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. 1992 Feb;60(2):485–490. doi: 10.1128/iai.60.2.485-490.1992

An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine.

V M Gordon 1, S C Whipp 1, H W Moon 1, A D O'Brien 1, J E Samuel 1
PMCID: PMC257653  PMID: 1730480

Abstract

Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv). Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight. Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity. Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity. Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response. Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions. The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form. An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv. Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K). These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively. The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT-IIvE167D. When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold. Pigs that were vaccinated with purified, mutant toxin designated SLT-IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions. These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED.

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Selected References

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

  1. Boyd B., Richardson S., Gariepy J. Serological responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin. Infect Immun. 1991 Mar;59(3):750–757. doi: 10.1128/iai.59.3.750-757.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clugston R. E., Nielsen N. O., Smith D. L. Experimental edema disease of swine (E. coli enterotoxemia). 3. Pathology and pathogenesis. Can J Comp Med. 1974 Jan;38(1):34–43. [PMC free article] [PubMed] [Google Scholar]
  3. Endo Y., Tsurugi K., Yutsudo T., Takeda Y., Ogasawara T., Igarashi K. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur J Biochem. 1988 Jan 15;171(1-2):45–50. doi: 10.1111/j.1432-1033.1988.tb13756.x. [DOI] [PubMed] [Google Scholar]
  4. Frankel A., Schlossman D., Welsh P., Hertler A., Withers D., Johnston S. Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):415–420. doi: 10.1128/mcb.9.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Frankel A., Welsh P., Richardson J., Robertus J. D. Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes. Mol Cell Biol. 1990 Dec;10(12):6257–6263. doi: 10.1128/mcb.10.12.6257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gentry M. K., Dalrymple J. M. Quantitative microtiter cytotoxicity assay for Shigella toxin. J Clin Microbiol. 1980 Sep;12(3):361–366. doi: 10.1128/jcm.12.3.361-366.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hovde C. J., Calderwood S. B., Mekalanos J. J., Collier R. J. Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2568–2572. doi: 10.1073/pnas.85.8.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jackson M. P., Deresiewicz R. L., Calderwood S. B. Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits. J Bacteriol. 1990 Jun;172(6):3346–3350. doi: 10.1128/jb.172.6.3346-3350.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Karmali M. A. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38. doi: 10.1128/cmr.2.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Karmali M. A., Petric M., Lim C., Fleming P. C., Arbus G. S., Lior H. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis. 1985 May;151(5):775–782. doi: 10.1093/infdis/151.5.775. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. MacLeod D. L., Gyles C. L., Wilcock B. P. Reproduction of edema disease of swine with purified Shiga-like toxin-II variant. Vet Pathol. 1991 Jan;28(1):66–73. doi: 10.1177/030098589102800109. [DOI] [PubMed] [Google Scholar]
  13. Marques L. R., Moore M. A., Wells J. G., Wachsmuth I. K., O'Brien A. D. Production of Shiga-like toxin by Escherichia coli. J Infect Dis. 1986 Aug;154(2):338–341. doi: 10.1093/infdis/154.2.338. [DOI] [PubMed] [Google Scholar]
  14. Mata L. J., Gangarosa E. J., Cáceres A., Perera D. R., Mejicanos M. L. Epidemic Shiga bacillus dysentery in Central America. I. Etiologic investigations in Guatemala, 1969. J Infect Dis. 1970 Sep;122(3):170–180. doi: 10.1093/infdis/122.3.170. [DOI] [PubMed] [Google Scholar]
  15. Morrison D. M., Tyrrell D. L., Jewell L. D. Colonic biopsy in verotoxin-induced hemorrhagic colitis and thrombotic thrombocytopenic purpura (TTP). Am J Clin Pathol. 1986 Jul;86(1):108–112. doi: 10.1093/ajcp/86.1.108. [DOI] [PubMed] [Google Scholar]
  16. O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O'Brien A. D., LaVeck G. D. Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli. Infect Immun. 1983 May;40(2):675–683. doi: 10.1128/iai.40.2.675-683.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Samuel J. E., Perera L. P., Ward S., O'Brien A. D., Ginsburg V., Krivan H. C. Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants. Infect Immun. 1990 Mar;58(3):611–618. doi: 10.1128/iai.58.3.611-618.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schlossman D., Withers D., Welsh P., Alexander A., Robertus J., Frankel A. Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes. Mol Cell Biol. 1989 Nov;9(11):5012–5021. doi: 10.1128/mcb.9.11.5012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Storsaeter J., Olin P., Renemar B., Lagergård T., Norberg R., Romanus V., Tiru M. Mortality and morbidity from invasive bacterial infections during a clinical trial of acellular pertussis vaccines in Sweden. Pediatr Infect Dis J. 1988 Sep;7(9):637–645. doi: 10.1097/00006454-198809000-00008. [DOI] [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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