Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Jun;63(6):2356–2360. doi: 10.1128/iai.63.6.2356-2360.1995

Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit.

M R Fontana 1, R Manetti 1, V Giannelli 1, C Magagnoli 1, A Marchini 1, R Olivieri 1, M Domenighini 1, R Rappuoli 1, M Pizza 1
PMCID: PMC173311  PMID: 7768621

Abstract

Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated. Six of them encoded proteins that were fully assembled in the AB5 structure and were nontoxic; these proteins were CT-D53 (Val-53-->Asp), CT-K63 (Ser-63-->Lys), CT-K97 (Val-97-->Lys), CT-K104 (Tyr-104-->Lys), CT-S106 (Pro-106-->Ser), and the double mutant CT-D53/K63 (Val-53-->Asp, Ser-63-->Lys). Two of the mutations encoded proteins that were assembled into the AB5 structure but were still toxic; these proteins were CT-H54 (Arg-54-->His) and CT-N107 (His-107-->Asn). Finally, one of the mutant proteins, CT-E114 (Ser-114-->Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized. The CT-K63 mutant, which was the most efficient in assembly of the AB5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits. This molecule may be useful for the construction of improved vaccines against cholera.

Full Text

The Full Text of this article is available as a PDF (607.6 KB).

Selected References

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

  1. Bhattacharya M. K., Bhattacharya S. K., Garg S., Saha P. K., Dutta D., Nair G. B., Deb B. C., Das K. P. Outbreak of Vibrio cholerae non-O1 in India and Bangladesh. Lancet. 1993 May 22;341(8856):1346–1347. doi: 10.1016/0140-6736(93)90855-b. [DOI] [PubMed] [Google Scholar]
  2. Black R. E., Levine M. M., Clements M. L., Young C. R., Svennerholm A. M., Holmgren J. Protective efficacy in humans of killed whole-vibrio oral cholera vaccine with and without the B subunit of cholera toxin. Infect Immun. 1987 May;55(5):1116–1120. doi: 10.1128/iai.55.5.1116-1120.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burnette W. N., Mar V. L., Platler B. W., Schlotterbeck J. D., McGinley M. D., Stoney K. S., Rohde M. F., Kaslow H. R. Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity. Infect Immun. 1991 Nov;59(11):4266–4270. doi: 10.1128/iai.59.11.4266-4270.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clemens J. D., Harris J. R., Sack D. A., Chakraborty J., Ahmed F., Stanton B. F., Khan M. U., Kay B. A., Huda N., Khan M. R. Field trial of oral cholera vaccines in Bangladesh: results of one year of follow-up. J Infect Dis. 1988 Jul;158(1):60–69. doi: 10.1093/infdis/158.1.60. [DOI] [PubMed] [Google Scholar]
  5. Clemens J. D., Sack D. A., Harris J. R., Chakraborty J., Neogy P. K., Stanton B., Huda N., Khan M. U., Kay B. A., Khan M. R. Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis. 1988 Aug;158(2):372–377. doi: 10.1093/infdis/158.2.372. [DOI] [PubMed] [Google Scholar]
  6. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Domenighini M., Magagnoli C., Pizza M., Rappuoli R. Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol Microbiol. 1994 Oct;14(1):41–50. doi: 10.1111/j.1365-2958.1994.tb01265.x. [DOI] [PubMed] [Google Scholar]
  8. Donta S. T., Moon H. W., Whipp S. C. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science. 1974 Jan 25;183(4122):334–336. doi: 10.1126/science.183.4122.334. [DOI] [PubMed] [Google Scholar]
  9. Field M., Rao M. C., Chang E. B. Intestinal electrolyte transport and diarrheal disease (1). N Engl J Med. 1989 Sep 21;321(12):800–806. doi: 10.1056/NEJM198909213211206. [DOI] [PubMed] [Google Scholar]
  10. Finkelstein R. A., Burks M. F., Zupan A., Dallas W. S., Jacob C. O., Ludwig D. S. Epitopes of the cholera family of enterotoxins. Rev Infect Dis. 1987 May-Jun;9(3):544–561. doi: 10.1093/clinids/9.3.544. [DOI] [PubMed] [Google Scholar]
  11. Gill D. M., Woolkalis M. J. Cholera toxin-catalyzed [32P]ADP-ribosylation of proteins. Methods Enzymol. 1991;195:267–280. doi: 10.1016/0076-6879(91)95172-g. [DOI] [PubMed] [Google Scholar]
  12. Glineur C., Locht C. Importance of ADP-ribosylation in the morphological changes of PC12 cells induced by cholera toxin. Infect Immun. 1994 Oct;62(10):4176–4185. doi: 10.1128/iai.62.10.4176-4185.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harford S., Dykes C. W., Hobden A. N., Read M. J., Halliday I. J. Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene. Eur J Biochem. 1989 Aug 1;183(2):311–316. doi: 10.1111/j.1432-1033.1989.tb14930.x. [DOI] [PubMed] [Google Scholar]
  14. Hirabayashi Y., Tamura S. I., Suzuki Y., Nagamine T., Aizawa C., Shimada K., Kurata T. H-2-unrestricted adjuvant effect of cholera toxin B subunit on murine antibody responses to influenza virus haemagglutinin. Immunology. 1991 Mar;72(3):329–335. [PMC free article] [PubMed] [Google Scholar]
  15. Holmes R. K., Twiddy E. M. Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits. Infect Immun. 1983 Dec;42(3):914–923. doi: 10.1128/iai.42.3.914-923.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holmgren J., Svennerholm A. M., Clemens J., Sack D., Black R., Levine M. An oral B subunit-whole cell vaccine against cholera: from concept to successful field trial. Adv Exp Med Biol. 1987;216B:1649–1660. [PubMed] [Google Scholar]
  17. Holmgren J., Svennerholm A. M., Jertborn M., Clemens J., Sack D. A., Salenstedt R., Wigzell H. An oral B subunit: whole cell vaccine against cholera. Vaccine. 1992;10(13):911–914. doi: 10.1016/0264-410x(92)90324-d. [DOI] [PubMed] [Google Scholar]
  18. Häse C. C., Thai L. S., Boesman-Finkelstein M., Mar V. L., Burnette W. N., Kaslow H. R., Stevens L. A., Moss J., Finkelstein R. A. Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs. Infect Immun. 1994 Aug;62(8):3051–3057. doi: 10.1128/iai.62.8.3051-3057.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lai C. Y., Cancedda F., Duffy L. K. ADP-ribosyl transferase activity of cholera toxin polypeptide A1 and the effect of limited trypsinolysis. Biochem Biophys Res Commun. 1981 Oct 15;102(3):1021–1027. doi: 10.1016/0006-291x(81)91640-5. [DOI] [PubMed] [Google Scholar]
  20. Lebens M., Johansson S., Osek J., Lindblad M., Holmgren J. Large-scale production of Vibrio cholerae toxin B subunit for use in oral vaccines. Biotechnology (N Y) 1993 Dec;11(13):1574–1578. doi: 10.1038/nbt1293-1574. [DOI] [PubMed] [Google Scholar]
  21. Levine M. M., Kaper J. B., Herrington D., Ketley J., Losonsky G., Tacket C. O., Tall B., Cryz S. Safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103-HgR. Lancet. 1988 Aug 27;2(8609):467–470. doi: 10.1016/s0140-6736(88)90120-1. [DOI] [PubMed] [Google Scholar]
  22. Levine M. M., Levine O. S. Changes in human ecology and behavior in relation to the emergence of diarrheal diseases, including cholera. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2390–2394. doi: 10.1073/pnas.91.7.2390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lindholm L., Holmgren J., Wikström M., Karlsson U., Andersson K., Lycke N. Monoclonal antibodies to cholera toxin with special reference to cross-reactions with Escherichia coli heat-labile enterotoxin. Infect Immun. 1983 May;40(2):570–576. doi: 10.1128/iai.40.2.570-576.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lobet Y., Cluff C. W., Cieplak W., Jr Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin. Infect Immun. 1991 Sep;59(9):2870–2879. doi: 10.1128/iai.59.9.2870-2879.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mandal B. K. Epidemic cholera due to a novel strain of V. cholerae non-01--the beginning of a new pandemic? J Infect. 1993 Sep;27(2):115–117. doi: 10.1016/0163-4453(93)94539-n. [DOI] [PubMed] [Google Scholar]
  26. Mekalanos J. J. Production and purification of cholera toxin. Methods Enzymol. 1988;165:169–175. doi: 10.1016/s0076-6879(88)65027-0. [DOI] [PubMed] [Google Scholar]
  27. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Okamoto K., Okamoto K., Miyama A., Tsuji T., Honda T., Miwatani T. Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin. J Bacteriol. 1988 May;170(5):2208–2211. doi: 10.1128/jb.170.5.2208-2211.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pearson G. D., Mekalanos J. J. Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1982 May;79(9):2976–2980. doi: 10.1073/pnas.79.9.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peltola H., Siitonen A., Kyrönseppä H., Simula I., Mattila L., Oksanen P., Kataja M. J., Cadoz M. Prevention of travellers' diarrhoea by oral B-subunit/whole-cell cholera vaccine. Lancet. 1991 Nov 23;338(8778):1285–1289. doi: 10.1016/0140-6736(91)92590-x. [DOI] [PubMed] [Google Scholar]
  32. Peterson J. W., Hejtmancik K. E., Markel D. E., Craig J. P., Kurosky A. Antigenic specificity of neutralizing antibody to cholera toxin. Infect Immun. 1979 Jun;24(3):774–779. doi: 10.1128/iai.24.3.774-779.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pizza M., Bugnoli M., Manetti R., Covacci A., Rappuoli R. The subunit S1 is important for pertussis toxin secretion. J Biol Chem. 1990 Oct 15;265(29):17759–17763. [PubMed] [Google Scholar]
  34. Pizza M., Domenighini M., Hol W., Giannelli V., Fontana M. R., Giuliani M. M., Magagnoli C., Peppoloni S., Manetti R., Rappuoli R. Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis. Mol Microbiol. 1994 Oct;14(1):51–60. doi: 10.1111/j.1365-2958.1994.tb01266.x. [DOI] [PubMed] [Google Scholar]
  35. Pizza M., Fontana M. R., Giuliani M. M., Domenighini M., Magagnoli C., Giannelli V., Nucci D., Hol W., Manetti R., Rappuoli R. A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit. J Exp Med. 1994 Dec 1;180(6):2147–2153. doi: 10.1084/jem.180.6.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rappaport R. S., Rubin B. A., Tint H. Development of a purified cholera toxoid. I. Purification of toxin. Infect Immun. 1974 Feb;9(2):294–303. doi: 10.1128/iai.9.2.294-303.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Remmers E. F., Colwell R. R., Goldsby R. A. Production and characterization of monoclonal antibodies to cholera toxin. Infect Immun. 1982 Jul;37(1):70–76. doi: 10.1128/iai.37.1.70-76.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simanjuntak C. H., O'Hanley P., Punjabi N. H., Noriega F., Pazzaglia G., Dykstra P., Kay B., Suharyono, Budiarso A., Rifai A. R. Safety, immunogenicity, and transmissibility of single-dose live oral cholera vaccine strain CVD 103-HgR in 24- to 59-month-old Indonesian children. J Infect Dis. 1993 Nov;168(5):1169–1176. doi: 10.1093/infdis/168.5.1169. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Spangler B. D. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev. 1992 Dec;56(4):622–647. doi: 10.1128/mr.56.4.622-647.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Svennerholm A. M., Holmgren J. Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid. Infect Immun. 1976 Mar;13(3):735–740. doi: 10.1128/iai.13.3.735-740.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsuji T., Inoue T., Miyama A., Noda M. Glutamic acid-112 of the A subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP-ribosyltransferase activity. FEBS Lett. 1991 Oct 21;291(2):319–321. doi: 10.1016/0014-5793(91)81311-u. [DOI] [PubMed] [Google Scholar]
  44. Tsuji T., Inoue T., Miyama A., Okamoto K., Honda T., Miwatani T. A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity. J Biol Chem. 1990 Dec 25;265(36):22520–22525. [PubMed] [Google Scholar]
  45. Waldor M. K., Mekalanos J. J. Emergence of a new cholera pandemic: molecular analysis of virulence determinants in Vibrio cholerae O139 and development of a live vaccine prototype. J Infect Dis. 1994 Aug;170(2):278–283. doi: 10.1093/infdis/170.2.278. [DOI] [PubMed] [Google Scholar]
  46. Weber J. T., Mintz E. D., Cañizares R., Semiglia A., Gomez I., Sempértegui R., Dávila A., Greene K. D., Puhr N. D., Cameron D. N. Epidemic cholera in Ecuador: multidrug-resistance and transmission by water and seafood. Epidemiol Infect. 1994 Feb;112(1):1–11. doi: 10.1017/s0950268800057368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilson M. M., Chelala C. Cholera is walking south. JAMA. 1994 Oct 19;272(15):1226–1227. [PubMed] [Google Scholar]
  48. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES