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. 1997 Dec;65(12):4943–4950. doi: 10.1128/iai.65.12.4943-4950.1997

Role of receptor binding in toxicity, immunogenicity, and adjuvanticity of Escherichia coli heat-labile enterotoxin.

J J Guidry 1, L Cárdenas 1, E Cheng 1, J D Clements 1
PMCID: PMC175713  PMID: 9393780

Abstract

The role of receptor binding in the toxicity, immunogenicity, and adjuvanticity of the heat-labile enterotoxin of Escherichia coli (LT) was examined by comparing native LT and LT(G33D), a B-subunit receptor binding mutant, with respect to the ability to bind to galactose and to GM1, toxicity on mouse Y-1 adrenal tumor cells, the ability to stimulate adenylate cyclase in Caco-2 cells, enterotoxicity in the patent mouse model, and oral immunogenicity and adjuvanticity. In contrast to native LT, LT(G33D) was unable to bind to the galactosyl moiety of Sepharose 4B or GM1 but did retain the lectin-like ability to bind to immobilized galactose on 6% agarose beads. LT(G33D) had no enterotoxicity in the patent mouse model but exhibited residual toxicity on mouse Y-1 adrenal tumor cells and had an ability equivalent to that of native LT to stimulate adenylate cyclase in Caco-2 cells (5,000 versus 6,900 pmol per mg of protein). In addition, LT(G33D) was unable to serve as an effective oral adjuvant for induction of immunoglobulin G or A directed against a coadministered antigen. Furthermore, LT(G33D) elicited negligible serum and mucosal antibody responses against itself. These data indicate that the toxicity, immunogenicity, and oral adjuvanticity of LT are dependent upon binding of the B subunit to ganglioside GM1.

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Selected References

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  1. Angström J., Teneberg S., Karlsson K. A. Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11859–11863. doi: 10.1073/pnas.91.25.11859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergquist C., Lagergård T., Lindblad M., Holmgren J. Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates. Infect Immun. 1995 May;63(5):2021–2025. doi: 10.1128/iai.63.5.2021-2025.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Bromander A. K., Kjerrulf M., Holmgren J., Lycke N. Cholera toxin enhances alloantigen presentation by cultured intestinal epithelial cells. Scand J Immunol. 1993 Apr;37(4):452–458. doi: 10.1111/j.1365-3083.1993.tb03318.x. [DOI] [PubMed] [Google Scholar]
  5. Cieplak W., Jr, Messer R. J., Konkel M. E., Grant C. C. Role of a potential endoplasmic reticulum retention sequence (RDEL) and the Golgi complex in the cytotonic activity of Escherichia coli heat-labile enterotoxin. Mol Microbiol. 1995 May;16(4):789–800. doi: 10.1111/j.1365-2958.1995.tb02440.x. [DOI] [PubMed] [Google Scholar]
  6. Clarke C. J., Wilson A. D., Williams N. A., Stokes C. R. Mucosal priming of T-lymphocyte responses to fed protein antigens using cholera toxin as an adjuvant. Immunology. 1991 Mar;72(3):323–328. [PMC free article] [PubMed] [Google Scholar]
  7. Clements J. D., Finkelstein R. A. Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures. Infect Immun. 1979 Jun;24(3):760–769. doi: 10.1128/iai.24.3.760-769.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Clements J. D., Yancey R. J., Finkelstein R. A. Properties of homogeneous heat-labile enterotoxin from Escherichia coli. Infect Immun. 1980 Jul;29(1):91–97. doi: 10.1128/iai.29.1.91-97.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Czerkinsky C., Russell M. W., Lycke N., Lindblad M., Holmgren J. Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues. Infect Immun. 1989 Apr;57(4):1072–1077. doi: 10.1128/iai.57.4.1072-1077.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cárdenas L., Clements J. D. Development of mucosal protection against the heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a genetic fusion delivered by a bacterial vector. Infect Immun. 1993 Nov;61(11):4629–4636. doi: 10.1128/iai.61.11.4629-4636.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dertzbaugh M. T., Elson C. O. Comparative effectiveness of the cholera toxin B subunit and alkaline phosphatase as carriers for oral vaccines. Infect Immun. 1993 Jan;61(1):48–55. doi: 10.1128/iai.61.1.48-55.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dertzbaugh M. T., Elson C. O. Reduction in oral immunogenicity of cholera toxin B subunit by N-terminal peptide addition. Infect Immun. 1993 Feb;61(2):384–390. doi: 10.1128/iai.61.2.384-390.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dickinson B. L., Clements J. D. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect Immun. 1995 May;63(5):1617–1623. doi: 10.1128/iai.63.5.1617-1623.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Douce G., Fontana M., Pizza M., Rappuoli R., Dougan G. Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin. Infect Immun. 1997 Jul;65(7):2821–2828. doi: 10.1128/iai.65.7.2821-2828.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elson C. O. Cholera toxin and its subunits as potential oral adjuvants. Curr Top Microbiol Immunol. 1989;146:29–33. doi: 10.1007/978-3-642-74529-4_3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Grant C. C., Messer R. J., Cieplak W., Jr Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin. Infect Immun. 1994 Oct;62(10):4270–4278. doi: 10.1128/iai.62.10.4270-4278.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Holmgren J., Czerkinsky C., Lycke N., Svennerholm A. M. Strategies for the induction of immune responses at mucosal surfaces making use of cholera toxin B subunit as immunogen, carrier, and adjuvant. Am J Trop Med Hyg. 1994;50(5 Suppl):42–54. [PubMed] [Google Scholar]
  22. Holmgren J. Receptors for cholera toxin and Escherichia coli heat-labile enterotoxin revisited. Prog Brain Res. 1994;101:163–177. doi: 10.1016/s0079-6123(08)61947-0. [DOI] [PubMed] [Google Scholar]
  23. Hörnquist E., Lycke N. Cholera toxin adjuvant greatly promotes antigen priming of T cells. Eur J Immunol. 1993 Sep;23(9):2136–2143. doi: 10.1002/eji.1830230914. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Lencer W. I., Constable C., Moe S., Jobling M. G., Webb H. M., Ruston S., Madara J. L., Hirst T. R., Holmes R. K. Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL. J Cell Biol. 1995 Nov;131(4):951–962. doi: 10.1083/jcb.131.4.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lencer W. I., Moe S., Rufo P. A., Madara J. L. Transcytosis of cholera toxin subunits across model human intestinal epithelia. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10094–10098. doi: 10.1073/pnas.92.22.10094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liang X. P., Lamm M. E., Nedrud J. G. Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus. J Immunol. 1988 Sep 1;141(5):1495–1501. [PubMed] [Google Scholar]
  29. Lycke N., Karlsson U., Sjölander A., Magnusson K. E. The adjuvant action of cholera toxin is associated with an increased intestinal permeability for luminal antigens. Scand J Immunol. 1991 Jun;33(6):691–698. doi: 10.1111/j.1365-3083.1991.tb02542.x. [DOI] [PubMed] [Google Scholar]
  30. Merritt E. A., Sixma T. K., Kalk K. H., van Zanten B. A., Hol W. G. Galactose-binding site in Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT). Mol Microbiol. 1994 Aug;13(4):745–753. doi: 10.1111/j.1365-2958.1994.tb00467.x. [DOI] [PubMed] [Google Scholar]
  31. Nashar T. O., Webb H. M., Eaglestone S., Williams N. A., Hirst T. R. Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):226–230. doi: 10.1073/pnas.93.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nedrud J. G., Sigmund N. Cholera toxin as a mucosal adjuvant: III. Antibody responses to nontarget dietary antigens are not increased. Reg Immunol. 1990;3(5):217–222. [PubMed] [Google Scholar]
  33. Orlandi P. A., Curran P. K., Fishman P. H. Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action. J Biol Chem. 1993 Jun 5;268(16):12010–12016. [PubMed] [Google Scholar]
  34. Richardson S. H., Giles J. C., Kruger K. S. Sealed adult mice: new model for enterotoxin evaluation. Infect Immun. 1984 Feb;43(2):482–486. doi: 10.1128/iai.43.2.482-486.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sack D. A., Sack R. B. Test for enterotoxigenic Escherichia coli using Y-1 adrenal cells in miniculture. Infect Immun. 1975 Feb;11(2):334–336. doi: 10.1128/iai.11.2.334-336.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sixma T. K., Kalk K. H., van Zanten B. A., Dauter Z., Kingma J., Witholt B., Hol W. G. Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin. J Mol Biol. 1993 Apr 5;230(3):890–918. doi: 10.1006/jmbi.1993.1209. [DOI] [PubMed] [Google Scholar]
  39. Sixma T. K., Pronk S. E., Kalk K. H., Wartna E. S., van Zanten B. A., Witholt B., Hol W. G. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature. 1991 May 30;351(6325):371–377. doi: 10.1038/351371a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Snider D. P., Marshall J. S., Perdue M. H., Liang H. Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin. J Immunol. 1994 Jul 15;153(2):647–657. [PubMed] [Google Scholar]
  42. Takahashi I., Marinaro M., Kiyono H., Jackson R. J., Nakagawa I., Fujihashi K., Hamada S., Clements J. D., Bost K. L., McGhee J. R. Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin. J Infect Dis. 1996 Mar;173(3):627–635. doi: 10.1093/infdis/173.3.627. [DOI] [PubMed] [Google Scholar]
  43. Tamura S., Funato H., Nagamine T., Aizawa C., Kurata T. Effectiveness of cholera toxin B subunit as an adjuvant for nasal influenza vaccination despite pre-existing immunity to CTB. Vaccine. 1989 Dec;7(6):503–505. doi: 10.1016/0264-410x(89)90273-9. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Xu-Amano J., Jackson R. J., Fujihashi K., Kiyono H., Staats H. F., McGhee J. R. Helper Th1 and Th2 cell responses following mucosal or systemic immunization with cholera toxin. Vaccine. 1994 Aug;12(10):903–911. doi: 10.1016/0264-410x(94)90033-7. [DOI] [PubMed] [Google Scholar]
  47. Xu-Amano J., Kiyono H., Jackson R. J., Staats H. F., Fujihashi K., Burrows P. D., Elson C. O., Pillai S., McGhee J. R. Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues. J Exp Med. 1993 Oct 1;178(4):1309–1320. doi: 10.1084/jem.178.4.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yamamoto S., Kiyono H., Yamamoto M., Imaoka K., Fujihashi K., Van Ginkel F. W., Noda M., Takeda Y., McGhee J. R. A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5267–5272. doi: 10.1073/pnas.94.10.5267. [DOI] [PMC free article] [PubMed] [Google Scholar]

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