title: Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis.
abstract: 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.
authors.affiliation: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840-2999,USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Heinzen
authors.forename: M A
authors.initials: MA
authors.lastname: Scidmore
authors.forename: D D
authors.initials: DD
authors.lastname: Rockey
authors.forename: T
authors.initials: T
authors.lastname: Hackstadt
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002692:Chlamydia trachomatis / Q000378:metabolism / Q000648:ultrastructure*; D002522:Chlorocebus aethiops; D016997:Coxiella burnetii / Q000378:metabolism / Q000648:ultrastructure*; D004705:Endocytosis; D005089:Exocytosis; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D011817:Rabbits; D013107:Sphingolipids / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D013107:Sphingolipids