Abstract: The continued emergence of antibiotic resistance has led to a global health crisis, leading to a predicted 10 million deaths per year by 2050 without significant intervention. Targeting bacterial virulence determinants with novel antibiotics rather than essential proteins would limit resistance development. We will utilize two powerful model systems, Listeria monocytogenes infection in the Danio rerio (zebrafish) model, to identify bacterial virulence factors that will serve as novel targets for antibiotic development. L. monocytogenes is a human and livestock pathogen that is the causative agent of Listeriosis. Perhaps more importantly, it is also a well-studied, genetically tractable model pathogen that has been used for decades to dissect novel mechanisms of bacterial pathogenesis, innate immune activation and cell biology. The zebrafish is a similarly well studied model host organism with robust genetic tools, high fecundity and translucent larvae that make them ideal models for infectious disease and innate immunity studies. In this proposal we will identify novel antibiotic targets in L. monocytogenes using a high-throughput transposon insertion sequencing (TIS) negative selection approach. In aim 1 we will execute our L. monocytogenes TIS screen in macrophage cell culture, identifying genes that are required for bacterial invasion, phagosomal escape, and cytosolic survival and replication in a cell autonomous manner. In aim 2 we will repeat the TIS screen in intact zebrafish larvae, identifying genes required for infection in vivo. Comparing cell culture essential genes with those identified in vivo will highlight those genes necessary for infection in the context of an immune system. Finally, to fully exploit the genetic tractability and high throughput nature of the zebrafish, and to identify bacterial virulence determinants required for defense against specific components of the host immune response, we will perform TIS in transgenic zebrafish deficient in components of the phagocyte oxidase complex or the inflammasome complex. Upon completion of these aims we will have developed and executed a whole genome screen to identify novel virulence factors in L. monocytogenes required for cell intrinsic virulence, in vivo virulence, and virulence selectively necessary to combat reactive oxygen species and inflammasome activation. Future studies will expand on these proof of principle experiments to identify virulence factors required for other aspects of innate immunity (antimicrobial peptides, nutritional immunity, specific cytokine functions, etc) as well as utilizing the hits identified in our screens to screen for small molecule inhibitors as novel antibiotics.