Coxiella burnetii, the causative agent of human Q fever, is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors manipulates host cell processes to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Through the implementation of genetic techniques to manipulate Coxiella, we have established that specific Dot/Icm effectors are essential for CCV biogenesis.
Functional characterization of one essential effector, Cig57, has revealed that Coxiella actively recruits clathrin to the CCV and that this is required for intracellular success of this pathogen. Cig57 subverts clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line, using CRISPR/Cas9, confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen.