PROJECT SUMMARY Rickettsiales are a diverse order of obligate, intracellular bacteria often transmitted by arthropods and pose a substantial threat to public health. Rickettsial organisms manipulate a wide array of host cell processes to gain entry and establish a replicative niche, while simultaneously avoiding host cell defenses. How rickettsial pathogens mediate this complex network of interactions is not well understood. A central feature of all rickettsial pathogens is the Type 4 Secretion System (T4SS), which secretes effector proteins into the cytosol and manipulates biological processes in the host cell. However, few T4SS translocated effectors have been identified or characterized from rickettsial organisms owing, in part, to the lack of genetic tools available among these bacteria. Their obligate intracellular nature has historically hindered genetic manipulation of Rickettsiales. Recently, we achieved targeted gene deletion in the rickettsial pathogen A. phagocytophilum using allelic exchange by homologous recombination. In the R61 portion of this proposal we will leverage this technique to add to the A. phagocytophilum genetic toolbox by developing 1) a T4SS effector translocation assay for use in the organism of study, 2) conditional expression systems, and 3) luminescent reporters. Using these tools in the R33 phase we will: 4) characterize the A. phagocytophilum T4SS effector repertoire, 5) evaluate the contributions of essential A. phagocytophilum effectors to infection, and 6) monitor the expression kinetics of effectors in vivo. The objectives of this proposal will expand the tractability of A. phagocytophilum and our understanding of the host-pathogen interactions it navigates during infection through elucidation to the T4 effector repertoire, and an understanding of how, when and where these genes are regulated. The impact of this proposal will extend to other rickettsial organisms, as these approaches can be adapted for use in related intracellular pathogens.