Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Dec;63(12):4715–4720. doi: 10.1128/iai.63.12.4715-4720.1995

Contribution of individual disulfide bonds to biological action of Escherichia coli heat-stable enterotoxin B.

Y L Arriaga 1, B A Harville 1, L A Dreyfus 1
PMCID: PMC173676  PMID: 7591127

Abstract

Heat-stable enterotoxins (STs) of Escherichia coli are peptides which alter normal gut physiology by stimulating the loss of water and electrolytes. The action of heat-stable toxin B (STb) is associated with an increase in levels of lumenal 5-hydroxytryptamine and prostaglandin E2, known mediators of intestinal secretion. In addition, the toxin is responsible for elevation of cytosolic calcium ion levels in cultured cells. STb is a 48-amino-acid basic peptide containing four cysteine residues and two disulfide bonds. Previous work indicates that disulfide bonds are required for intestinal secretory activity, and yet the relative contribution of the two bonds to toxin stability and action is presently unclear. Site-directed mutagenesis was used to alter the cysteine residues of STb to assess the role of the individual disulfide bonds in toxin activity. Our results indicate that loss of a single disulfide bond was sufficient to abolish the intestinal secretory and G protein-coupled calcium ion influx activities associated with STb toxicity. Loss of toxin action was not a function of increased sensitivity of STb mutants to proteolysis, since mutant toxins displayed proteolytic decay rates equivalent to that of wild-type STb. Circular dichroism spectroscopy of mutant STb toxins indicated that single-disulfide-bond elimination did not apparently affect the toxin secondary structure of one mutant, STbC33S,C71S. In contrast, the alpha-helical content of the other disulfide bond mutant, STbC44S,C59G, was significantly altered, as was that of reduced and alkylated authentic STb. Since both Cys-Cys mutant STbs were completely nontoxic, the absence of biological activity cannot be explained by dramatic secondary structural changes alone; keys to the conformational requirements for STb toxicity undoubtedly reside in the three-dimensional structure of this peptide.

Full Text

The Full Text of this article is available as a PDF (257.0 KB).

Selected References

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

  1. Andrade M. A., Chacón P., Merelo J. J., Morán F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 1993 Jun;6(4):383–390. doi: 10.1093/protein/6.4.383. [DOI] [PubMed] [Google Scholar]
  2. Compton L. A., Johnson W. C., Jr Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem. 1986 May 15;155(1):155–167. doi: 10.1016/0003-2697(86)90241-1. [DOI] [PubMed] [Google Scholar]
  3. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dreyfus L. A., Frantz J. C., Robertson D. C. Chemical properties of heat-stable enterotoxins produced by enterotoxigenic Escherichia coli of different host origins. Infect Immun. 1983 Nov;42(2):539–548. doi: 10.1128/iai.42.2.539-548.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dreyfus L. A., Harville B., Howard D. E., Shaban R., Beatty D. M., Morris S. J. Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB). Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3202–3206. doi: 10.1073/pnas.90.8.3202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dreyfus L. A., Urban R. G., Whipp S. C., Slaughter C., Tachias K., Kupersztoch Y. M., Drefus L. A. Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity. Mol Microbiol. 1992 Aug;6(16):2397–2406. doi: 10.1111/j.1365-2958.1992.tb01414.x. [DOI] [PubMed] [Google Scholar]
  7. Field M., Graf L. H., Jr, Laird W. J., Smith P. L. Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2800–2804. doi: 10.1073/pnas.75.6.2800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fujii Y., Hayashi M., Hitotsubashi S., Fuke Y., Yamanaka H., Okamoto K. Purification and characterization of Escherichia coli heat-stable enterotoxin II. J Bacteriol. 1991 Sep;173(17):5516–5522. doi: 10.1128/jb.173.17.5516-5522.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujii Y., Okamuro Y., Hitotsubashi S., Saito A., Akashi N., Okamoto K. Effect of alterations of basic amino acid residues of Escherichia coli heat-stable enterotoxin II on enterotoxicity. Infect Immun. 1994 Jun;62(6):2295–2301. doi: 10.1128/iai.62.6.2295-2301.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garbers D. L. Guanylyl cyclase receptors and their endocrine, paracrine, and autocrine ligands. Cell. 1992 Oct 2;71(1):1–4. doi: 10.1016/0092-8674(92)90258-e. [DOI] [PubMed] [Google Scholar]
  11. Gariépy J., Judd A. K., Schoolnik G. K. Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8907–8911. doi: 10.1073/pnas.84.24.8907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gariépy J., Lane A., Frayman F., Wilbur D., Robien W., Schoolnik G. K., Jardetzky O. Structure of the toxic domain of the Escherichia coli heat-stable enterotoxin ST I. Biochemistry. 1986 Dec 2;25(24):7854–7866. doi: 10.1021/bi00372a011. [DOI] [PubMed] [Google Scholar]
  13. Guerrant R. L., Hughes J. M., Chang B., Robertson D. C., Murad F. Activation of intestinal guanylate cyclase by heat-stable enterotoxin of Escherichia coli: studies of tissue specificity, potential receptors, and intermediates. J Infect Dis. 1980 Aug;142(2):220–228. doi: 10.1093/infdis/142.2.220. [DOI] [PubMed] [Google Scholar]
  14. Harville B. A., Dreyfus L. A. Involvement of 5-hydroxytryptamine and prostaglandin E2 in the intestinal secretory action of Escherichia coli heat-stable enterotoxin B. Infect Immun. 1995 Mar;63(3):745–750. doi: 10.1128/iai.63.3.745-750.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hitotsubashi S., Fujii Y., Yamanaka H., Okamoto K. Some properties of purified Escherichia coli heat-stable enterotoxin II. Infect Immun. 1992 Nov;60(11):4468–4474. doi: 10.1128/iai.60.11.4468-4474.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huott P. A., Liu W., McRoberts J. A., Giannella R. A., Dharmsathaphorn K. Mechanism of action of Escherichia coli heat stable enterotoxin in a human colonic cell line. J Clin Invest. 1988 Aug;82(2):514–523. doi: 10.1172/JCI113626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kennedy D. J., Greenberg R. N., Dunn J. A., Abernathy R., Ryerse J. S., Guerrant R. L. Effects of Escherichia coli heat-stable enterotoxin STb on intestines of mice, rats, rabbits, and piglets. Infect Immun. 1984 Dec;46(3):639–643. doi: 10.1128/iai.46.3.639-643.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kupersztoch Y. M., Tachias K., Moomaw C. R., Dreyfus L. A., Urban R., Slaughter C., Whipp S. Secretion of methanol-insoluble heat-stable enterotoxin (STB): energy- and secA-dependent conversion of pre-STB to an intermediate indistinguishable from the extracellular toxin. J Bacteriol. 1990 May;172(5):2427–2432. doi: 10.1128/jb.172.5.2427-2432.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peterson J. W., Whipp S. C. Comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of Escherichia coli. Infect Immun. 1995 Apr;63(4):1452–1461. doi: 10.1128/iai.63.4.1452-1461.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schulz S., Green C. K., Yuen P. S., Garbers D. L. Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell. 1990 Nov 30;63(5):941–948. doi: 10.1016/0092-8674(90)90497-3. [DOI] [PubMed] [Google Scholar]
  21. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  22. Shimonishi Y., Hidaka Y., Koizumi M., Hane M., Aimoto S., Takeda T., Miwatani T., Takeda Y. Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli. FEBS Lett. 1987 May 4;215(1):165–170. doi: 10.1016/0014-5793(87)80134-5. [DOI] [PubMed] [Google Scholar]
  23. Sukumar M., Rizo J., Wall M., Dreyfus L. A., Kupersztoch Y. M., Gierasch L. M. The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism. Protein Sci. 1995 Sep;4(9):1718–1729. doi: 10.1002/pro.5560040907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Urban R. G., Pipper E. M., Dreyfus L. A., Whipp S. C. High-level production of Escherichia coli STb heat-stable enterotoxin and quantification by a direct enzyme-linked immunosorbent assay. J Clin Microbiol. 1990 Nov;28(11):2383–2388. doi: 10.1128/jcm.28.11.2383-2388.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  26. Weikel C. S., Guerrant R. L. STb enterotoxin of Escherichia coli: cyclic nucleotide-independent secretion. Ciba Found Symp. 1985;112:94–115. doi: 10.1002/9780470720936.ch6. [DOI] [PubMed] [Google Scholar]
  27. Whipp S. C. Assay for enterotoxigenic Escherichia coli heat-stable toxin b in rats and mice. Infect Immun. 1990 Apr;58(4):930–934. doi: 10.1128/iai.58.4.930-934.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. White K. N., Metzger H. Translocation of protein kinase C in rat basophilic leukemic cells induced by phorbol ester or by aggregation of IgE receptors. J Immunol. 1988 Aug 1;141(3):942–947. [PubMed] [Google Scholar]

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

RESOURCES