Project Summary Pathogenic bacteria have developed an array of strategies to undermine the host's defense mechanisms. In the context of non-Typhoidal Salmonella Typhimurium, a notable strategy involves the confinement of a single bacterium within a host vacuole called the Salmonella Containing Vacuole (SCV). This approach potentially provides an evolutionary advantage: by ensuring one bacterium per SCV, the host must target each SCV individually to eliminate the bacterial load, as opposed to confronting a vacuole harboring multiple bacteria clustered together. This mechanism could potentially extend the time required to combat the infection. Moreover, a single bacterium residing within an SCV exploits all accessible nutrients for replication and division. Conversely, multiple bacteria clustered within a single vacuole may engage in nutrient competition. Hence, understanding the fundamental mechanism of bacterial division in conjunction with vacuolar scission is imperative for gaining deeper insights into the pathogenic strategies employed by Salmonella enterica. Prior to this proposal we engineered a series of genetically minimal pathogenic strains that eliminates redundancy within the complex SPI-2 effector gene repertoire of Salmonella enterica serovar Typhimurium. Using this unique resource, here we will determine how a small network of SPI-2 T3SS effector proteins coordinate the complex events involved in SCV membrane scission and bacterial division within the host cell. This includes determining the location and host substrates of effector proteins at the SCV membrane using single cell particle tracking and live cell imaging (Aim 1). We will also investigate the molecular mechanisms of individual effector proteins that target a novel Rab-family GTPase defense system of the host (Aim 2). The resulting cellular and biochemical theories will be tested in murine models of systemic disease that are designed to evaluate effector protein functions at single cell resol