Coxiella burnetii actively express effectors that likely remodel the host cell for replication and are essential for pathogenesis are likely type 4 secretion system-dependent (T4SS), similar to Dot/Icm secretion in Legionella pneumophila. Identification of T4SS substrates of C. burnetii has been facilitated using Legionella, but studies in C. burnetii have not been done. Recent advances including extracellular growth medium, transposon mutagenesis and a stable shuttle vector provide a unique opportunity to change the experimental paradigm with this agent. Objectives are to identify T4SS-dependent effector functions by: 1) Identification of novel T4SS system-dependent substrates. The working hypothesis is that genome-wide screens of ORFs from C. burnetii will identify a large collection of T4SS substrates (candidate effectors). 2) Establish a potential effector function for selected T4SS substrate. The working hypothesis is that examination of putative T4SS dependent substrates using a series of in vitro and in situ assays to identify localization, binding partner and cellular activity will identify critical effectors in the pathogenic process. We have placed a major priority in this aim on molecular characterization of nuclear localized and Fic-domain containing effectors. 3) Determine a role in virulence for T4SS substrates in vitro and in vivo using knockout mutagenesis. The working hypothesis is that genetic inactivation of key T4SS substrates will result in reduced virulence. These data will be used to expand our understanding of the pathogenic process in Q fever and identify targets for anti-C. burnetii therapy. PUBLIC HEALTH RELEVANCE: This is a comprehensive study, including knockout mutagenesis, with novel approaches and depth of study unparalleled with the agent. It is our expectation that these studies will result in identification of several novel T4SS dependent effector molecules, including both acute and chronic pathotype-specific determinants. The ability to perform these studies only recently became possible with advancement in cultivation and mutagenesis procedures for C. burnetii. These data will expand our understanding of the pathogenic process in Q fever and identify targets for anti-C. burnetii therapy.