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. 1996 Sep;64(9):3713–3727. doi: 10.1128/iai.64.9.3713-3727.1996

The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins.

T Taraska 1, D M Ward 1, R S Ajioka 1, P B Wyrick 1, S R Davis-Kaplan 1, C H Davis 1, J Kaplan 1
PMCID: PMC174285  PMID: 8751921

Abstract

Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newly internalized bacteria are surrounded by host plasma membrane; however, the source of membrane for the expansion of the inclusion is unknown. To determine if the membrane for the mature inclusion was derived by fusion with cellular organelles, we stained infected cells with fluorescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presence of endoplasmic reticulum, Golgi, late endosomal, or lysosomal proteins in the inclusion. These data suggest that the expansion of the inclusion membrane, beginning 24 h postinoculation, does not occur by the addition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of the expanding inclusion membrane, antibodies were produced against isolated membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by both immunoprecipitation of [35S]methionine-labeled extracts and Western blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the inclusion, allowing antibodies access to the outer surface of the inclusion membrane. Immunofluorescent staining demonstrated a ring-like fluorescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. These studies demonstrate that the inclusion membrane is made up, in part, of Chlamydia-specified proteins and not of existing host membrane proteins.

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Selected References

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  1. Ajioka R. S., Kaplan J. Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique. J Cell Biol. 1987 Jan;104(1):77–85. doi: 10.1083/jcb.104.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander J. J. Separation of protein synthesis in meningopneumonitisgent from that in L cells by differential susceptibility to cycloheximide. J Bacteriol. 1968 Feb;95(2):327–332. doi: 10.1128/jb.95.2.327-332.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davidson H. W., Balch W. E. Use of two-stage incubations to define sequential intermediates in endoplasmic reticulum to Golgi transport. Methods Enzymol. 1992;219:261–267. doi: 10.1016/0076-6879(92)19027-4. [DOI] [PubMed] [Google Scholar]
  6. Dluzewski A. R., Mitchell G. H., Fryer P. R., Griffiths S., Wilson R. J., Gratzer W. B. Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells. J Cell Sci. 1992 Jul;102(Pt 3):527–532. doi: 10.1242/jcs.102.3.527. [DOI] [PubMed] [Google Scholar]
  7. Eissenberg L. G., Wyrick P. B., Davis C. H., Rumpp J. W. Chlamydia psittaci elementary body envelopes: ingestion and inhibition of phagolysosome fusion. Infect Immun. 1983 May;40(2):741–751. doi: 10.1128/iai.40.2.741-751.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Fennell D. F., Whatley R. E., McIntyre T. M., Prescott S. M., Zimmerman G. A. Endothelial cells reestablish functional integrity after reversible permeabilization. Arterioscler Thromb. 1991 Jan-Feb;11(1):97–106. doi: 10.1161/01.atv.11.1.97. [DOI] [PubMed] [Google Scholar]
  10. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  11. Griffiths G., Warren G., Quinn P., Mathieu-Costello O., Hoppeler H. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies. J Cell Biol. 1984 Jun;98(6):2133–2141. doi: 10.1083/jcb.98.6.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hatch T. P., Al-Hossainy E., Silverman J. A. Adenine nucleotide and lysine transport in Chlamydia psittaci. J Bacteriol. 1982 May;150(2):662–670. doi: 10.1128/jb.150.2.662-670.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hedman K., Pastan I., Willingham M. C. The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin. J Histochem Cytochem. 1986 Aug;34(8):1069–1077. doi: 10.1177/34.8.3734417. [DOI] [PubMed] [Google Scholar]
  15. Hodinka R. L., Davis C. H., Choong J., Wyrick P. B. Ultrastructural study of endocytosis of Chlamydia trachomatis by McCoy cells. Infect Immun. 1988 Jun;56(6):1456–1463. doi: 10.1128/iai.56.6.1456-1463.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hodinka R. L., Wyrick P. B. Ultrastructural study of mode of entry of Chlamydia psittaci into L-929 cells. Infect Immun. 1986 Dec;54(3):855–863. doi: 10.1128/iai.54.3.855-863.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Horwitz M. A., Maxfield F. R. Legionella pneumophila inhibits acidification of its phagosome in human monocytes. J Cell Biol. 1984 Dec;99(6):1936–1943. doi: 10.1083/jcb.99.6.1936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horwitz M. A. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med. 1983 Dec 1;158(6):2108–2126. doi: 10.1084/jem.158.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jahraus A., Storrie B., Griffiths G., Desjardins M. Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment. J Cell Sci. 1994 Jan;107(Pt 1):145–157. doi: 10.1242/jcs.107.1.145. [DOI] [PubMed] [Google Scholar]
  20. Jensen J. B. Ultrastructure of the invasion of Eimeria magna sporozoites into cultured cells. J Protozool. 1975 Aug;22(3):411–415. doi: 10.1111/j.1550-7408.1975.tb05193.x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kara U. A., Stenzel D. J., Ingram L. T., Kidson C. The parasitophorous vacuole membrane of Plasmodium falciparum: demonstration of vesicle formation using an immunoprobe. Eur J Cell Biol. 1988 Apr;46(1):9–17. [PubMed] [Google Scholar]
  23. Kimata I., Tanabe K. Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell. J Cell Sci. 1987 Sep;88(Pt 2):231–239. doi: 10.1242/jcs.88.2.231. [DOI] [PubMed] [Google Scholar]
  24. Lamb J. E., Ray F., Ward J. H., Kushner J. P., Kaplan J. Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells. J Biol Chem. 1983 Jul 25;258(14):8751–8758. [PubMed] [Google Scholar]
  25. MacDonald R. G., Tepper M. A., Clairmont K. B., Perregaux S. B., Czech M. P. Serum form of the rat insulin-like growth factor II/mannose 6-phosphate receptor is truncated in the carboxyl-terminal domain. J Biol Chem. 1989 Feb 25;264(6):3256–3261. [PubMed] [Google Scholar]
  26. 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]
  27. Matsumoto A. Isolation and electron microscopic observations of intracytoplasmic inclusions containing Chlamydia psittaci. J Bacteriol. 1981 Jan;145(1):605–612. doi: 10.1128/jb.145.1.605-612.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nichols B. A., Chiappino M. L., O'Connor G. R. Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J Ultrastruct Res. 1983 Apr;83(1):85–98. doi: 10.1016/s0022-5320(83)90067-9. [DOI] [PubMed] [Google Scholar]
  29. Perou C. M., Kaplan J. Chediak-Higashi syndrome is not due to a defect in microtubule-based lysosomal mobility. J Cell Sci. 1993 Sep;106(Pt 1):99–107. doi: 10.1242/jcs.106.1.99. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Rockey D. D., Rosquist J. L. Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body. Infect Immun. 1994 Jan;62(1):106–112. doi: 10.1128/iai.62.1.106-112.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Roth M. G., Doyle C., Sambrook J., Gething M. J. Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway. J Cell Biol. 1986 Apr;102(4):1271–1283. doi: 10.1083/jcb.102.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schachter J. The intracellular life of Chlamydia. Curr Top Microbiol Immunol. 1988;138:109–139. [PubMed] [Google Scholar]
  34. Sibley L. D., Krahenbuhl J. L., Adams G. M., Weidner E. Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins. J Cell Biol. 1986 Sep;103(3):867–874. doi: 10.1083/jcb.103.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sibley L. D., Krahenbuhl J. L. Modification of host cell phagosomes by Toxoplasma gondii involves redistribution of surface proteins and secretion of a 32 kDa protein. Eur J Cell Biol. 1988 Oct;47(1):81–87. [PubMed] [Google Scholar]
  36. Sibley L. D., Weidner E., Krahenbuhl J. L. Phagosome acidification blocked by intracellular Toxoplasma gondii. 1985 May 30-Jun 5Nature. 315(6018):416–419. doi: 10.1038/315416a0. [DOI] [PubMed] [Google Scholar]
  37. Snider M. D., Rogers O. C. Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I. J Cell Biol. 1986 Jul;103(1):265–275. doi: 10.1083/jcb.103.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Speer C. A., Whitmire W. M. Shedding of the immunodominant P20 surface antigen of Eimeria bovis sporozoites. Infect Immun. 1989 Mar;57(3):999–1001. doi: 10.1128/iai.57.3.999-1001.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stewart M. J., Schulman S., Vanderberg J. P. Rhoptry secretion of membranous whorls by Plasmodium falciparum merozoites. Am J Trop Med Hyg. 1986 Jan;35(1):37–44. doi: 10.4269/ajtmh.1986.35.37. [DOI] [PubMed] [Google Scholar]
  40. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  41. Uster P. S., Pagano R. E. Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells. J Cell Biol. 1986 Oct;103(4):1221–1234. doi: 10.1083/jcb.103.4.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wiese C., Wilson K. L. Nuclear membrane dynamics. Curr Opin Cell Biol. 1993 Jun;5(3):387–394. doi: 10.1016/0955-0674(93)90002-8. [DOI] [PubMed] [Google Scholar]
  43. Wyrick P. B., Choong J., Davis C. H., Knight S. T., Royal M. O., Maslow A. S., Bagnell C. R. Entry of genital Chlamydia trachomatis into polarized human epithelial cells. Infect Immun. 1989 Aug;57(8):2378–2389. doi: 10.1128/iai.57.8.2378-2389.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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