Identification of C. burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation

Coxiella burnetii is a Gram-negative intracellular pathogen that encodes a specialized type IVb secretion system (T4SS) which is essential for intracellular replication, Coxiella-containing vacuole (CCV) formation, modulation of apoptosis, and effector translocation. To identify T4SS candidate substrates, we used an enhanced bioinformatics guided approach. Expression of 234 T4SS candidate substrates as TEM1 ?-lactamase fusions identified 53 substrates that were translocated in a Dot/Icm-dependent manner. Large scale screens aimed at identifying localization and function revealed that several of these substrates traffic to distinct subcellular compartments and interfere with crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Of these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells, but displayed normal growth in axenic culture. Given their confirmed role in intracellular replication and CCV formation, we named 5 of these substrates CirA-E (Coxiella effector for intracellular replication). To identify the pathways targeted by these crucial substrates, S. cerevisiae strains were co-transformed with pYEp13 yeast genomic library and the T4SS substrates. Using this approach we identified multiple members of the Rho family of GTPases as suppressors of Cbu0041 (CirA) toxicity. Overexpression in mammalian cells resulted in cell rounding, detachment, and reduced stress fibers. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation.