Summary The Francisella constitute a genus of Gram-negative bacteria that occupy intracellular niches within a wide range of metazoan hosts. This group includes Francisella tularensis subsp. tularensis, a highly virulent human pathogen that has been classified as a Tier 1 select agent due to its low infectious dose, aerosol route of inoculation, and aggressive course of infection. Several studies have suggested that the key virulence factor of these bacteria is the Francisella pathogenicity island (FPI), which encodes a variant of the bacterial type VI secretion system (T6SS). However, the FPI is conserved among Francisella spp with diverse host ranges and variable capacity to cause disease. In preliminary data accompanying this proposal, we demonstrate that genes encoding substrate effector proteins of the FPI T6SS can be found at distal locations in the genome. We further find that although these effectors are variably present in Francisella spp, they play important, direct roles in promoting the virulence strategies of the bacteria that harbor them. These findings lead us to hypothesize that the FPI-encoded T6SS serves as a versatile platform for the delivery of diverse effector molecules encoded elsewhere within the genome. In our first aim we will define the molecular mechanism by which one T6SS effector of F. tularensis, OpiA, facilitates intracellular replication. Importantly, the mode of action of an FPI effector protein has not been identified to date. We show herein that OpiA is the founding member of a family of bacterial phosphatidylinositol kinases with representatives in diverse pathogens including Vibrio, Legionella, and Rickettsia spp. These data and additional preliminary findings suggest that OpiA acts by manipulating host cell membrane trafficking. In our second aim, we seek to identify the proteins required for targeting effectors to the T6SS in Francisella, as well as to define the mechanism by which the secretory apparatus and its substrates are coordinately regulated. Finally, in the third aim we propose to identify additional substrates of FPI-encoded T6SSs belonging to multiple F. tularensis subspecies and to characterize their contribution to the pathogenesis of F. tularensis subsp. tularensis. Collectively, the proposed research will provide insight into the virulence mechanisms of an important group of human and animal pathogens.