Abstract: Bacterial pathogens require specific adaptations to survive and thrive in their replicative niche. A subset of important human pathogens, including Shigella Spp. Rickettsia spp and Burkholderia spp., utilize the cytosol of host cells as their primary replication niche. Although the host cell cytosol is an inhospitable environment for bacteria, the stresses encountered in this environment and the mechanisms pathogens utilize to overcome these stresses are not well understood. The goal of this proposal is to utilize the cytosolic pathogen Listeria monocytogenes (Lm) to understand how host cells recognize and respond to bacteria in the cytosol and in turn how cytosolic pathogens have adapted to life in the cytosol. Lm is a Gram positive foodborne pathogen that causes the rare but often fatal disease Listeriosis. To identify mechanisms by which Lm has adapted to survive and thrive in the mammalian cytosol, we have utilized a series of forward genetic screens to identify genes required for Lm cytosolic survival. These screens highlighted robust cell wall stress responses and specific metabolic adaptations as essential for cytosolic survival and virulence. Specifically, we demonstrated that the PASTA kinase PrkA is a master regulator of cell wall stress responses essential for cytosolic survival and virulence in vivo. We defined the phosphoproteome of PrkA and defined the role of one phosphosubstrate, ReoM, in regulating peptidoglycan synthesis in response to cell wall stress during infection. GlmR is another PrkA substrate required for cell wall stress responses, cytosolic survival and virulence in vivo and we defined its role as an accessory uridyltransferase. Finally, an additional gene of unknown function, yvcJ, was identified through our genetic screens and shown to be involved in cell wall stress responses, cytosolic survival and virulence. How the remaining PrkA dependent phosphoproteins coordinate cell wall stress responses, how phosphorylation of GlmR regulates its function, how YvcJ contributes to cell wall stress responses and finally the mechanism by which the host imparts cell wall stress on bacteria in the cytosol are all unknown. These questions are the focus of Aim 1. Our cytosolic survival screens also revealed multiple genes involved in central metabolism, notably the pyruvate dehydrogenase complex and genes required for DHNA biosynthesis. Mutants lacking these pathways secrete lactate instead of acetate as the major metabolic byproduct and interventions that restore acetate production rescue cytosolic survival of DHNA deficient strains. Additionally, we created a series of mutants missing genes essential for Lm glycerol utilization, hexose phosphate utilization, or both. As the only described carbon sources utilized by Lm during infection we were surprised to find minimal defects in ex vivo virulence and only 1-2 log reductions in virulence in mice. How increased concentrations of cytosolic lactate during Lm infection alter...