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. 1996 Jan 9;93(1):226–230. doi: 10.1073/pnas.93.1.226

Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets.

T O Nashar 1, H M Webb 1, S Eaglestone 1, N A Williams 1, T R Hirst 1
PMCID: PMC40211  PMID: 8552610

Abstract

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

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Selected References

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

  1. Amin T., Hirst T. R. Purification of the B-subunit oligomer of Escherichia coli heat-labile enterotoxin by heterologous expression and secretion in a marine vibrio. Protein Expr Purif. 1994 Apr;5(2):198–204. doi: 10.1006/prep.1994.1031. [DOI] [PubMed] [Google Scholar]
  2. Clements J. D., Hartzog N. M., Lyon F. L. Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens. Vaccine. 1988 Jun;6(3):269–277. doi: 10.1016/0264-410x(88)90223-x. [DOI] [PubMed] [Google Scholar]
  3. Craig S. W., Cuatrecasas P. Mobility of cholera toxin receptors on rat lymphocyte membranes. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3844–3848. doi: 10.1073/pnas.72.10.3844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elson C. O., Ealding W. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J Immunol. 1984 Dec;133(6):2892–2897. [PubMed] [Google Scholar]
  5. Elson C. O., Ealding W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol. 1984 Jun;132(6):2736–2741. [PubMed] [Google Scholar]
  6. Elson C. O., Ealding W., Lefkowitz J. A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions. J Immunol Methods. 1984 Feb 24;67(1):101–108. doi: 10.1016/0022-1759(84)90089-9. [DOI] [PubMed] [Google Scholar]
  7. Elson C. O., Holland S. P., Dertzbaugh M. T., Cuff C. F., Anderson A. O. Morphologic and functional alterations of mucosal T cells by cholera toxin and its B subunit. J Immunol. 1995 Feb 1;154(3):1032–1040. [PubMed] [Google Scholar]
  8. Elson C. O., Solomon S. Activation of cholera toxin-specific T cells in vitro. Infect Immun. 1990 Nov;58(11):3711–3716. doi: 10.1128/iai.58.11.3711-3716.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Francis M. L., Ryan J., Jobling M. G., Holmes R. K., Moss J., Mond J. J. Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation. J Immunol. 1992 Apr 1;148(7):1999–2005. [PubMed] [Google Scholar]
  10. Fukuta S., Magnani J. L., Twiddy E. M., Holmes R. K., Ginsburg V. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb. Infect Immun. 1988 Jul;56(7):1748–1753. doi: 10.1128/iai.56.7.1748-1753.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hakomori S. Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J Biol Chem. 1990 Nov 5;265(31):18713–18716. [PubMed] [Google Scholar]
  12. Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 Jul 30;292(5822):413–417. doi: 10.1038/292413a0. [DOI] [PubMed] [Google Scholar]
  13. Holmgren J., Fredman P., Lindblad M., Svennerholm A. M., Svennerholm L. Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin. Infect Immun. 1982 Nov;38(2):424–433. doi: 10.1128/iai.38.2.424-433.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holmgren J., Lindholm L., Lönnroth I. Interaction of cholera toxin and toxin derivatives with lymphocytes. I. Binding properties and interference with lectin-induced cellular stimulation. J Exp Med. 1974 Apr 1;139(4):801–819. doi: 10.1084/jem.139.4.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hörnqvist E., Goldschmidt T. J., Holmdahl R., Lycke N. Host defense against cholera toxin is strongly CD4+ T cell dependent. Infect Immun. 1991 Oct;59(10):3630–3638. doi: 10.1128/iai.59.10.3630-3638.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jobling M. G., Holmes R. K. Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis. Mol Microbiol. 1991 Jul;5(7):1755–1767. doi: 10.1111/j.1365-2958.1991.tb01925.x. [DOI] [PubMed] [Google Scholar]
  17. Julius M., Maroun C. R., Haughn L. Distinct roles for CD4 and CD8 as co-receptors in antigen receptor signalling. Immunol Today. 1993 Apr;14(4):177–183. doi: 10.1016/0167-5699(93)90282-p. [DOI] [PubMed] [Google Scholar]
  18. Lassila O., Vainio O., Matzinger P. Can B cells turn on virgin T cells? Nature. 1988 Jul 21;334(6179):253–255. doi: 10.1038/334253a0. [DOI] [PubMed] [Google Scholar]
  19. Merritt E. A., Sarfaty S., van den Akker F., L'Hoir C., Martial J. A., Hol W. G. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 1994 Feb;3(2):166–175. doi: 10.1002/pro.5560030202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller A., Lider O., Roberts A. B., Sporn M. B., Weiner H. L. Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):421–425. doi: 10.1073/pnas.89.1.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nashar T. O., Amin T., Marcello A., Hirst T. R. Current progress in the development of the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin as carriers for the oral delivery of heterologous antigens and epitopes. Vaccine. 1993;11(2):235–240. doi: 10.1016/0264-410x(93)90023-q. [DOI] [PubMed] [Google Scholar]
  22. Nashar T. O., Hirst T. R. Immunoregulatory role of H-2 and intra-H-2 alleles on antibody responses to recombinant preparations of B-subunits of Escherichia coli heat-labile enterotoxin (rEtxB) and cholera toxin (rCtxB). Vaccine. 1995 Jun;13(9):803–810. doi: 10.1016/0264-410x(94)00077-z. [DOI] [PubMed] [Google Scholar]
  23. Pineau N., Aucouturier P., Brugier J. C., Preud'homme J. L. Jacalin: a lectin mitogenic for human CD4 T lymphocytes. Clin Exp Immunol. 1990 Jun;80(3):420–425. doi: 10.1111/j.1365-2249.1990.tb03304.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sandkvist M., Hirst T. R., Bagdasarian M. Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin. J Bacteriol. 1987 Oct;169(10):4570–4576. doi: 10.1128/jb.169.10.4570-4576.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sixma T. K., Pronk S. E., Kalk K. H., van Zanten B. A., Berghuis A. M., Hol W. G. Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography. Nature. 1992 Feb 6;355(6360):561–564. doi: 10.1038/355561a0. [DOI] [PubMed] [Google Scholar]
  26. Spiegel S., Fishman P. H. Gangliosides as bimodal regulators of cell growth. Proc Natl Acad Sci U S A. 1987 Jan;84(1):141–145. doi: 10.1073/pnas.84.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  28. Tsuji T., Honda T., Miwatani T., Wakabayashi S., Matsubara H. Analysis of receptor-binding site in Escherichia coli enterotoxin. J Biol Chem. 1985 Jul 15;260(14):8552–8558. [PubMed] [Google Scholar]
  29. Williams N. A., Hill T. J., Hooper D. C. Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to herpes simplex virus in vitro. Immunology. 1991 Jan;72(1):34–39. [PMC free article] [PubMed] [Google Scholar]
  30. Wilson A. D., Bailey M., Williams N. A., Stokes C. R. The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocyanin using cholera toxin as an adjuvant. Eur J Immunol. 1991 Oct;21(10):2333–2339. doi: 10.1002/eji.1830211007. [DOI] [PubMed] [Google Scholar]
  31. Woogen S. D., Ealding W., Elson C. O. Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin. J Immunol. 1987 Dec 1;139(11):3764–3770. [PubMed] [Google Scholar]
  32. Yankelevich B., Brown E., Mazumder A. Prevention of acute graft-versus-host disease by treatment with a novel immunosuppressant. Cholera toxin B subunit. J Immunol. 1995 Apr 1;154(7):3611–3617. [PubMed] [Google Scholar]
  33. Yu J., Webb H., Hirst T. R. A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. Mol Microbiol. 1992 Jul;6(14):1949–1958. doi: 10.1111/j.1365-2958.1992.tb01368.x. [DOI] [PubMed] [Google Scholar]

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