salmonella susceptibility to in vitro nitric oxide donors with susceptibility to cell-derived nitric oxide and virulence. S-nitrosoglutathione, SIN-1, and DETA/NO will be used as donors of nitrosonium (NO+), peroxynitrite (OONO-), and NO radical (NO-), respectively. Bacterial genes which influence NO susceptibility or whose regulation is influenced by NO will be mutated, mapped, cloned, sequenced, and complemented. In preliminary work, a considerable number of isogenic mutants with altered susceptibility to specific redox forms of NO have already been identified and characterized. The susceptibility of mutants to cell-derived NO and virulence of these strains in mice will be examined to define the biological significance of specific redox forms of NO. Genetic manipulation of intracellular homocysteine levels and the use of homocysteine-regulated gene fusions will be employed to test the provocative thesis that microbial homocysteine synthesis antagonizes the intraphagosomal antimicrobial activity of NO. Although this project exploits the advanced knowledge of Salmonella genetics in utilizing this specific model system, the implications of this work are by no means limited to salmonellosis. The preliminary findings have already identified novel pathways used by endogenous antimicrobial mediators, and novel mechanisms of microbial resistance to these mediators. The proposed studies will provide further insights into the molecular basis of NO cytotoxicity and the potential importance of this activity in a wide range of infections, particularly those caused by intracellular pathogens.