Skip to main content
Infection and Immunity logoLink to Infection and Immunity
. 1997 Sep;65(9):3857–3866. doi: 10.1128/iai.65.9.3857-3866.1997

Binding of vitronectin to opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells.

O G Gómez-Duarte 1, M Dehio 1, C A Guzmán 1, G S Chhatwal 1, C Dehio 1, T F Meyer 1
PMCID: PMC175551  PMID: 9284164

Abstract

Neisseria gonorrhoeae induces local infections in the human genitourinary tract and can disseminate to other organs to cause severe disease. Blood-derived factors present in the genital mucosa have been suggested to facilitate the spread of N. gonorrhoeae in disseminated gonococcal infections. Using gentamicin invasion assays and confocal microscopy, we observed a strong stimulatory effect of fetal calf serum (FCS) on the gonococcal invasion of HeLa cells. FCS-mediated invasion was dependent on the expression of the epithelial cell invasion-associated Opa protein (plasmid-encoded Opa50 or its chromosomal homolog Opa30), while N. gonorrhoeae expressing noninvasive Opa proteins (Opa(51-60)) or no Opa protein (Opa-) was not invasive even in the presence of FCS. Incubation of N. gonorrhoeae MS11 with biotinylated FCS revealed a 78-kDa protein as the prominent protein binding to Opa50- or Opa30-expressing gonococci. This protein was recognized by antibodies against vitronectin (VN) in Western blots. Purified human or bovine VN efficiently bound to Opa50-expressing gonococci, while binding to noninvasive Opa- or Opa52-expressing gonococci was significantly lower. Binding of VN was inhibited by heparin in a concentration-dependent manner, indicating that the heparin binding sites present in VN or Opa50 may play an essential role in this interaction. Based on gentamicin invasion assays and confocal microscopy studies, VN binding was associated with an increased invasion of Opa50- and Opa30-expressing gonococci into HeLa cells. The ability of VN to mediate entry into epithelial cells may constitute an important event in the pathogenesis of local as well as disseminated gonococcal infections.

Full Text

The Full Text of this article is available as a PDF (573.2 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. Arko R. J., Chen C. Y., Schalla W. O., Sarafian S. K., Taylor C. L., Knapp J. S., Morse S. A. Binding of S protein by Neisseria gonorrhoeae and potential role in invasion. J Clin Microbiol. 1991 Jan;29(1):70–75. doi: 10.1128/jcm.29.1.70-75.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bessen D., Gotschlich E. C. Interactions of gonococci with HeLa cells: attachment, detachment, replication, penetration, and the role of protein II. Infect Immun. 1986 Oct;54(1):154–160. doi: 10.1128/iai.54.1.154-160.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhat K. S., Gibbs C. P., Barrera O., Morrison S. G., Jähnig F., Stern A., Kupsch E. M., Meyer T. F., Swanson J. The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes. Mol Microbiol. 1991 Aug;5(8):1889–1901. doi: 10.1111/j.1365-2958.1991.tb00813.x. [DOI] [PubMed] [Google Scholar]
  5. Bihimaier A., Römling U., Meyer T. F., Tümmler B., Gibbs C. P. Physical and genetic map of the Neisseria gonorrhoeae strain MS11-N198 chromosome. Mol Microbiol. 1991 Oct;5(10):2529–2539. doi: 10.1111/j.1365-2958.1991.tb02099.x. [DOI] [PubMed] [Google Scholar]
  6. Blake M. S., Blake C. M., Apicella M. A., Mandrell R. E. Gonococcal opacity: lectin-like interactions between Opa proteins and lipooligosaccharide. Infect Immun. 1995 Apr;63(4):1434–1439. doi: 10.1128/iai.63.4.1434-1439.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen T., Belland R. J., Wilson J., Swanson J. Adherence of pilus- Opa+ gonococci to epithelial cells in vitro involves heparan sulfate. J Exp Med. 1995 Aug 1;182(2):511–517. doi: 10.1084/jem.182.2.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chhatwal G. S., Preissner K. T., Müller-Berghaus G., Blobel H. Specific binding of the human S protein (vitronectin) to streptococci, Staphylococcus aureus, and Escherichia coli. Infect Immun. 1987 Aug;55(8):1878–1883. doi: 10.1128/iai.55.8.1878-1883.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Declerck P. J., De Mol M., Alessi M. C., Baudner S., Pâques E. P., Preissner K. T., Müller-Berghaus G., Collen D. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin). J Biol Chem. 1988 Oct 25;263(30):15454–15461. [PubMed] [Google Scholar]
  10. Elsinghorst E. A. Measurement of invasion by gentamicin resistance. Methods Enzymol. 1994;236:405–420. doi: 10.1016/0076-6879(94)36030-8. [DOI] [PubMed] [Google Scholar]
  11. Fussenegger M., Kahrs A. F., Facius D., Meyer T. F. Tetrapac (tpc), a novel genotype of Neisseria gonorrhoeae affecting epithelial cell invasion, natural transformation competence and cell separation. Mol Microbiol. 1996 Mar;19(6):1357–1372. doi: 10.1111/j.1365-2958.1996.tb02479.x. [DOI] [PubMed] [Google Scholar]
  12. Griffiss J. M., Jarvis G. A., O'Brien J. P., Eads M. M., Schneider H. Lysis of Neisseria gonorrhoeae initiated by binding of normal human IgM to a hexosamine-containing lipooligosaccharide epitope(s) is augmented by strain-specific, properdin-binding-dependent alternative complement pathway activation. J Immunol. 1991 Jul 1;147(1):298–305. [PubMed] [Google Scholar]
  13. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  14. Handsfield H. H. Disseminated gonococcal infection. Clin Obstet Gynecol. 1975 Mar;18(1):131–142. doi: 10.1097/00003081-197503000-00012. [DOI] [PubMed] [Google Scholar]
  15. Hayman E. G., Pierschbacher M. D., Suzuki S., Ruoslahti E. Vitronectin--a major cell attachment-promoting protein in fetal bovine serum. Exp Cell Res. 1985 Oct;160(2):245–258. doi: 10.1016/0014-4827(85)90173-9. [DOI] [PubMed] [Google Scholar]
  16. Hess S., Kanse S. M., Kost C., Preissner K. T. The versatility of adhesion receptor ligands in haemostasis: morpho-regulatory functions of vitronectin. Thromb Haemost. 1995 Jul;74(1):258–265. [PubMed] [Google Scholar]
  17. Holmes K. K., Counts G. W., Beaty H. N. Disseminated gonococcal infection. Ann Intern Med. 1971 Jun;74(6):979–993. doi: 10.7326/0003-4819-74-6-979. [DOI] [PubMed] [Google Scholar]
  18. Jarvis G. A. Analysis of C3 deposition and degradation on Neisseria meningitidis and Neisseria gonorrhoeae. Infect Immun. 1994 May;62(5):1755–1760. doi: 10.1128/iai.62.5.1755-1760.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kahrs A. F., Bihlmaier A., Facius D., Meyer T. F. Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants. Mol Microbiol. 1994 Jun;12(5):819–831. doi: 10.1111/j.1365-2958.1994.tb01068.x. [DOI] [PubMed] [Google Scholar]
  20. Kohl P. K. Epidemiology of sexually transmitted diseases. What does it tell us? Sex Transm Dis. 1994 Mar-Apr;21(2 Suppl):S81–S83. [PubMed] [Google Scholar]
  21. Kronvall G., Myhre E. B., Björck L., Berggård I. Binding of aggregated human beta2-microglobulin to surface protein structure in group A, C, and G streptococci. Infect Immun. 1978 Oct;22(1):136–142. doi: 10.1128/iai.22.1.136-142.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kupsch E. M., Knepper B., Kuroki T., Heuer I., Meyer T. F. Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells. EMBO J. 1993 Feb;12(2):641–650. doi: 10.1002/j.1460-2075.1993.tb05697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Laurière M. A semidry electroblotting system efficiently transfers both high- and low-molecular-weight proteins separated by SDS-PAGE. Anal Biochem. 1993 Jul;212(1):206–211. doi: 10.1006/abio.1993.1313. [DOI] [PubMed] [Google Scholar]
  25. Limper A. H., Standing J. E., Hoffman O. A., Castro M., Neese L. W. Vitronectin binds to Pneumocystis carinii and mediates organism attachment to cultured lung epithelial cells. Infect Immun. 1993 Oct;61(10):4302–4309. doi: 10.1128/iai.61.10.4302-4309.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lydon M. J., Foulger C. A. Cell-substratum interactions: serum spreading factor. Biomaterials. 1988 Nov;9(6):525–527. doi: 10.1016/0142-9612(88)90049-x. [DOI] [PubMed] [Google Scholar]
  27. Makino S., van Putten J. P., Meyer T. F. Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells. EMBO J. 1991 Jun;10(6):1307–1315. doi: 10.1002/j.1460-2075.1991.tb07649.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Milis L., Morris C. A., Sheehan M. C., Charlesworth J. A., Pussell B. A. Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9. Clin Exp Immunol. 1993 Apr;92(1):114–119. doi: 10.1111/j.1365-2249.1993.tb05956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nowicki S., Martens M. G., Nowicki B. J. Gonococcal infection in a nonhuman host is determined by human complement C1q. Infect Immun. 1995 Dec;63(12):4790–4794. doi: 10.1128/iai.63.12.4790-4794.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Panetti T. S., McKeown-Longo P. J. Receptor-mediated endocytosis of vitronectin is regulated by its conformational state. J Biol Chem. 1993 Jun 5;268(16):11988–11993. [PubMed] [Google Scholar]
  31. Panetti T. S., McKeown-Longo P. J. The alpha v beta 5 integrin receptor regulates receptor-mediated endocytosis of vitronectin. J Biol Chem. 1993 Jun 5;268(16):11492–11495. [PubMed] [Google Scholar]
  32. Panetti T. S., Wilcox S. A., Horzempa C., McKeown-Longo P. J. Alpha v beta 5 integrin receptor-mediated endocytosis of vitronectin is protein kinase C-dependent. J Biol Chem. 1995 Aug 4;270(31):18593–18597. doi: 10.1074/jbc.270.31.18593. [DOI] [PubMed] [Google Scholar]
  33. Petersen B. H., Graham J. A., Brooks G. F. Human deficiency of the eighth component of complement. The requirement of C8 for serum Neisseria gonorrhoeae bactericidal activity. J Clin Invest. 1976 Feb;57(2):283–290. doi: 10.1172/JCI108279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piot P., Islam M. Q. Sexually transmitted diseases in the 1990s. Global epidemiology and challenges for control. Sex Transm Dis. 1994 Mar-Apr;21(2 Suppl):S7–13. [PubMed] [Google Scholar]
  35. 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]
  36. Preissner K. T., Grulich-Henn J., Ehrlich H. J., Declerck P., Justus C., Collen D., Pannekoek H., Müller-Berghaus G. Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin. J Biol Chem. 1990 Oct 25;265(30):18490–18498. [PubMed] [Google Scholar]
  37. Richardson W. P., Sadoff J. C. Induced engulfment of Neisseria gonorrhoeae by tissue culture cells. Infect Immun. 1988 Sep;56(9):2512–2514. doi: 10.1128/iai.56.9.2512-2514.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ringnér M., Valkonen K. H., Wadström T. Binding of vitronectin and plasminogen to Helicobacter pylori. FEMS Immunol Med Microbiol. 1994 Jun;9(1):29–34. doi: 10.1111/j.1574-695X.1994.tb00470.x. [DOI] [PubMed] [Google Scholar]
  39. Robertson B. D., Frosch M., van Putten J. P. The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide. Mol Microbiol. 1993 May;8(5):891–901. doi: 10.1111/j.1365-2958.1993.tb01635.x. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Simon D., Rest R. F. Escherichia coli expressing a Neisseria gonorrhoeae opacity-associated outer membrane protein invade human cervical and endometrial epithelial cell lines. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5512–5516. doi: 10.1073/pnas.89.12.5512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stockmann A., Hess S., Declerck P., Timpl R., Preissner K. T. Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein. J Biol Chem. 1993 Oct 25;268(30):22874–22882. [PubMed] [Google Scholar]
  44. Sweet R. L., Draper D. L., Hadley W. K. Etiology of acute salpingitis: influence of episode number and duration of symptoms. Obstet Gynecol. 1981 Jul;58(1):62–68. [PubMed] [Google Scholar]
  45. Sweet R. L. Sexually transmitted diseases. Pelvic inflammatory disease and infertility in women. Infect Dis Clin North Am. 1987 Mar;1(1):199–215. [PubMed] [Google Scholar]
  46. Tomasini B. R., Mosher D. F. Vitronectin. Prog Hemost Thromb. 1991;10:269–305. [PubMed] [Google Scholar]
  47. Tramont E. C. Inhibition of adherence of Neisseria gonorrhoeae by human genital secretions. J Clin Invest. 1977 Jan;59(1):117–124. doi: 10.1172/JCI108608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tran Van Nhieu G., Isberg R. R. Isolation and identification of eukaryotic receptors promoting bacterial internalization. Methods Enzymol. 1994;236:307–318. doi: 10.1016/0076-6879(94)36023-5. [DOI] [PubMed] [Google Scholar]
  49. Ullberg M., Kuusela P., Kristiansen B. E., Kronvall G. Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoeae and formation of surface-associated plasmin. J Infect Dis. 1992 Dec;166(6):1329–1334. doi: 10.1093/infdis/166.6.1329. [DOI] [PubMed] [Google Scholar]
  50. Underwood P. A., Bennett F. A. A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin. J Cell Sci. 1989 Aug;93(Pt 4):641–649. doi: 10.1242/jcs.93.4.641. [DOI] [PubMed] [Google Scholar]
  51. Virji M., Everson J. S. Comparative virulence of opacity variants of Neisseria gonorrhoeae strain P9. Infect Immun. 1981 Mar;31(3):965–970. doi: 10.1128/iai.31.3.965-970.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Virji M., Makepeace K., Moxon E. R. Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions. Mol Microbiol. 1994 Oct;14(1):173–184. doi: 10.1111/j.1365-2958.1994.tb01277.x. [DOI] [PubMed] [Google Scholar]
  53. Woods A., Couchman J. R. Heparan sulfate proteoglycans and signalling in cell adhesion. Adv Exp Med Biol. 1992;313:87–96. doi: 10.1007/978-1-4899-2444-5_9. [DOI] [PubMed] [Google Scholar]
  54. Woods A., Couchman J. R. Protein kinase C involvement in focal adhesion formation. J Cell Sci. 1992 Feb;101(Pt 2):277–290. doi: 10.1242/jcs.101.2.277. [DOI] [PubMed] [Google Scholar]
  55. de Boer H. C., Preissner K. T., Bouma B. N., de Groot P. G. Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complex into the subendothelial matrix. J Biol Chem. 1995 Dec 22;270(51):30733–30740. doi: 10.1074/jbc.270.51.30733. [DOI] [PubMed] [Google Scholar]
  56. van Putten J. P., Paul S. M. Binding of syndecan-like cell surface proteoglycan receptors is required for Neisseria gonorrhoeae entry into human mucosal cells. EMBO J. 1995 May 15;14(10):2144–2154. doi: 10.1002/j.1460-2075.1995.tb07208.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES