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. 1997 Nov;65(11):4539–4547. doi: 10.1128/iai.65.11.4539-4547.1997

Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins.

J E Raulston 1
PMCID: PMC175652  PMID: 9353031

Abstract

Iron is a well-established mediator of virulence in several bacterial pathogens, yet little is known about the role of iron in infectious disease processes caused by obligate intracellular bacterial pathogens. In this study, the effect of iron limitation was examined for the sexually transmitted infectious agent Chlamydia trachomatis in an in vitro model of human genital infection using the intracellular iron-chelating reagent deferoxamine mesylate (Desferal). Iron restriction caused a significant reduction in infectivity of C. trachomatis elementary bodies (EB) harvested from Desferal-exposed polarized epithelial cells when compared to that of EB harvested from iron-sufficient control cell cultures. Replacement of the Desferal exposure medium with medium containing iron-saturated transferrin restored chlamydial infectivity, whereas replacement with growth medium alone had no effect. The following three prominent morphological features were observed by electron microscopic examination of chlamydia-infected cells exposed to Desferal: (i) inclusions containing chlamydiae greatly delayed in maturation, (ii) substantial blebbing within chlamydial inclusions, and (iii) electron-dense material surrounding inclusions. Protein analyses of highly purified EB by two-dimensional polyacrylamide gel electrophoresis revealed that there were at least 19 candidate iron-repressible proteins in C. trachomatis and at least one protein which was iron inducible. One putative iron-repressible protein was confirmed by Western blot (immunoblot) analysis to be the chlamydial heat shock protein 60 (hsp60). The enhanced production of this antigen by chlamydiae as a result of iron limitation is of particular importance since there is a well-documented association between chlamydial hsp60 and destructive immunopathological sequelae in infected patients.

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Selected References

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  1. Aisen P. The transferrin receptor and the release of iron from transferrin. Adv Exp Med Biol. 1994;356:31–40. doi: 10.1007/978-1-4615-2554-7_4. [DOI] [PubMed] [Google Scholar]
  2. Ala'Aldeen D. A. Transferrin receptors of Neisseria meningitidis: promising candidates for a broadly cross-protective vaccine. J Med Microbiol. 1996 Apr;44(4):237–243. doi: 10.1099/00222615-44-4-237. [DOI] [PubMed] [Google Scholar]
  3. Alford C. E., King T. E., Jr, Campbell P. A. Role of transferrin, transferrin receptors, and iron in macrophage listericidal activity. J Exp Med. 1991 Aug 1;174(2):459–466. doi: 10.1084/jem.174.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balla G., Jacob H. S., Balla J., Rosenberg M., Nath K., Apple F., Eaton J. W., Vercellotti G. M. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem. 1992 Sep 5;267(25):18148–18153. [PubMed] [Google Scholar]
  5. Bavoil P., Stephens R. S., Falkow S. A soluble 60 kiloDalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL. Mol Microbiol. 1990 Mar;4(3):461–469. doi: 10.1111/j.1365-2958.1990.tb00612.x. [DOI] [PubMed] [Google Scholar]
  6. Beatty W. L., Byrne G. I., Morrison R. P. Morphologic and antigenic characterization of interferon gamma-mediated persistent Chlamydia trachomatis infection in vitro. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3998–4002. doi: 10.1073/pnas.90.9.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beatty W. L., Byrne G. I., Morrison R. P. Repeated and persistent infection with Chlamydia and the development of chronic inflammation and disease. Trends Microbiol. 1994 Mar;2(3):94–98. doi: 10.1016/0966-842x(94)90542-8. [DOI] [PubMed] [Google Scholar]
  8. Bini L., Sanchez-Campillo M., Santucci A., Magi B., Marzocchi B., Comanducci M., Christiansen G., Birkelund S., Cevenini R., Vretou E. Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting. Electrophoresis. 1996 Jan;17(1):185–190. doi: 10.1002/elps.1150170130. [DOI] [PubMed] [Google Scholar]
  9. Brunham R. C., Peeling R. W. Chlamydia trachomatis antigens: role in immunity and pathogenesis. Infect Agents Dis. 1994 Oct;3(5):218–233. [PubMed] [Google Scholar]
  10. Byrd T. F., Horwitz M. A. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J Clin Invest. 1989 May;83(5):1457–1465. doi: 10.1172/JCI114038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper C. E., Lynagh G. R., Hoyes K. P., Hider R. C., Cammack R., Porter J. B. The relationship of intracellular iron chelation to the inhibition and regeneration of human ribonucleotide reductase. J Biol Chem. 1996 Aug 23;271(34):20291–20299. doi: 10.1074/jbc.271.34.20291. [DOI] [PubMed] [Google Scholar]
  12. Cornelissen C. N., Sparling P. F. Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens. Mol Microbiol. 1994 Dec;14(5):843–850. doi: 10.1111/j.1365-2958.1994.tb01320.x. [DOI] [PubMed] [Google Scholar]
  13. Danve B., Lissolo L., Mignon M., Dumas P., Colombani S., Schryvers A. B., Quentin-Millet M. J. Transferrin-binding proteins isolated from Neisseria meningitidis elicit protective and bactericidal antibodies in laboratory animals. Vaccine. 1993 Sep;11(12):1214–1220. doi: 10.1016/0264-410x(93)90045-y. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Esparza I., Brock J. H. Release of iron by resident and stimulated mouse peritoneal macrophages following ingestion and degradation of transferrin-antitransferrin immune complexes. Br J Haematol. 1981 Dec;49(4):603–614. doi: 10.1111/j.1365-2141.1981.tb07270.x. [DOI] [PubMed] [Google Scholar]
  16. Garcia-del Portillo F., Foster J. W., Maguire M. E., Finlay B. B. Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells. Mol Microbiol. 1992 Nov;6(22):3289–3297. doi: 10.1111/j.1365-2958.1992.tb02197.x. [DOI] [PubMed] [Google Scholar]
  17. Gray-Owen S. D., Schryvers A. B. Bacterial transferrin and lactoferrin receptors. Trends Microbiol. 1996 May;4(5):185–191. doi: 10.1016/0966-842x(96)10025-1. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Heinzen R. A., Hackstadt T. The Chlamydia trachomatis parasitophorous vacuolar membrane is not passively permeable to low-molecular-weight compounds. Infect Immun. 1997 Mar;65(3):1088–1094. doi: 10.1128/iai.65.3.1088-1094.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hickey E. K., Cianciotto N. P. An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases. Infect Immun. 1997 Jan;65(1):133–143. doi: 10.1128/iai.65.1.133-143.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Husson M. O., Legrand D., Spik G., Leclerc H. Iron acquisition by Helicobacter pylori: importance of human lactoferrin. Infect Immun. 1993 Jun;61(6):2694–2697. doi: 10.1128/iai.61.6.2694-2697.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kelver M. E., Kaul A., Nowicki B., Findley W. E., Hutchens T. W., Nagamani M. Estrogen regulation of lactoferrin expression in human endometrium. Am J Reprod Immunol. 1996 Nov;36(5):243–247. doi: 10.1111/j.1600-0897.1996.tb00171.x. [DOI] [PubMed] [Google Scholar]
  23. Klausner R. D., Rouault T. A., Harford J. B. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell. 1993 Jan 15;72(1):19–28. doi: 10.1016/0092-8674(93)90046-s. [DOI] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lipschitz D. A., Dugard J., Simon M. O., Bothwell T. H., Charlton R. W. The site of action of desferrioxamine. Br J Haematol. 1971 Apr;20(4):395–404. doi: 10.1111/j.1365-2141.1971.tb07051.x. [DOI] [PubMed] [Google Scholar]
  26. Litwin C. M., Calderwood S. B. Role of iron in regulation of virulence genes. Clin Microbiol Rev. 1993 Apr;6(2):137–149. doi: 10.1128/cmr.6.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maslow A. S., Davis C. H., Choong J., Wyrick P. B. Estrogen enhances attachment of Chlamydia trachomatis to human endometrial epithelial cells in vitro. Am J Obstet Gynecol. 1988 Oct;159(4):1006–1014. doi: 10.1016/s0002-9378(88)80189-3. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Mickelsen P. A., Sparling P. F. Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from transferrin and iron compounds. Infect Immun. 1981 Aug;33(2):555–564. doi: 10.1128/iai.33.2.555-564.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mietzner T. A., Luginbuhl G. H., Sandstrom E., Morse S. A. Identification of an iron-regulated 37,000-dalton protein in the cell envelope of Neisseria gonorrhoeae. Infect Immun. 1984 Aug;45(2):410–416. doi: 10.1128/iai.45.2.410-416.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morrison R. P., Belland R. J., Lyng K., Caldwell H. D. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein. J Exp Med. 1989 Oct 1;170(4):1271–1283. doi: 10.1084/jem.170.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Morrison R. P., Lyng K., Caldwell H. D. Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kD protein. J Exp Med. 1989 Mar 1;169(3):663–675. doi: 10.1084/jem.169.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Murray H. W., Granger A. M., Teitelbaum R. F. Gamma interferon-activated human macrophages and Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani: antimicrobial role of limiting intracellular iron. Infect Immun. 1991 Dec;59(12):4684–4686. doi: 10.1128/iai.59.12.4684-4686.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Newman S. L., Gootee L., Brunner G., Deepe G. S., Jr Chloroquine induces human macrophage killing of Histoplasma capsulatum by limiting the availability of intracellular iron and is therapeutic in a murine model of histoplasmosis. J Clin Invest. 1994 Apr;93(4):1422–1429. doi: 10.1172/JCI117119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Núez M. T., Gaete V., Watkins J. A., Glass J. Mobilization of iron from endocytic vesicles. The effects of acidification and reduction. J Biol Chem. 1990 Apr 25;265(12):6688–6692. [PubMed] [Google Scholar]
  36. Otto B. R., Verweij-van Vught A. M., MacLaren D. M. Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit Rev Microbiol. 1992;18(3):217–233. doi: 10.3109/10408419209114559. [DOI] [PubMed] [Google Scholar]
  37. Pollack S. Receptor-mediated iron uptake and intracellular iron transport. Am J Hematol. 1992 Feb;39(2):113–118. doi: 10.1002/ajh.2830390208. [DOI] [PubMed] [Google Scholar]
  38. Raulston J. E., Davis C. H., Schmiel D. H., Morgan M. W., Wyrick P. B. Molecular characterization and outer membrane association of a Chlamydia trachomatis protein related to the hsp70 family of proteins. J Biol Chem. 1993 Nov 5;268(31):23139–23147. [PubMed] [Google Scholar]
  39. Robbins E., Fant J., Norton W. Intracellular iron-binding macromolecules in HeLa cells. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3708–3712. doi: 10.1073/pnas.69.12.3708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schachter J., Wyrick P. B. Culture and isolation of Chlamydia trachomatis. Methods Enzymol. 1994;236:377–390. doi: 10.1016/0076-6879(94)36028-6. [DOI] [PubMed] [Google Scholar]
  41. Schramm N., Bagnell C. R., Wyrick P. B. Vesicles containing Chlamydia trachomatis serovar L2 remain above pH 6 within HEC-1B cells. Infect Immun. 1996 Apr;64(4):1208–1214. doi: 10.1128/iai.64.4.1208-1214.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Scidmore M. A., Fischer E. R., Hackstadt T. Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion. J Cell Biol. 1996 Jul;134(2):363–374. doi: 10.1083/jcb.134.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tao X., Schiering N., Zeng H. Y., Ringe D., Murphy J. R. Iron, DtxR, and the regulation of diphtheria toxin expression. Mol Microbiol. 1994 Oct;14(2):191–197. doi: 10.1111/j.1365-2958.1994.tb01280.x. [DOI] [PubMed] [Google Scholar]
  44. Taraska T., Ward D. M., Ajioka R. S., Wyrick P. B., Davis-Kaplan S. R., Davis C. H., Kaplan J. The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins. Infect Immun. 1996 Sep;64(9):3713–3727. doi: 10.1128/iai.64.9.3713-3727.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Williams P., Griffiths E. Bacterial transferrin receptors--structure, function and contribution to virulence. Med Microbiol Immunol. 1992;181(6):301–322. doi: 10.1007/BF00191543. [DOI] [PubMed] [Google Scholar]
  46. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  47. Wyrick P. B., Brownridge E. A., Ivins B. E. Interaction of Chlamydia psittaci with mouse peritoneal macrophages. Infect Immun. 1978 Mar;19(3):1061–1067. doi: 10.1128/iai.19.3.1061-1067.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Wyrick P. B., Davis C. H., Knight S. T., Choong J., Raulston J. E., Schramm N. An in vitro human epithelial cell culture system for studying the pathogenesis of Chlamydia trachomatis. Sex Transm Dis. 1993 Sep-Oct;20(5):248–256. doi: 10.1097/00007435-199309000-00002. [DOI] [PubMed] [Google Scholar]
  50. Wyrick P. B., Gerbig D. G., Jr, Knight S. T., Raulston J. E. Accelerated development of genital Chlamydia trachomatis serovar E in McCoy cells grown on microcarrier beads. Microb Pathog. 1996 Jan;20(1):31–40. doi: 10.1006/mpat.1996.0003. [DOI] [PubMed] [Google Scholar]
  51. Zhu L. J., Bagchi M. K., Bagchi I. C. Ferritin heavy chain is a progesterone-inducible marker in the uterus during pregnancy. Endocrinology. 1995 Sep;136(9):4106–4115. doi: 10.1210/endo.136.9.7649119. [DOI] [PubMed] [Google Scholar]
  52. de Lorenzo V., Wee S., Herrero M., Neilands J. B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987 Jun;169(6):2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. de Silva D. M., Askwith C. C., Kaplan J. Molecular mechanisms of iron uptake in eukaryotes. Physiol Rev. 1996 Jan;76(1):31–47. doi: 10.1152/physrev.1996.76.1.31. [DOI] [PubMed] [Google Scholar]
  54. van Ooij C., Apodaca G., Engel J. Characterization of the Chlamydia trachomatis vacuole and its interaction with the host endocytic pathway in HeLa cells. Infect Immun. 1997 Feb;65(2):758–766. doi: 10.1128/iai.65.2.758-766.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]

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