The goal of the research plan outlined in this proposal is to understand how two bacterial pathogens, Rickettsia parkeri and Listeria monocytogenes, hijack host pathways of intercellular communication to promote spread; a process that is crucial for their virulence. These pathogens live in the host cell cytosol and spread from cell to cell by mobilizing the host’s actin cytoskeleton for intracellular motility and transport to the plasma membrane. Once at the cell periphery, they induce the formation of membrane protrusions that are engulfed by neighboring cells. Despite recent advances, we lack critical information about how these stages of spread occur. Based on my preliminary work, this proposal will investigate the specific hypothesis that bacterial pathogens promote spread by hijacking proteins normally involved in cell adhesion, membrane remodeling, and endocytosis; processes critically involved in intercellular communication. Furthermore, I hypothesize that distinct host pathways are hijacked by each pathogen. I propose the following experiments to test these hypotheses. In Aim 1, I will identify which endocytic, cell adhesion, and membrane remodeling proteins specifically promote spread and their mechanisms of action. To provide further mechanistic insight, in Aim 2 I will utilize live cell imaging to determine the dynamics of host protein recruitment, and super-resolution imaging to define the subcellular architecture of those host proteins studied in Aim 1. This will reveal the order in which host proteins are recruited and their spatial organization during spread, providing key insights into their mechanisms of action. Finally, to examine how bacterial effector proteins hijack host factors to promote spread, in Aim 3 I will follow-up on the exciting discovery that the R. parkeri secreted effector Sca4 inhibits the cell adhesion protein vinculin to promote spread. I will investigate how and when Sca4 is secreted and where it targets vinculin in the host cell. As a whole, this work will delineate how bacterial effectors and host pathways cooperate to enable spread, and reveal similarities and differences in the molecular strategies of spread for diverse pathogens. Discerning the targets of spread should also improve our understanding of basic cellular mechanisms, like vesicular traffic and intercellular communication, and how these go awry in disease.