Carlson College of Veterinary Medicine

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.