Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Jun;65(6):2420–2427. doi: 10.1128/iai.65.6.2420-2427.1997

Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

H A Harvey 1, M R Ketterer 1, A Preston 1, D Lubaroff 1, R Williams 1, M A Apicella 1
PMCID: PMC175335  PMID: 9169783

Abstract

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.

Full Text

The Full Text of this article is available as a PDF (972.4 KB).

Selected References

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

  1. Apicella M. A., Ketterer M., Lee F. K., Zhou D., Rice P. A., Blake M. S. The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae. J Infect Dis. 1996 Mar;173(3):636–646. doi: 10.1093/infdis/173.3.636. [DOI] [PubMed] [Google Scholar]
  2. Chen J. C., Bavoil P., Clark V. L. Enhancement of the invasive ability of Neisseria gonorrhoeae by contact with HecIB, an adenocarcinoma endometrial cell line. Mol Microbiol. 1991 Jun;5(6):1531–1538. doi: 10.1111/j.1365-2958.1991.tb00800.x. [DOI] [PubMed] [Google Scholar]
  3. Cohen M. S., Cannon J. G., Jerse A. E., Charniga L. M., Isbey S. F., Whicker L. G. Human experimentation with Neisseria gonorrhoeae: rationale, methods, and implications for the biology of infection and vaccine development. J Infect Dis. 1994 Mar;169(3):532–537. doi: 10.1093/infdis/169.3.532. [DOI] [PubMed] [Google Scholar]
  4. Goldberg M. B., Sansonetti P. J. Shigella subversion of the cellular cytoskeleton: a strategy for epithelial colonization. Infect Immun. 1993 Dec;61(12):4941–4946. doi: 10.1128/iai.61.12.4941-4946.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grassmé H. U., Ireland R. M., van Putten J. P. Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton. Infect Immun. 1996 May;64(5):1621–1630. doi: 10.1128/iai.64.5.1621-1630.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jones S., Portnoy D. A. Intracellular growth of bacteria. Methods Enzymol. 1994;236:463–467. doi: 10.1016/0076-6879(94)36034-0. [DOI] [PubMed] [Google Scholar]
  7. McGee Z. A., Johnson A. P., Taylor-Robinson D. Pathogenic mechanisms of Neisseria gonorrhoeae: observations on damage to human fallopian tubes in organ culture by gonococci of colony type 1 or type 4. J Infect Dis. 1981 Mar;143(3):413–422. doi: 10.1093/infdis/143.3.413. [DOI] [PubMed] [Google Scholar]
  8. McGee Z. A., Stephens D. S., Hoffman L. H., Schlech W. F., 3rd, Horn R. G. Mechanisms of mucosal invasion by pathogenic Neisseria. Rev Infect Dis. 1983 Sep-Oct;5 (Suppl 4):S708–S714. doi: 10.1093/clinids/5.supplement_4.s708. [DOI] [PubMed] [Google Scholar]
  9. Novotny P., Short J. A., Walker P. D. An electron-microscope study of naturally occurring and cultured cells of Neisseria Gonorrhoeae. J Med Microbiol. 1975 Aug;8(3):413–427. doi: 10.1099/00222615-8-3-413. [DOI] [PubMed] [Google Scholar]
  10. Phillips S. G., Phillips D. M., Kabat E. A., Miller O. J. Human semen as a source of epithelial cells for culture. In Vitro. 1978 Aug;14(8):639–650. doi: 10.1007/BF02616161. [DOI] [PubMed] [Google Scholar]
  11. Porat N., Apicella M. A., Blake M. S. A lipooligosaccharide-binding site on HepG2 cells similar to the gonococcal opacity-associated surface protein Opa. Infect Immun. 1995 Jun;63(6):2164–2172. doi: 10.1128/iai.63.6.2164-2172.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Porat N., Apicella M. A., Blake M. S. Neisseria gonorrhoeae utilizes and enhances the biosynthesis of the asialoglycoprotein receptor expressed on the surface of the hepatic HepG2 cell line. Infect Immun. 1995 Apr;63(4):1498–1506. doi: 10.1128/iai.63.4.1498-1506.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ramsey K. H., Schneider H., Cross A. S., Boslego J. W., Hoover D. L., Staley T. L., Kuschner R. A., Deal C. D. Inflammatory cytokines produced in response to experimental human gonorrhea. J Infect Dis. 1995 Jul;172(1):186–191. doi: 10.1093/infdis/172.1.186. [DOI] [PubMed] [Google Scholar]
  14. Reznikoff C. A., Loretz L. J., Pesciotta D. M., Oberley T. D., Ignjatovic M. M. Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium. J Cell Physiol. 1987 Jun;131(3):285–301. doi: 10.1002/jcp.1041310302. [DOI] [PubMed] [Google Scholar]
  15. Schneider H., Cross A. S., Kuschner R. A., Taylor D. N., Sadoff J. C., Boslego J. W., Deal C. D. Experimental human gonococcal urethritis: 250 Neisseria gonorrhoeae MS11mkC are infective. J Infect Dis. 1995 Jul;172(1):180–185. doi: 10.1093/infdis/172.1.180. [DOI] [PubMed] [Google Scholar]
  16. Schneider H., Griffiss J. M., Boslego J. W., Hitchcock P. J., Zahos K. M., Apicella M. A. Expression of paragloboside-like lipooligosaccharides may be a necessary component of gonococcal pathogenesis in men. J Exp Med. 1991 Dec 1;174(6):1601–1605. doi: 10.1084/jem.174.6.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schwan E. T., Robertson B. D., Brade H., van Putten J. P. Gonococcal rfaF mutants express Rd2 chemotype LPS and do not enter epithelial host cells. Mol Microbiol. 1995 Jan;15(2):267–275. doi: 10.1111/j.1365-2958.1995.tb02241.x. [DOI] [PubMed] [Google Scholar]
  18. Shaw J. H., Falkow S. Model for invasion of human tissue culture cells by Neisseria gonorrhoeae. Infect Immun. 1988 Jun;56(6):1625–1632. doi: 10.1128/iai.56.6.1625-1632.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Stone J. L., Vernon S. K., Warren G. H. Cultivation and survival studies of Neisseria gonorrhoeae in a human diploid cell strain. Yale J Biol Med. 1974 Dec;47(4):291–296. [PMC free article] [PubMed] [Google Scholar]
  20. Tjia K. F., van Putten J. P., Pels E., Zanen H. C. The interaction between Neisseria gonorrhoeae and the human cornea in organ culture. An electron microscopic study. Graefes Arch Clin Exp Ophthalmol. 1988;226(4):341–345. doi: 10.1007/BF02172964. [DOI] [PubMed] [Google Scholar]
  21. Waitkins S. A., Flynn J. Intracellular growth and type variation of Neisseria gonorrhoeae in tissue cell-cultures. J Med Microbiol. 1973 Aug;6(3):399–403. doi: 10.1099/00222615-6-3-399. [DOI] [PubMed] [Google Scholar]
  22. Ward M. E., Watt P. J. Adherence of Neisseria gonorrhoeae to urethral mucosal cells: an electron-microscopic study of human gonorrhea. J Infect Dis. 1972 Dec;126(6):601–605. doi: 10.1093/infdis/126.6.601. [DOI] [PubMed] [Google Scholar]
  23. Ward M. E., Watt P. J., Robertson J. N. The human fallopian tube: a laboratory model for gonococcal infection. J Infect Dis. 1974 Jun;129(6):650–659. doi: 10.1093/infdis/129.6.650. [DOI] [PubMed] [Google Scholar]
  24. van Putten J. P., Grassmé H. U., Robertson B. D., Schwan E. T. Function of lipopolysaccharide in the invasion of Neisseria gonorrhoeae into human mucosal cells. Prog Clin Biol Res. 1995;392:49–58. [PubMed] [Google Scholar]
  25. van Putten J. P. Phase variation of lipopolysaccharide directs interconversion of invasive and immuno-resistant phenotypes of Neisseria gonorrhoeae. EMBO J. 1993 Nov;12(11):4043–4051. doi: 10.1002/j.1460-2075.1993.tb06088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van Putten J. P., Robertson B. D. Molecular mechanisms and implications for infection of lipopolysaccharide variation in Neisseria. Mol Microbiol. 1995 Jun;16(5):847–853. doi: 10.1111/j.1365-2958.1995.tb02312.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES