Fusogenicity of the Coxiella burnetii parasitophorous vacuole

This study investigated whether C. burnetii protein synthesis and replication is required for maintenance of the fusogenic character of the Coxiella parasitophorous vacuole (PV). Vero cells were infected with C. burnetii, (Nine Mile strain in phase II) at a multiplicity of infection of approximately 10 and simultaneously treated with bacteriostatic concentrations of chloramphenicol or carbenicillin. At 96 h post-infection, cells were viewed by phase contrast microscopy for PV maturation. Mature, spacious PV containing multiple nonreplicating C. burnetii were clearly visible in infected Vero cells treated with the cell wall inhibitor carbenicillin. Conversely, mature, spacious PV did not form in cells treated with the protein synthesis inhibitor chloramphenicol. Rather, immunofluorescence microscopy revealed individual C. burnetii in small, tight PV scattered throughout the cytoplasm. Like mature PV, these PV localized with the lysosomal glycoprotein LAMP-1, but not the early endosome protein EEA.1. This result suggests that de novo C. burnetii protein synthesis, but not replication, is required for homotypic fusion and maturation of nascent C. burnetii PV. We next examined whether sustained C. burnetii protein synthesis is necessary for maintenance of PV fusogenicity. J774A.1 murine macrophage-like cells with mature C. burnetii PV were incubated with latex beads and the trafficking of beads to PV was quantified. Fusion of PV with bead-laden vacuoles was severely inhibited in cells treated with chloramphenicol. These results suggest that sustained C. burnetii protein synthesis is required for PV fusion with other vacuoles of the endocytic pathway.