Skip to main content

Genetic Detoxification of Bacterial Toxins

  • Protocol
Vaccine Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 4))

Abstract

Several pathogens, such as Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Vibrio cholerae, enterotoxigenic Escherichia co1i (1), and even some emerging pathogens, such as Helicobacter pylori (2), produce potent toxins that are responsible for the pathology caused by the bacterium. In most cases the disease, and often even the infection, can be prevented by a vaccine that induces immunity against the toxin. In order to be used in vaccines, the dangerous toxins need to be depleted of their toxic activity in an effective and irreversible manner. The most effective way to inactivate toxins for inclusion in vaccines was developed by Ramon in 1924 by using formaldehyde treatment at 37°C to detoxify diphtheria toxin (3). This method was then used to inactivate other toxins and also viral and bacterial suspensions. Even today, widely used vaccines, such as diphtheria, tetanus, inactivated polio, and whole cell pertussis, and even some of the newly developed acellular pertussis vaccines are produced using formaldehyde or other chemical treatments to inactivate the toxin and/or kill the microorganisms that are present in the vaccine (4, 5).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rappuoh, R. and Pizza, M. (1991) Structure and evolutionary aspects of ADP-ribosylating toxins, in Sourcebook of Bacterial Protein Toxins (Alouf, J and Freer, J., eds), Academic, New York, pp. 1–20

    Google Scholar 

  2. Telford, J. L., Ghiara, P., Dell’Orco, M., Comanducci, M., Burroni, D., Bugnoli, M., Tecce, M. F., Censini, S., Covacci, A., Xiang Z., Papini, E., Montecucco, C., Parente, L., and Rappuoli, R. (1994) Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J. Exp. Med. 179, 1653–1658

    Article  PubMed  CAS  Google Scholar 

  3. Ramon, G. (1924) Sur la toxine et sur l’anatoxine diphtheriques. Ann. Inst Pasteur 38, 1–10.

    CAS  Google Scholar 

  4. Rappuoh, R. (1990) New and improved vaccines against diphtheria and tetanus, in New Generation Vaccines (Woodrow, G. and Levine, M. M., eds.), Marcel Dekker, New York, pp. 251–268.

    Google Scholar 

  5. Rappuoli, R. (1994) Toxin inactivation and antigen stabilization: two different uses of formaldehyde-short review. Vuccine 12, 579–581.

    Article  CAS  Google Scholar 

  6. Pizza, M., Covacci, A., Bartoloni, A., Perugini, M., Nencioni, L., De Magistris, M. T., Villa, L., Nucci, D., Manetti, R., Bugnoli, M., Giovanrtom, F., Olivieri, R., Barbieri, J T., Sato, H, and Rappuoh, R. (1989) Mutants of pertussis toxin surtable for vaccine development, Science 246, 497–500.

    Article  PubMed  CAS  Google Scholar 

  7. Rappuoli, R., Podda, A., Pizza, M., Covacci, A., Bartolom, A., De Magistris, M. T., and Nencioni, L. (1992) Progress towards the development of new vaccines against whooping cough Vaccine 10, 1027–1032

    Article  PubMed  CAS  Google Scholar 

  8. Podda, A., Deluca, E. C, Contu, B., Furlan, R., Maida, A., Moiraghi, A., et al. (1994) Comparative study of a whole-cell pertussis vaccine and a recombinant acellular pertussis vaccine. J. Pedzatr. 124, 921–926.

    Article  CAS  Google Scholar 

  9. Pizza, M., Domenighini, M., Hol, W., Giannelli, V., Fontana, M. R., Giuliani, M. M., Magagnoli, C., Peppoloni, S., Manetti, R., and Rappuoh, R. (1994) Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis Mol Microbial. 14, 51–60.

    Article  CAS  Google Scholar 

  10. Pizza, M., Fontana, M. R., Giuliani, M. M., Domebnighini, M., Magagnoli, C., Giannelli, V., Nucci, D., Hol, W., Manetti, R., and Rappuoh, R. (1994) A genetically detoxified derivative of heat-labile E. coli enterotoxin induces neutralizing antibodies agamst the A subunit. J. Exp. Med. 6, 2147–2153.

    Article  Google Scholar 

  11. Deuce, G., Turcotte, C., Cropley, I., Robets, M., Pizza, M., Domenighini, M., Rappuoli, R., and Dougan, G. (1995) Mutants of Escherichia coli heat-labile toxin lacking ADP-nbosyltransferase activity act as nontoxic, mucosal adjuvants. Proc. Natl. Acad. Sci. USA 92, 1644–1648

    Article  Google Scholar 

  12. Tamura, M., Nogimori, K., Katada, T., Ui, M., and Ishii, S. (1982) Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry 21, 5516–5520.

    Article  PubMed  CAS  Google Scholar 

  13. Sixma, T. K., Pronk, S. E., Kalk, K. H., Wartna, E S., van Zanten, B A. M., Witholt, B.,and Hol, W. G. J. (1991) Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature 351, 371–377.

    CAS  Google Scholar 

  14. Stein, P. E., Boodhoo, A., Armstrong, G. D., Cockle, S. A., Klein, M. H., and Read, R. J. (1994) The crystal structure of pertussis toxin. Structure 2, 45–57.

    Article  PubMed  CAS  Google Scholar 

  15. Domenighini, M., Montecucco, C., Ripka, W. C., and Rappuoli, R. (1991) Computer modeling of the NAD binding sate of ADP-ribosylating toxins: active site structure and mechanism of NAD binding. Mol. Microbial 5, 23–31

    Article  CAS  Google Scholar 

  16. Domenighini, M., Magagnoli, C., Pizza, M, and Rappuoli, R. (1994) Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol. Mtcrobiol. 14, 41–50.

    Article  CAS  Google Scholar 

  17. Nicosia, A., Perugini, M., Franzini, C., Casagli, M. C., Borri, M. G., Antoni, G., Almoni, M., Nero, P., Ratti, G., and Rappuoli, R. (1986) Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication Proc. Natl. Acad. Sci. USA 83, 4631–4635.

    Article  PubMed  CAS  Google Scholar 

  18. Locht, C. and Keith, J. M. (1986) Pertussis toxin gene: nucleotide sequence and genetic organization. Science 232, 1258–1264.

    Article  PubMed  CAS  Google Scholar 

  19. Mekalanos, J. J., Swartz, D. J., Pearson, G. D., Harford, N., Groyne, F., and de Wilde, M. (1983) Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature 306, 551–557.

    Article  PubMed  CAS  Google Scholar 

  20. Dallas, W. S. and Falkow, S. (1980) Amino acid homology between cholera toxin and Eschertchia coli heat labile toxin Nature 288, 499–501.

    Article  PubMed  CAS  Google Scholar 

  21. Zoller, M. J. and Smith, M. (1982) 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. 10, 6487–6500

    Article  PubMed  CAS  Google Scholar 

  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  23. Pittman, M. (1984) Genus Bordetella, in Bergey’s Manual of Systemic Bacteriology (Krief, N. R. and Holt, J. G., eds.), William and Wilkins, Baltimore, pp. 338

    Google Scholar 

  24. Stainer, D. W. and Scholte, M J. (1971) A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbtol. 63, 211–220

    Google Scholar 

  25. Stibitz, S. and Yang, M. S. (1991) Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis. J. Bacteriol 173, 4288–4291.

    PubMed  CAS  Google Scholar 

  26. Simon, R., Priefer, U., and Puhler, A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gramnegative bacteria. BioTechnology 1, 784–791.

    Article  CAS  Google Scholar 

  27. Lebens, M., Johansson, S, Osek, J., Lindblad, M., and Holmgren, J. (1993) Largescale production of Vibrio-cholerae toxin-B subunit for use in oral vaccines BioTechnology 11, 1574–1578

    Article  PubMed  CAS  Google Scholar 

  28. Donta, S. T., Moon, H. W., and Whipp, S. C. (1973) Detection and use of heatlabile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture Science 183, 334–335.

    Article  Google Scholar 

  29. Hewlett, E. L., Sauer, K. T., Myers, G. A., Cowell, J. L., and Guerrant, R. (1983) Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Inject. Immun. 40, 1198–1203.

    CAS  Google Scholar 

  30. Stibitz, S, Black, W., and Falkow, S. (1986) The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene 50, 133–140.

    Article  PubMed  CAS  Google Scholar 

  31. Pronk, S. E., Hofstra, H., Groendijk, H., Kingma, J., Swarte, M. B. A., Dorner, F., Drenth, J., Hol, W. G. J., and Witholt, B. (1985) Heat-labile enterotoxin of Escherichia coli: characterization of different crystal forms. J. Biol. Chem. 260, 13,580–13,584.

    PubMed  CAS  Google Scholar 

  32. Pearson, G. D. N. and Mekalanos, J. J. (1982) Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12. Proc. Natl. Acad. Sci. USA 79, 2976–2980.

    Article  PubMed  CAS  Google Scholar 

  33. Dente, L., Cesarem, G., and Cortese, R. (1983) pEMBL: a new family of single stranded plasmids. Nuclezc Acids Res. 11, 1645–1655.

    Article  CAS  Google Scholar 

  34. Mekalanos, J. (1988) Production and purification of cholera toxin. Meth. Enzymol. 165, 169–175.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1996 Humana Press Inc, Totowa, NJ

About this protocol

Cite this protocol

Pizza, M., Fontana, M.R., Scarlato, V., Rappuoli, R. (1996). Genetic Detoxification of Bacterial Toxins. In: Robinson, A., Farrar, G.H., Wiblin, C.N. (eds) Vaccine Protocols. Methods in Molecular Medicine™, vol 4. Humana Press. https://doi.org/10.1385/0-89603-334-1:91

Download citation

  • DOI: https://doi.org/10.1385/0-89603-334-1:91

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-334-4

  • Online ISBN: 978-1-59259-588-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics