Summary: Bacterial pathogens require specific adaptations to survive and replicate in a host organism. There are many important bacterial pathogens that spend at least part of their infectious life cycle in the host cytosol. The mammalian cytosol is normally kept completely free of invading bacteria through innate and cell autonomous defenses. Despite the importance of this host-pathogen interface, we know little about what factors bacteria require to survive in this specially protected compartment. To uncover these factors, we use a model gram-positive cytosolic pathogen, Listeria monocytogenes. Two factors we have found which L. monocytogenes requires to survive in host cells and to cause disease are a eukaryotic-like Ser/Thr kinase, PrkA, and a protein of previously unknown function, GlmR (YvcK). Both proteins are widely conserved in other gram-positive species. In this work I have found that these proteins play important roles in maintaining the bacterial cell wall and that these roles are essential for bacterial virulence.