Macrophages normally engulf and kill bacteria by producing reactive oxygen species (ROS) and other antimicrobial substances. Salmonella Typhimurium is a major food-borne pathogen capable of surviving within macrophages. The periplasmic superoxide dismutase, SodCI, directly and specifically protects Salmonella against the primary phagocytic ROS, superoxide. But the mechanism by which phagocytic superoxide damages or kills bacteria is unknown. Dogma states that bacterial DNA is a primary target of oxidative damage in the macrophage, consistent with known effects of ROS produced endogenously in the bacterial cytoplasm. Our recent results have led to a paradigm shift. We have shown that, contrary to dogma, the targets of the phagocytic oxidative burst are not the bacterial DNA or other cytoplasmic molecules. Rather, phagocytic superoxide damages an extracytoplasmic bacterial target. Our goal is to understand the physiological basis of bacterial sensitivity to phagocytic superoxide and the mechanisms by which pathogenic bacteria protect against this innate immune response. Novel genetic and biochemical approaches are proposed to study the physiological roles of phagocytic superoxide. We hypothesize that by identifying genes that genetically interact with sodCI, we will gain insight into the mechanism(s) by which superoxide damages the bacterial cell and how Salmonella resists this damage. In Aim 1, a variation on a high- throughput transposon mutagenesis technique that we call "differential TnSeq" will be used to identify genes that show synthetic, epistatic, or suppressive genetic interaction with sodCI. In Aim 2, the identified genes will be further characterized using in vivo and in vitro assays. To facilitate these studies, we have developed a novel in vitro device that, for the first time, allows the production of superoxide in the laboratory at levels that mimic thos produced in the phagocyte. These innovative approaches, along with the co-investigators' combined expertise in bacterial genetics, pathogenesis, physiology, and biochemistry of oxidative stress, make us uniquely qualified to address this fundamental aspect of innate immunity. Completion of the specific aims will increase our ability to prevent and treat, not only Salmonella infection, but also diseases caused by other pathogens.