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. 1993 Jun;61(6):2445–2452. doi: 10.1128/iai.61.6.2445-2452.1993

Concomitant loss of conformation and superantigenic activity of staphylococcal enterotoxin B deletion mutant proteins.

B Metzroth 1, T Marx 1, M Linnig 1, B Fleischer 1
PMCID: PMC280867  PMID: 8500880

Abstract

The T-cell-stimulating activity of staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases. To investigate the immunologically active domains of the SEB molecule, we have produced truncated fragments of recombinant SEB by C-terminal and N-terminal deletions. The fragments were expressed as fusion proteins with protein A, including a cleavage site to remove the protein A part. Mutant proteins were tested for the ability to stimulate human resting T cells and SEB-reactive T-cell clones. Deletion of only 9 amino acids from the C terminus leads to complete loss of T-cell-stimulating activity. Removing further amino acids from the SEB molecule did not lead to a reexpression of T-cell-mitogenic activity. A mutant protein, however, in which the 9 C-terminal amino acids were replaced with a tail of 68 amino acids encoded by the vector was fully active. Two mutant proteins with N-terminal deletions of 60 and 81 amino acids were inactive as well. A neutralizing monoclonal antibody against a conformational epitope lost binding with all the inactive mutant proteins only, whereas a monoclonal antibody recognizing an epitope involved in emetic activity reacted with all mutant proteins. These results suggest that even small deletions at the C terminus affect the three-dimensional conformation of the SEB molecule.

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Selected References

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  1. Alber G., Hammer D. K., Fleischer B. Relationship between enterotoxic- and T lymphocyte-stimulating activity of staphylococcal enterotoxin B. J Immunol. 1990 Jun 15;144(12):4501–4506. [PubMed] [Google Scholar]
  2. Bamberger U., Scheuber P. H., Sailer-Kramer B., Bartsch K., Hartmann A., Beck G., Hammer D. K. Anti-idiotypic antibodies that inhibit immediate-type skin reactions in unsensitized monkeys on challenge with staphylococcal enterotoxin. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7054–7058. doi: 10.1073/pnas.83.18.7054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blomster-Hautamaa D. A., Novick R. P., Schlievert P. M. Localization of biologic functions of toxic shock syndrome toxin-1 by use of monoclonal antibodies and cyanogen bromide-generated toxin fragments. J Immunol. 1986 Dec 1;137(11):3572–3576. [PubMed] [Google Scholar]
  4. Bohach G. A., Handley J. P., Schlievert P. M. Biological and immunological properties of the carboxyl terminus of staphylococcal enterotoxin C1. Infect Immun. 1989 Jan;57(1):23–28. doi: 10.1128/iai.57.1.23-28.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bröker M. Improved "ATG vector" series for bacterial synthesis of proteins and protein fragments. Biotechniques. 1988 Sep;6(8):734–734. [PubMed] [Google Scholar]
  6. Buelow R., O'Hehir R. E., Schreifels R., Kummerehl T. J., Riley G., Lamb J. R. Localization of the immunologic activity in the superantigen Staphylococcal enterotoxin B using truncated recombinant fusion proteins. J Immunol. 1992 Jan 1;148(1):1–6. [PubMed] [Google Scholar]
  7. Chintagumpala M. M., Mollick J. A., Rich R. R. Staphylococcal toxins bind to different sites on HLA-DR. J Immunol. 1991 Dec 1;147(11):3876–3881. [PubMed] [Google Scholar]
  8. Couch J. L., Soltis M. T., Betley M. J. Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene. J Bacteriol. 1988 Jul;170(7):2954–2960. doi: 10.1128/jb.170.7.2954-2960.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edwin C., Kass E. H. Identification of functional antigenic segments of toxic shock syndrome toxin 1 by differential immunoreactivity and by differential mitogenic responses of human peripheral blood mononuclear cells, using active toxin fragments. Infect Immun. 1989 Jul;57(7):2230–2236. doi: 10.1128/iai.57.7.2230-2236.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fleischer B., Bailey C. J. Recombinant epidermolytic (exfoliative) toxin A of Staphylococcus aureus is not a superantigen. Med Microbiol Immunol. 1992;180(6):273–278. doi: 10.1007/BF00191548. [DOI] [PubMed] [Google Scholar]
  11. Fleischer B., Gerardy-Schahn R., Metzroth B., Carrel S., Gerlach D., Köhler W. An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction. J Immunol. 1991 Jan 1;146(1):11–17. [PubMed] [Google Scholar]
  12. Fleischer B., Schmidt K. H., Gerlach D., Köhler W. Separation of T-cell-stimulating activity from streptococcal M protein. Infect Immun. 1992 May;60(5):1767–1770. doi: 10.1128/iai.60.5.1767-1770.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fleischer B., Schrezenmeier H. T cell stimulation by staphylococcal enterotoxins. Clonally variable response and requirement for major histocompatibility complex class II molecules on accessory or target cells. J Exp Med. 1988 May 1;167(5):1697–1707. doi: 10.1084/jem.167.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fraser J. D. High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature. 1989 May 18;339(6221):221–223. doi: 10.1038/339221a0. [DOI] [PubMed] [Google Scholar]
  15. Grossman D., Cook R. G., Sparrow J. T., Mollick J. A., Rich R. R. Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes. J Exp Med. 1990 Dec 1;172(6):1831–1841. doi: 10.1084/jem.172.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hufnagle W. O., Tremaine M. T., Betley M. J. The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule. Infect Immun. 1991 Jun;59(6):2126–2134. doi: 10.1128/iai.59.6.2126-2134.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Irwin M. J., Hudson K. R., Fraser J. D., Gascoigne N. R. Enterotoxin residues determining T-cell receptor V beta binding specificity. Nature. 1992 Oct 29;359(6398):841–843. doi: 10.1038/359841a0. [DOI] [PubMed] [Google Scholar]
  18. Jones C. L., Khan S. A. Nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus. J Bacteriol. 1986 Apr;166(1):29–33. doi: 10.1128/jb.166.1.29-33.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kappler J., Kotzin B., Herron L., Gelfand E. W., Bigler R. D., Boylston A., Carrel S., Posnett D. N., Choi Y., Marrack P. V beta-specific stimulation of human T cells by staphylococcal toxins. Science. 1989 May 19;244(4906):811–813. doi: 10.1126/science.2524876. [DOI] [PubMed] [Google Scholar]
  20. MacDonald H. R., Baschieri S., Lees R. K. Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo. Eur J Immunol. 1991 Aug;21(8):1963–1966. doi: 10.1002/eji.1830210827. [DOI] [PubMed] [Google Scholar]
  21. Marrack P., Blackman M., Kushnir E., Kappler J. The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells. J Exp Med. 1990 Feb 1;171(2):455–464. doi: 10.1084/jem.171.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marrack P., Kappler J. The staphylococcal enterotoxins and their relatives. Science. 1990 May 11;248(4956):705–711. doi: 10.1126/science.2185544. [DOI] [PubMed] [Google Scholar]
  23. Nilsson B., Abrahmsén L., Uhlén M. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 1985 Apr;4(4):1075–1080. doi: 10.1002/j.1460-2075.1985.tb03741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Noskova V. P., Ezepchuk YuV, Noskov A. N. Topology of the functions in molecule of staphylococcal enterotoxin Type A. Int J Biochem. 1984;16(2):201–206. doi: 10.1016/0020-711x(84)90073-9. [DOI] [PubMed] [Google Scholar]
  25. Pontzer C. H., Russell J. K., Johnson H. M. Structural basis for differential binding of staphylococcal enterotoxin A and toxic shock syndrome toxin 1 to class II major histocompatibility molecules. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):125–128. doi: 10.1073/pnas.88.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reck B., Scheuber P. H., Londong W., Sailer-Kramer B., Bartsch K., Hammer D. K. Protection against the staphylococcal enterotoxin-induced intestinal disorder in the monkey by anti-idiotypic antibodies. Proc Natl Acad Sci U S A. 1988 May;85(9):3170–3174. doi: 10.1073/pnas.85.9.3170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rust C. J., Verreck F., Vietor H., Koning F. Specific recognition of staphylococcal enterotoxin A by human T cells bearing receptors with the V gamma 9 region. Nature. 1990 Aug 9;346(6284):572–574. doi: 10.1038/346572a0. [DOI] [PubMed] [Google Scholar]
  28. Spero L., Morlock B. A. Biological activities of the peptides of staphylococcal enterotoxin C formed by limited tryptic hydrolysis. J Biol Chem. 1978 Dec 25;253(24):8787–8791. [PubMed] [Google Scholar]
  29. Swaminathan S., Furey W., Pletcher J., Sax M. Crystal structure of staphylococcal enterotoxin B, a superantigen. Nature. 1992 Oct 29;359(6398):801–806. doi: 10.1038/359801a0. [DOI] [PubMed] [Google Scholar]

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