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. 1991 Jan;59(1):198–203. doi: 10.1128/iai.59.1.198-203.1991

Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain.

J D Dubreuil 1, J M Fairbrother 1, R Lallier 1, S Larivière 1
PMCID: PMC257726  PMID: 1987033

Abstract

Production of heat-stable enterotoxin b (STb) by porcine Escherichia coli strains belonging to serogroup O115 was evaluated in ligated intestinal segments of adult rats. The conditions for optimal production and detection of STb were studied by using the STb-producing strain 4247. As STb production was similar in complex Trypticase soy broth and minimal Davis medium, the latter was used for the fermentation of strain 4247 and the production of STb in large quantities. STb was then purified to apparent homogeneity by sequential ultrafiltration, ultracentrifugation, and preparative gel electrophoresis. The enterotoxin was purified more than 500-fold and exhibited a molecular weight of approximately 5,000 as determined by urea-sodium dodecyl sulfate gel electrophoresis. Purified STb retained such chemical characteristics as resistance to heating (60 degrees C/30 min) and sensitivity to trypsin. A rabbit polyclonal antiserum was produced against the purified toxin. Numerous booster doses were required to obtain a significant enzyme-linked immunosorbent assay titer, suggesting that STb is a poor immunogen. Nevertheless, the antiserum was used successfully to discriminate between culture supernatants of STb-positive and STb-negative O115 E. coli strains, thus demonstrating the immunogenicity of purified STb.

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Selected References

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  1. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Broes A., Fairbrother J. M., Mainil J., Harel J., Lariviere S. Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype O8:KX105 and O8:K"2829" strains isolated from piglets with diarrhea. J Clin Microbiol. 1988 Nov;26(11):2402–2409. doi: 10.1128/jcm.26.11.2402-2409.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown W. E., Wold F. Alkyl isocyanates as active-site-specific reagents for serine proteases. Reaction properties. Biochemistry. 1973 Feb 27;12(5):828–834. doi: 10.1021/bi00729a007. [DOI] [PubMed] [Google Scholar]
  4. Burgess M. N., Bywater R. J., Cowley C. M., Mullan N. A., Newsome P. M. Biological evaluation of a methanol-soluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves. Infect Immun. 1978 Aug;21(2):526–531. doi: 10.1128/iai.21.2.526-531.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dubreuil J. D., Kostrzynska M., Austin J. W., Trust T. J. Antigenic differences among Campylobacter fetus S-layer proteins. J Bacteriol. 1990 Sep;172(9):5035–5043. doi: 10.1128/jb.172.9.5035-5043.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fairbrother J. M., Broes A., Jacques M., Larivière S. Pathogenicity of Escherichia coli O115:K"V165" strains isolated from pigs with diarrhea. Am J Vet Res. 1989 Jul;50(7):1029–1036. [PubMed] [Google Scholar]
  7. Handl C., Rönnberg B., Nilsson B., Olsson E., Jonsson H., Flock J. I. Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II. J Clin Microbiol. 1988 Aug;26(8):1555–1560. doi: 10.1128/jcm.26.8.1555-1560.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Lawrence R. M., Huang P. T., Glick J., Oppenheim J. D., Maas W. K. Expression of the cloned gene for enterotoxin STb of Escherichia coli. Infect Immun. 1990 Apr;58(4):970–977. doi: 10.1128/iai.58.4.970-977.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lazure C., Seidah N. G., Chrétien M., Lallier R., St-Pierre S. Primary structure determination of Escherichia coli heat-stable enterotoxin of porcine origin. Can J Biochem Cell Biol. 1983 May;61(5):287–292. doi: 10.1139/o83-039. [DOI] [PubMed] [Google Scholar]
  12. Lee C. H., Moseley S. L., Moon H. W., Whipp S. C., Gyles C. L., So M. Characterization of the gene encoding heat-stable toxin II and preliminary molecular epidemiological studies of enterotoxigenic Escherichia coli heat-stable toxin II producers. Infect Immun. 1983 Oct;42(1):264–268. doi: 10.1128/iai.42.1.264-268.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Monckton R. P., Hasse D. Detection of enterotoxigenic Escherichia coli in piggeries in Victoria by DNA hybridisation using K88, K99, LT, ST1 and ST2 probes. Vet Microbiol. 1988 Mar;16(3):273–281. doi: 10.1016/0378-1135(88)90031-4. [DOI] [PubMed] [Google Scholar]
  14. Moon H. W., Schneider R. A., Moseley S. L. Comparative prevalence of four enterotoxin genes among Escherichia coli isolated from swine. Am J Vet Res. 1986 Feb;47(2):210–212. [PubMed] [Google Scholar]
  15. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  16. Picken R. N., Mazaitis A. J., Maas W. K., Rey M., Heyneker H. Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli. Infect Immun. 1983 Oct;42(1):269–275. doi: 10.1128/iai.42.1.269-275.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sheng H. Z., Martenson R. E., Carnegie P. R., Bernard C. C. Electroimmunoblotting of small peptides separated on urea-dodecyl sulphate (SUDS) gels. Application to myelin basic protein. J Immunol Methods. 1988 Feb 24;107(1):13–22. doi: 10.1016/0022-1759(88)90003-8. [DOI] [PubMed] [Google Scholar]
  18. Swank R. T., Munkres K. D. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem. 1971 Feb;39(2):462–477. doi: 10.1016/0003-2697(71)90436-2. [DOI] [PubMed] [Google Scholar]
  19. Söderlind O., Thafvelin B., Möllby R. Virulence factors in Escherichia coli strains isolated from Swedish piglets with diarrhea. J Clin Microbiol. 1988 May;26(5):879–884. doi: 10.1128/jcm.26.5.879-884.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Weikel C. S., Nellans H. N., Guerrant R. L. In vivo and in vitro effects of a novel enterotoxin, STb, produced by Escherichia coli. J Infect Dis. 1986 May;153(5):893–901. doi: 10.1093/infdis/153.5.893. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Whipp S. C. Protease degradation of Escherichia coli heat-stable, mouse-negative, pig-positive enterotoxin. Infect Immun. 1987 Sep;55(9):2057–2060. doi: 10.1128/iai.55.9.2057-2060.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wilson R. A., Francis D. H. Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis. Am J Vet Res. 1986 Feb;47(2):213–217. [PubMed] [Google Scholar]
  25. de Castro A. F., Gatti M. S., Serafim M. B., Brito J. R., de Barcellos D. S. Significance of thermostable enterotoxin produced by porcine enteropathogenic Escherichia coli. Vet Rec. 1984 Nov 17;115(20):518–519. doi: 10.1136/vr.115.20.518. [DOI] [PubMed] [Google Scholar]

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