Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Feb;65(2):537–543. doi: 10.1128/iai.65.2.537-543.1997

Identification of D motif epitopes in Staphylococcus aureus fibronectin-binding protein for the production of antibody inhibitors of fibronectin binding.

Q Sun 1, G M Smith 1, C Zahradka 1, M J McGavin 1
PMCID: PMC176093  PMID: 9009310

Abstract

A fibronectin-binding protein (FnBP) adhesin of Staphylococcus aureus possesses three 37- or 38-amino-acid motifs (D1, D2, and D3) that can each bind fibronectin (Fn) with low affinity and that in tandem comprise D1-3, a high-affinity Fn-binding domain. To identify epitopes for the generation of adhesion-blocking antibodies, rabbits were immunized with recombinant D1-3 or with a glutathione S-transferase fusion protein, GSTD1-3. Affinity-purified antibodies from the D1-3 immunization were poor inhibitors of Fn binding to S. aureus and recognized several different epitopes, with a preference for clusters of acidic amino acids that do not contribute to Fn binding. Antibodies generated with GSTD1-3 as an immunogen were more effective inhibitors, but concentrations in excess of 20 microg x ml-1 did not promote more than 50% inhibition. These antibodies were highly specific for amino acids 21 to 34 of D1 (D1(21-34)), which contain a sequence that is essential for Fn binding and are identical to D2 at 12 of 14 residues. Neither antibody preparation recognized D3(20-33) of the D3 motif, where the only homology to D1(21-34) and D2(21-34) comprises a sequence motif, GG(X3,4)(I/V)DF, that is critical to Fn binding. However, antibodies specific for both D1(21-34) and D3(20-33) could be obtained by using synthetic peptides corresponding to these sequences as immunogens. F(ab')2 fragments derived from these antibodies each caused 40 to 50% inhibition of Fn binding to S. aureus, and their ability to bind to purified FnBP was eliminated by competing Fn. However, mixtures of the two F(ab')2 preparations did not provide additive or synergistic inhibition of Fn binding. Therefore, inhibition of Fn binding to S. aureus requires antibodies specific for D1(21-34) and D3(20-33), but a mixture of antibodies specific for both sequences did not provide complete inhibition.

Full Text

The Full Text of this article is available as a PDF (249.1 KB).

Selected References

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

  1. Bethell G. S., Ayers J. S., Hancock W. S., Hearn M. T. A novel method of activation of cross-linked agaroses with 1,1'-carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J Biol Chem. 1979 Apr 25;254(8):2572–2574. [PubMed] [Google Scholar]
  2. Boyle M. D. Variation of multifunctional surface binding proteins--a virulence strategy for group A streptococci? J Theor Biol. 1995 Apr 21;173(4):415–426. doi: 10.1006/jtbi.1995.0073. [DOI] [PubMed] [Google Scholar]
  3. Ciborowski P., Flock J. I., Wadström T. Immunological response to a Staphylococcus aureus fibronectin-binding protein. J Med Microbiol. 1992 Dec;37(6):376–381. doi: 10.1099/00222615-37-6-376. [DOI] [PubMed] [Google Scholar]
  4. Cleary P., Retnoningrum D. Group A streptococcal immunoglobulin-binding proteins: adhesins, molecular mimicry or sensory proteins? Trends Microbiol. 1994 Apr;2(4):131–136. doi: 10.1016/0966-842x(94)90600-9. [DOI] [PubMed] [Google Scholar]
  5. Fattom A. I., Naso R. Staphylococcal vaccines: a realistic dream. Ann Med. 1996 Feb;28(1):43–46. doi: 10.3109/07853899608999073. [DOI] [PubMed] [Google Scholar]
  6. Fattom A. I., Sarwar J., Ortiz A., Naso R. A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge. Infect Immun. 1996 May;64(5):1659–1665. doi: 10.1128/iai.64.5.1659-1665.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fedson D. S., Shapiro E. D., LaForce F. M., Mufson M. A., Musher D. M., Spika J. S., Breiman R. F., Broome C. V. Pneumococcal vaccine after 15 years of use. Another view. Arch Intern Med. 1994 Nov 28;154(22):2531–2535. [PubMed] [Google Scholar]
  8. Fröman G., Switalski L. M., Speziale P., Hök M. Isolation and characterization of a fibronectin receptor from Staphylococcus aureus. J Biol Chem. 1987 May 15;262(14):6564–6571. [PubMed] [Google Scholar]
  9. Greene C., McDevitt D., Francois P., Vaudaux P. E., Lew D. P., Foster T. J. Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes. Mol Microbiol. 1995 Sep;17(6):1143–1152. doi: 10.1111/j.1365-2958.1995.mmi_17061143.x. [DOI] [PubMed] [Google Scholar]
  10. Güner E. S. Complement evasion by the Lyme disease spirochete Borrelia burgdorferi grown in host-derived tissue co-cultures: role of fibronectin in complement-resistance. Experientia. 1996 Apr 15;52(4):364–372. doi: 10.1007/BF01919542. [DOI] [PubMed] [Google Scholar]
  11. Hanski E., Caparon M. Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6172–6176. doi: 10.1073/pnas.89.13.6172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. House-Pompeo K., Xu Y., Joh D., Speziale P., Hök M. Conformational changes in the fibronectin binding MSCRAMMs are induced by ligand binding. J Biol Chem. 1996 Jan 19;271(3):1379–1384. doi: 10.1074/jbc.271.3.1379. [DOI] [PubMed] [Google Scholar]
  13. Huff S., Matsuka Y. V., McGavin M. J., Ingham K. C. Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy. J Biol Chem. 1994 Jun 3;269(22):15563–15570. [PubMed] [Google Scholar]
  14. Jönsson K., Signäs C., Müller H. P., Lindberg M. Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene. Eur J Biochem. 1991 Dec 18;202(3):1041–1048. doi: 10.1111/j.1432-1033.1991.tb16468.x. [DOI] [PubMed] [Google Scholar]
  15. Kline J. B., Xu S., Bisno A. L., Collins C. M. Identification of a fibronectin-binding protein (GfbA) in pathogenic group G streptococci. Infect Immun. 1996 Jun;64(6):2122–2129. doi: 10.1128/iai.64.6.2122-2129.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuypers J. M., Proctor R. A. Reduced adherence to traumatized rat heart valves by a low-fibronectin-binding mutant of Staphylococcus aureus. Infect Immun. 1989 Aug;57(8):2306–2312. doi: 10.1128/iai.57.8.2306-2312.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McGavin M. J., Gurusiddappa S., Lindgren P. E., Lindberg M., Raucci G., Hök M. Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus. Involvement of conserved residues in ligand binding. J Biol Chem. 1993 Nov 15;268(32):23946–23953. [PubMed] [Google Scholar]
  18. McGavin M. J., Raucci G., Gurusiddappa S., Hök M. Fibronectin binding determinants of the Staphylococcus aureus fibronectin receptor. J Biol Chem. 1991 May 5;266(13):8343–8347. [PubMed] [Google Scholar]
  19. Miekka S. I., Ingham K. C., Menache D. Rapid methods for isolation of human plasma fibronectin. Thromb Res. 1982 Jul 1;27(1):1–14. doi: 10.1016/0049-3848(82)90272-9. [DOI] [PubMed] [Google Scholar]
  20. Noble W. C., Virani Z., Cree R. G. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jun 1;72(2):195–198. doi: 10.1016/0378-1097(92)90528-v. [DOI] [PubMed] [Google Scholar]
  21. Okada N., Pentland A. P., Falk P., Caparon M. G. M protein and protein F act as important determinants of cell-specific tropism of Streptococcus pyogenes in skin tissue. J Clin Invest. 1994 Sep;94(3):965–977. doi: 10.1172/JCI117463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ozeri V., Tovi A., Burstein I., Natanson-Yaron S., Caparon M. G., Yamada K. M., Akiyama S. K., Vlodavsky I., Hanski E. A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix. EMBO J. 1996 Mar 1;15(5):989–998. [PMC free article] [PubMed] [Google Scholar]
  23. Patti J. M., Allen B. L., McGavin M. J., Hök M. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol. 1994;48:585–617. doi: 10.1146/annurev.mi.48.100194.003101. [DOI] [PubMed] [Google Scholar]
  24. Patti J. M., Hök M. Microbial adhesins recognizing extracellular matrix macromolecules. Curr Opin Cell Biol. 1994 Oct;6(5):752–758. doi: 10.1016/0955-0674(94)90104-x. [DOI] [PubMed] [Google Scholar]
  25. Rozalska B., Sakata N., Wadström T. Staphylococcus aureus fibronectin-binding proteins (FnBPs). Identification of antigenic epitopes using polyclonal antibodies. APMIS. 1994 Feb;102(2):112–118. [PubMed] [Google Scholar]
  26. Shapiro E. D., Berg A. T., Austrian R., Schroeder D., Parcells V., Margolis A., Adair R. K., Clemens J. D. The protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N Engl J Med. 1991 Nov 21;325(21):1453–1460. doi: 10.1056/NEJM199111213252101. [DOI] [PubMed] [Google Scholar]
  27. Sheagren J. N. Staphylococcus aureus. The persistent pathogen (second of two parts). N Engl J Med. 1984 May 31;310(22):1437–1442. doi: 10.1056/NEJM198405313102206. [DOI] [PubMed] [Google Scholar]
  28. Siber G. R. Pneumococcal disease: prospects for a new generation of vaccines. Science. 1994 Sep 2;265(5177):1385–1387. doi: 10.1126/science.8073278. [DOI] [PubMed] [Google Scholar]
  29. Signäs C., Raucci G., Jönsson K., Lindgren P. E., Anantharamaiah G. M., Hök M., Lindberg M. Nucleotide sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides. Proc Natl Acad Sci U S A. 1989 Jan;86(2):699–703. doi: 10.1073/pnas.86.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  31. Speziale P., Joh D., Visai L., Bozzini S., House-Pompeo K., Lindberg M., Hök M. A monoclonal antibody enhances ligand binding of fibronectin MSCRAMM (adhesin) from Streptococcus dysgalactiae. J Biol Chem. 1996 Jan 19;271(3):1371–1378. doi: 10.1074/jbc.271.3.1371. [DOI] [PubMed] [Google Scholar]
  32. Talay S. R., Valentin-Weigand P., Jerlström P. G., Timmis K. N., Chhatwal G. S. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells. Infect Immun. 1992 Sep;60(9):3837–3844. doi: 10.1128/iai.60.9.3837-3844.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vaudaux P., Pittet D., Haeberli A., Huggler E., Nydegger U. E., Lew D. P., Waldvogel F. A. Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen or fibrin. J Infect Dis. 1989 Nov;160(5):865–875. doi: 10.1093/infdis/160.5.865. [DOI] [PubMed] [Google Scholar]
  34. Wangsgard W. P., Meixell G. E., Dasgupta M., Blumenthal D. K. Activation and inhibition of phosphorylase kinase by monospecific antibodies raised against peptides from the regulatory domain of the gamma-subunit. J Biol Chem. 1996 Aug 30;271(35):21126–21133. doi: 10.1074/jbc.271.35.21126. [DOI] [PubMed] [Google Scholar]

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

RESOURCES