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. 1982 Jun;36(3):996–1005. doi: 10.1128/iai.36.3.996-1005.1982

Purification and Biological Characterization of Shiga Toxin from Shigella dysenteriae 1

J Edward Brown 1, Darrell E Griffin 1, Sara W Rothman 1, B P Doctor 1
PMCID: PMC551430  PMID: 7047403

Abstract

Shiga toxin has been purified in milligram quantities to near homogeneity from cell lysates of Shigella dysenteriae 1 strain 3818-0. Purification involved an initial ultracentrifugation, ammonium sulfate fractionation, chromatography on DEAE-cellulose and carboxymethyl cellulose, gel filtration, and preparative isoelectric focusing in sucrose gradients. The purified toxin was resolved by discontinuous polyacrylamide gel electrophoresis into a major cytotoxic protein band and a closely migrating, cytotoxic protease-nicked minor band. Antiserum generated by immunization with glutaraldehyde-inactivated toxin was shown to be monospecific against S. dysenteriae cell lysates. This highly purified toxin was cytotoxic to HeLa cells, enterotoxic in rabbit ileal loops, and lethal to mice. Monospecific antiserum to the toxin neutralized completely these toxin activities in both purified toxin preparations and crude shigella cell lysates.

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Selected References

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  1. Binder H. J., Whiting D. S. Inhibition of small-intestinal sugar and amino acid transport by the enterotoxin of Shigella dysenteriae I. Infect Immun. 1977 May;16(2):510–512. doi: 10.1128/iai.16.2.510-512.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown J. E., Rothman S. W., Doctor B. P. Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin. Infect Immun. 1980 Jul;29(1):98–107. doi: 10.1128/iai.29.1.98-107.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown J. E., Ussery M. A., Leppla S. H., Rothman S. W. Inhibition of protein synthesis by Shiga toxin: activation of the toxin and inhibition of peptide elongation. FEBS Lett. 1980 Aug 11;117(1):84–88. doi: 10.1016/0014-5793(80)80918-5. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Charney A. N., Gots R. E., Formal S. B., Giannella R. A. Activation of intestinal mucosal adenylate cyclase by Shigella dysenteriae I enterotoxin. Gastroenterology. 1976 Jun;70(6):1085–1090. [PubMed] [Google Scholar]
  6. Donowitz M., Keusch G. T., Binder H. J. Effect of Shigella enterotoxin on electrolyte transport in rabbit ileum. Gastroenterology. 1975 Dec;69(6):1230–1237. [PubMed] [Google Scholar]
  7. Eiklid K., Olsnes S. Interaction of Shigella shigae cytotoxin with receptors on sensitive and insensitive cells. J Recept Res. 1980;1(2):199–213. doi: 10.3109/10799898009044098. [DOI] [PubMed] [Google Scholar]
  8. FORMAL S. B., KUNDEL D., SCHNEIDER H., KUNEVN, SPRINZ H. Studies with Vibrio cholerae in the ligated loop of the rabbit intestine. Br J Exp Pathol. 1961 Oct;42:504–510. [PMC free article] [PubMed] [Google Scholar]
  9. Gemski P., Jr, Takeuchi A., Washington O., Formal S. B. Shigellosis due to Shigella dysenteriae. 1. Relative importance of mucosal invasion versus toxin production in pathogenesis. J Infect Dis. 1972 Nov;126(5):523–530. doi: 10.1093/infdis/126.5.523. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Keusch G. T., Grady G. F., Mata L. J., McIver J. The pathogenesis of Shigella diarrhea. I. Enterotoxin production by Shigella dysenteriae I. J Clin Invest. 1972 May;51(5):1212–1218. doi: 10.1172/JCI106915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keusch G. T., Jacewicz M., Hirschman S. Z. Quantitative microassay in cell culture for enterotoxin of Shigella dysenteriae. J Infect Dis. 1972 May;125(5):539–541. doi: 10.1093/infdis/125.5.539. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Labrec E. H., Schneider H., Magnani T. J., Formal S. B. EPITHELIAL CELL PENETRATION AS AN ESSENTIAL STEP IN THE PATHOGENESIS OF BACILLARY DYSENTERY. J Bacteriol. 1964 Nov;88(5):1503–1518. doi: 10.1128/jb.88.5.1503-1518.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McIver J., Grady G. F., Keusch G. T. Production and characterization of exotoxin(s) of Shigella dysenteriae type 1. J Infect Dis. 1975 May;131(5):559–566. doi: 10.1093/infdis/131.5.559. [DOI] [PubMed] [Google Scholar]
  16. O'Brien A. D., LaVeck G. D., Griffin D. E., Thompson M. R. Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography. Infect Immun. 1980 Oct;30(1):170–179. doi: 10.1128/iai.30.1.170-179.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Olenick J. G., Wolfe A. D. Shigella toxin inhibition of binding and translation of polyuridylic acid by Escherichia coli ribosomes. J Bacteriol. 1980 Mar;141(3):1246–1250. doi: 10.1128/jb.141.3.1246-1250.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Olsnes S., Eiklid K. Isolation and characterization of Shigella shigae cytotoxin. J Biol Chem. 1980 Jan 10;255(1):284–289. [PubMed] [Google Scholar]
  19. Reisbig R., Olsnes S., Eiklid K. The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit. J Biol Chem. 1981 Aug 25;256(16):8739–8744. [PubMed] [Google Scholar]
  20. Thompson M. R., Steinberg M. S., Gemski P., Formal S. B., Doctor B. P. Inhibition of in vitro protein synthesis by Shigella dysenteriae 1 toxin. Biochem Biophys Res Commun. 1976 Aug 9;71(3):783–788. doi: 10.1016/0006-291x(76)90899-8. [DOI] [PubMed] [Google Scholar]
  21. VAN HEYNINGEN W. E., GLADSTONE G. P. The neurotoxin of Shigella shigae. I. Production, purification and properties of the toxin. Br J Exp Pathol. 1953 Apr;34(2):202–216. [PMC free article] [PubMed] [Google Scholar]
  22. VICARI G., OLITZKI A. L., OLITZKI Z. The action of the thermolabile toxin of Shigella dysenteriae on cells cultivated in vitro. Br J Exp Pathol. 1960 Apr;41:179–189. [PMC free article] [PubMed] [Google Scholar]

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