DIFFERENTIAL INTERACTION WITH ENDOCYTIC AND EXOCYTIC PATHWAYS DISTINGUISH PARASITOPHOROUS VACUOLES OF COXIELLA-BURNETII AND CHLAMYDIA-TRACHOMATIS

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate int racellular parasites that occupy distinct vacuolar niches within eucar yotic 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 va cuolar environments of these pathogens. The acidic nature of the C. bu rnetii-containing vacuole was confirmed by its acquisition of the acid otropic base acridine orange (AO). The presence of the vacuolar-type ( H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demons trated by indirect immunofluorescence, and growth of C. burnetii was i nhibited by bafilomycin A(1) (Baf A), a specific inhibitor of the V-AT Pase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamy dial growth was not inhibited by Baf A or the lysosomotropic amines me thylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. b urnetii 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 FI TC-dex nor LY was sequestered by chlamydial inclusions. The late endos omal-prelysosomal marker cation-independent mannose 6-phosphate recept or was not detectable in the vacuolar membranes encompassing either pa rasite. 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. t rachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C-6- NBD-ceramide, to the chlamydial inclusion and incorporation into the b acterial cell wall. Similar trafficking of C-6-NBD-ceramide was not ev ident in C. burnetii-infected cells. Collectively, the data indicate t hat C. trachomatis replicates within a nonacidified vacuole that is di sconnected from endosome-lysosome trafficking but may receive lipid fr om exocytic vesicles derived from the trans-Golgi network. These obser vations are in sharp contrast to those for C. burnetii, which by all c riteria resides in a typical phagolysosome.