Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Oct;64(10):4269–4278. doi: 10.1128/iai.64.10.4269-4278.1996

Temporal analysis of the developing Chlamydia psittaci inclusion by use of fluorescence and electron microscopy.

D D Rockey 1, E R Fischer 1, T Hackstadt 1
PMCID: PMC174367  PMID: 8926099

Abstract

The chlamydiae are obligate intracellular parasites that develop and multiply within a vacuole (termed an inclusion) that does not fuse with lysosomes. Inclusion morphology varies dramatically among the different chlamydiae, particularly within the species Chlamydia psittaci. Some strains develop within a single vacuole, while the mature inclusion of other strains consists of several distinct lobes, each filled with chlamydial developmental forms. The development of this lobed structure was investigated in HeLa cells infected with the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci. We employed two recently described probes for the chlamydial inclusion to study the development of these unique lobed structures. The novel probes were an antiserum directed at a protein localized to the GPIC inclusion membrane (anti-IncA) and the fluorescent sphingolipid (N-[7-(4-nitrobenzo-2-oxa-1,3-)]) aminocaproyl sphingosine (NBD-ceramide). Lobed inclusions developed in cells infected at very low multiplicities of infection, suggesting that the structure is not a function of infection by more than one elementary body (EB). Double-label fluorescent-antibody analysis with anti-IncA and an antibody directed at a chlamydial outer membrane protein showed that, prior to 18 h postinfection (p.i.), the inclusion membrane and the chlamydial membrane were tightly associated. After 18 to 20 h p.i., the lobes began to expand and fill with developmental forms and the inclusion membrane and chlamydial membrane became distinct. At times from 8 to 48 h p.i., GPIC inclusions were shown to receive fluorescent derivatives of NBD-ceramide and to be localized to the perinuclear region of the host cell. Labeled lectins with affinity for carbohydrate moieties localized to the Golgi apparatus showed that the lobes of mature inclusions surround the Golgi apparatus. Labeling with NBD-ceramide and the Golgi apparatus-specific lectins therefore demonstrated a functional and physical association of the inclusion with the Golgi apparatus throughout the developmental cycle. Collectively, these results lead to a model for the development of the lobed chlamydial inclusion. We propose that the lobed structure is a result of division of inclusions occurring in parallel with the multiplication of reticulate bodies (RB) early in the developmental cycle. The division of inclusions slows or stops in mid-cycle, and dividing RB accumulate within the enlarging lobes. The RB then differentiate to EBs, the inclusion and cell are lysed, and EBs are freed to infect another cell.

Full Text

The Full Text of this article is available as a PDF (4.2 MB).

Selected References

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

  1. Anderson D. R., Hopps H. E., Barile M. F., Bernheim B. C. Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J Bacteriol. 1965 Nov;90(5):1387–1404. doi: 10.1128/jb.90.5.1387-1404.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Campbell S., Richmond S. J., Yates P. The development of Chlamydia trachomatis inclusions within the host eukaryotic cell during interphase and mitosis. J Gen Microbiol. 1989 May;135(5):1153–1165. doi: 10.1099/00221287-135-5-1153. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. HIGASHI N. ELECTRON MICROSCOPIC STUDIES ON THE MODE OF REPRODUCTION OF TRACHOMA VIRUS AND PSITTACOSIS VIRUS IN CELL CULTURES. Exp Mol Pathol. 1965 Feb;76:24–39. doi: 10.1016/0014-4800(65)90021-3. [DOI] [PubMed] [Google Scholar]
  6. Hackstadt T., Rockey D. D., Heinzen R. A., Scidmore M. A. Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. EMBO J. 1996 Mar 1;15(5):964–977. [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Heinzen R. A., Scidmore M. A., Rockey D. D., Hackstadt T. Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect Immun. 1996 Mar;64(3):796–809. doi: 10.1128/iai.64.3.796-809.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Lépinay A., Orfila J., Anteunis A., Boutry J. M., Orme-Roselli L., Robineaux R. Etude en microscopie électronique du développement et de la morphologie de Chlamydia psittaci dans les macrophages de souris. Ann Inst Pasteur (Paris) 1970 Aug;119(2):222–231. [PubMed] [Google Scholar]
  12. Matsumoto A., Bessho H., Uehira K., Suda T. Morphological studies of the association of mitochondria with chlamydial inclusions and the fusion of chlamydial inclusions. J Electron Microsc (Tokyo) 1991 Oct;40(5):356–363. [PubMed] [Google Scholar]
  13. McDonald K. Osmium ferricyanide fixation improves microfilament preservation and membrane visualization in a variety of animal cell types. J Ultrastruct Res. 1984 Feb;86(2):107–118. doi: 10.1016/s0022-5320(84)80051-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Schramm N., Wyrick P. B. Cytoskeletal requirements in Chlamydia trachomatis infection of host cells. Infect Immun. 1995 Jan;63(1):324–332. doi: 10.1128/iai.63.1.324-332.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Spears P., Storz J. Biotyping of Chlamydia psittaci based on inclusion morphology and response to diethylaminoethyl-dextran and cycloheximide. Infect Immun. 1979 Apr;24(1):224–232. doi: 10.1128/iai.24.1.224-232.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Virtanen I., Ekblom P., Laurila P. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. J Cell Biol. 1980 May;85(2):429–434. doi: 10.1083/jcb.85.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ward M. E. Chlamydial classification, development and structure. Br Med Bull. 1983 Apr;39(2):109–115. doi: 10.1093/oxfordjournals.bmb.a071800. [DOI] [PubMed] [Google Scholar]
  22. 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]

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

RESOURCES