Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Mar;64(3):796–809. doi: 10.1128/iai.64.3.796-809.1996

Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis.

R A Heinzen 1, M A Scidmore 1, D D Rockey 1, T Hackstadt 1
PMCID: PMC173840  PMID: 8641784

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.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akporiaye E. T., Rowatt J. D., Aragon A. A., Baca O. G. Lysosomal response of a murine macrophage-like cell line persistently infected with Coxiella burnetii. Infect Immun. 1983 Jun;40(3):1155–1162. doi: 10.1128/iai.40.3.1155-1162.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antoine J. C., Prina E., Jouanne C., Bongrand P. Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH. Infect Immun. 1990 Mar;58(3):779–787. doi: 10.1128/iai.58.3.779-787.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baca O. G., Li Y. P., Kumar H. Survival of the Q fever agent Coxiella burnetii in the phagolysosome. Trends Microbiol. 1994 Dec;2(12):476–480. doi: 10.1016/0966-842x(94)90651-3. [DOI] [PubMed] [Google Scholar]
  4. Baca O. G., Paretsky D. Q fever and Coxiella burnetii: a model for host-parasite interactions. Microbiol Rev. 1983 Jun;47(2):127–149. doi: 10.1128/mr.47.2.127-149.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beckers C. J., Dubremetz J. F., Mercereau-Puijalon O., Joiner K. A. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J Cell Biol. 1994 Nov;127(4):947–961. doi: 10.1083/jcb.127.4.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowman E. J., Siebers A., Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7972–7976. doi: 10.1073/pnas.85.21.7972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burton P. R., Kordová N., Paretsky D. Electron microscopic studies of the rickettsia Coxiella burneti: entry, lysosomal response, and fate of rickettsial DNA in L-cells. Can J Microbiol. 1971 Feb;17(2):143–150. doi: 10.1139/m71-025. [DOI] [PubMed] [Google Scholar]
  8. Caldwell H. D., Kromhout J., Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1981 Mar;31(3):1161–1176. doi: 10.1128/iai.31.3.1161-1176.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chanock S. J., el Benna J., Smith R. M., Babior B. M. The respiratory burst oxidase. J Biol Chem. 1994 Oct 7;269(40):24519–24522. [PubMed] [Google Scholar]
  10. Chen J. W., Murphy T. L., Willingham M. C., Pastan I., August J. T. Identification of two lysosomal membrane glycoproteins. J Cell Biol. 1985 Jul;101(1):85–95. doi: 10.1083/jcb.101.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clemens D. L., Horwitz M. A. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med. 1995 Jan 1;181(1):257–270. doi: 10.1084/jem.181.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eissenberg L. G., Wyrick P. B. Inhibition of phagolysosome fusion is localized to Chlamydia psittaci-laden vacuoles. Infect Immun. 1981 May;32(2):889–896. doi: 10.1128/iai.32.2.889-896.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. FURNESS G., GRAHAM D. M., REEVE P. The titration of trachoma and inclusion blennorrhoea viruses in cell cultures. J Gen Microbiol. 1960 Dec;23:613–619. doi: 10.1099/00221287-23-3-613. [DOI] [PubMed] [Google Scholar]
  14. Friis R. R. Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J Bacteriol. 1972 May;110(2):706–721. doi: 10.1128/jb.110.2.706-721.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hackstadt T., Messer R., Cieplak W., Peacock M. G. Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing. Infect Immun. 1992 Jan;60(1):159–165. doi: 10.1128/iai.60.1.159-165.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hackstadt T., Scidmore M. A., Rockey D. D. Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4877–4881. doi: 10.1073/pnas.92.11.4877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hackstadt T., Williams J. C. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc Natl Acad Sci U S A. 1981 May;78(5):3240–3244. doi: 10.1073/pnas.78.5.3240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hart P. D., Armstrong J. A., Brown C. A., Draper P. Ultrastructural study of the behavior of macrophages toward parasitic mycobacteria. Infect Immun. 1972 May;5(5):803–807. doi: 10.1128/iai.5.5.803-807.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hatch T. P., Miceli M., Silverman J. A. Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis. J Bacteriol. 1985 Jun;162(3):938–942. doi: 10.1128/jb.162.3.938-942.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joiner K. A., Fuhrman S. A., Miettinen H. M., Kasper L. H., Mellman I. Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science. 1990 Aug 10;249(4969):641–646. doi: 10.1126/science.2200126. [DOI] [PubMed] [Google Scholar]
  21. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  22. Lawn A. M., Blyth W. A., Taverne J. Interactions of TRIC agents with macrophages and BHK-21 cells observed by electron microscopy. J Hyg (Lond) 1973 Sep;71(3):515–528. doi: 10.1017/s0022172400046507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lipsky N. G., Pagano R. E. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J Cell Biol. 1985 Jan;100(1):27–34. doi: 10.1083/jcb.100.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lombardi D., Soldati T., Riederer M. A., Goda Y., Zerial M., Pfeffer S. R. Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J. 1993 Feb;12(2):677–682. doi: 10.1002/j.1460-2075.1993.tb05701.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mane S. M., Marzella L., Bainton D. F., Holt V. K., Cha Y., Hildreth J. E., August J. T. Purification and characterization of human lysosomal membrane glycoproteins. Arch Biochem Biophys. 1989 Jan;268(1):360–378. doi: 10.1016/0003-9861(89)90597-3. [DOI] [PubMed] [Google Scholar]
  26. McClarty G. Chlamydiae and the biochemistry of intracellular parasitism. Trends Microbiol. 1994 May;2(5):157–164. doi: 10.1016/0966-842x(94)90665-3. [DOI] [PubMed] [Google Scholar]
  27. Moreau P., Cassagne C. Phospholipid trafficking and membrane biogenesis. Biochim Biophys Acta. 1994 Dec 9;1197(3):257–290. doi: 10.1016/0304-4157(94)90010-8. [DOI] [PubMed] [Google Scholar]
  28. Moulder J. W. Comparative biology of intracellular parasitism. Microbiol Rev. 1985 Sep;49(3):298–337. doi: 10.1128/mr.49.3.298-337.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moulder J. W. Interaction of chlamydiae and host cells in vitro. Microbiol Rev. 1991 Mar;55(1):143–190. doi: 10.1128/mr.55.1.143-190.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Márquez-Sterling N., Herman I. M., Pesacreta T., Arai H., Terres G., Forgac M. Immunolocalization of the vacuolar-type (H+)-ATPase from clathrin-coated vesicles. Eur J Cell Biol. 1991 Oct;56(1):19–33. [PubMed] [Google Scholar]
  31. Pelham H. R., Munro S. Sorting of membrane proteins in the secretory pathway. Cell. 1993 Nov 19;75(4):603–605. doi: 10.1016/0092-8674(93)90479-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Poole B., Ohkuma S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol. 1981 Sep;90(3):665–669. doi: 10.1083/jcb.90.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Redd T., Thompson H. A. Secretion of proteins by Coxiella burnetii. Microbiology. 1995 Feb;141(Pt 2):363–369. doi: 10.1099/13500872-141-2-363. [DOI] [PubMed] [Google Scholar]
  34. Rockey D. D., Heinzen R. A., Hackstadt T. Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells. Mol Microbiol. 1995 Feb;15(4):617–626. doi: 10.1111/j.1365-2958.1995.tb02371.x. [DOI] [PubMed] [Google Scholar]
  35. Schachter J., Dawson C. R. The epidemiology of trachoma predicts more blindness in the future. Scand J Infect Dis Suppl. 1990;69:55–62. [PubMed] [Google Scholar]
  36. Schwab J. C., Beckers C. J., Joiner K. A. The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):509–513. doi: 10.1073/pnas.91.2.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Small P. L., Ramakrishnan L., Falkow S. Remodeling schemes of intracellular pathogens. Science. 1994 Feb 4;263(5147):637–639. doi: 10.1126/science.8303269. [DOI] [PubMed] [Google Scholar]
  38. Sturgill-Koszycki S., Schlesinger P. H., Chakraborty P., Haddix P. L., Collins H. L., Fok A. K., Allen R. D., Gluck S. L., Heuser J., Russell D. G. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science. 1994 Feb 4;263(5147):678–681. doi: 10.1126/science.8303277. [DOI] [PubMed] [Google Scholar]
  39. Swanson J. Fluorescent labeling of endocytic compartments. Methods Cell Biol. 1989;29:137–151. doi: 10.1016/s0091-679x(08)60192-2. [DOI] [PubMed] [Google Scholar]
  40. Söderlund G., Kihlström E. Effect of methylamine and monodansylcadaverine on the susceptibility of McCoy cells to Chlamydia trachomatis infection. Infect Immun. 1983 May;40(2):534–541. doi: 10.1128/iai.40.2.534-541.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wyrick P. B., Brownridge E. A. Growth of Chlamydia psittaci in macrophages. Infect Immun. 1978 Mar;19(3):1054–1060. doi: 10.1128/iai.19.3.1054-1060.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yoshimori T., Yamamoto A., Moriyama Y., Futai M., Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991 Sep 15;266(26):17707–17712. [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES