Phage/Salmonella interactions during oxidative stress - PROJECT ABSTRACT Nontyphoidal Salmonella are responsible for about 180 million cases of diarrhea and 300,000 deaths a year globally. Very young and very old people, cancer patients, immunocompromised individuals coinfected with HIV, and chronic granulomatous disease (CGD) patients bearing inactivating mutations in the phagocyte NADPH oxidase are some of the populations that are at high risk from suffering disseminated nontyphoidal Salmonella infections. The high susceptibility of CGD patients to disseminated nontyphoidal salmonellosis attest to the role reactive oxygen species produced in the respiratory burst of macrophages play in resistance to this facultative enteropathogen. We still have profound gaps in knowledge regarding the mechanisms by which reactive oxygen species mediate antimicrobial activity. Reactive oxygen species directly damage a variety of biomolecules. We have made the unprecedented discovey that reactive oxygen species produced by host phagocytic cells exert potent anti-Salmonella activity by inducing expression of the Gifsy-1 prophage terminase. The Gifsy-1 terminase gains unprecedented tRNase activity upon oxidation of cysteine residues forming a structural zinc finger. The oxidation of Gifsy-1 terminase results in the redox- dependent cleavage of the anticodon loop of tRNALeu. This is a very unexpected finding if we consider that the canonical functions of phage terminases are to cleave phage DNA and load the resulting single genomes into preformed viral capsids. Cumulatively, our data support the hypothesis that the oxidation of redox active cysteine residues in the zinc finger of the Gifsy-1 terminase stimulates the formation of an oligomer with tRNase activity, thus sensitizing Salmonella to the phagocyte NADPH oxidase. Aim 1 will characterize the mechanism by which gpA senses oxidative stress. Aim 2 will identify the mechanism by which oxidized gpA gains tRNase activity. Aim 3 will reveal the determinants responsible for the selective cleavage of tRNALeu by oxidized gpA. The knowledge generated using a combination of synergistic biophysics, biochemistry, bioinformatics, bacteriology, as well as bacterial and mouse genetics will illuminate key aspects of Salmonella pathogenesis, while providing a new framework for understanding the strategies used by bacteriophages to subvert their bacterial hosts during the mammalian immune response. Terminases are highly conserved in evolutionarily distant phages and herpesviruses. Hence, the knowledge gained in our investigations may be applicable to phylogenetically diverse prokaryotic and human viruses.
