SummaryThis study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal-like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii , an obligate intracellular bacterium. Large and spacious PV with multiple non-replicating C. burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively. Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol. Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP-1 positive vacuolar membrane. These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug. Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse. Collectively, these results demonstrate that C. burnetii protein synthesis, but not replication, is required for fusion between nascent C. burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV. However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.

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.

A cross-reactivity among some strains of Coxiella burnetii and chlamydiae with immune rabbit and mouse sera in ELISA and immunoblot analysis was observed. In the latter, the cross-reactivity disappeared after a treatment of C. burnetii or C. psittaci with proteinase K, which indicates that only proteins were involved. The observed cross-reactivity was not influenced by host chick embryo yolk sac proteins. After adsorption of immune rabbit sera with homologous corpuscular antigens the cross-reactivity disappeared. The possibility of influence of such cross-reactivity on serological diagnosis of C. burnetii or chlamydiae infections is discussed.