| dc.description.abstract | 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
β-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. | en |
