Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1993 Jul;61(7):2912–2918. doi: 10.1128/iai.61.7.2912-2918.1993

Purification and characterization of alpha-toxin produced by Clostridium novyi type A.

D W Ball 1, R L Van Tassell 1, M D Roberts 1, P E Hahn 1, D M Lyerly 1, T D Wilkins 1
PMCID: PMC280939  PMID: 8514395

Abstract

Our study describes the production, purification, and properties of alpha-toxin from Clostridium novyi type A 19402. The bacterium produced maximal amounts of alpha-toxin when grown at 37 degrees C for 72 h in dialysis flask cultures containing brain heart infusion supplemented with 0.75% Tween 80 and 2% glycerol. The alpha-toxin was purified by precipitation with polyethylene glycol 6000, followed by chromatography on Q-Sepharose, phenyl-agarose, and Mono-Q. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the toxin exhibited a single band with an M(r) of 200,000. The toxin also exhibited a single immunoprecipitin arc by crossed immunoelectrophoresis with antiserum against crude toxin. It was stable when stored at 4 degrees C and also following exposure to buffers with pHs in the range of 4 to 7. The toxin had a minimum lethal dose in mice of 5 to 10 ng, caused rounding of a variety of cells in tissue culture, and was negative in the rabbit ileal loop assay. The cytotoxic activity was inhibited by agents that affect receptor-mediated processes, and the toxin was less active on a CHO mutant cell line that is defective in endosomal acidification. The analysis of the amino acid composition revealed an unusually high proline content. The N-terminal sequence is Met-Leu-Ile-Thr-Arg-Glu-Gln-Leu-Met-Lys.

Full text

PDF
2912

Images in this article

2915Image
on p.2915

Selected References

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

  1. Bette P., Mauler F., Mohr C., Habermann E. Microinjection of alpha-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes. Toxicon. 1990;28(11):1368–1371. doi: 10.1016/0041-0101(90)90104-f. [DOI] [PubMed] [Google Scholar]
  2. Bette P., Oksche A., Mauler F., von Eichel-Streiber C., Popoff M. R., Habermann E. A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin. Toxicon. 1991;29(7):877–887. doi: 10.1016/0041-0101(91)90224-f. [DOI] [PubMed] [Google Scholar]
  3. Dove C. H., Wang S. Z., Price S. B., Phelps C. J., Lyerly D. M., Wilkins T. D., Johnson J. L. Molecular characterization of the Clostridium difficile toxin A gene. Infect Immun. 1990 Feb;58(2):480–488. doi: 10.1128/iai.58.2.480-488.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ehrich M., Van Tassell R. L., Libby J. M., Wilkins T. D. Production of Clostridium difficile antitoxin. Infect Immun. 1980 Jun;28(3):1041–1043. doi: 10.1128/iai.28.3.1041-1043.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fiorentini C., Thelestam M. Clostridium difficile toxin A and its effects on cells. Toxicon. 1991;29(6):543–567. doi: 10.1016/0041-0101(91)90050-2. [DOI] [PubMed] [Google Scholar]
  6. Krivan H. C., Clark G. F., Smith D. F., Wilkins T. D. Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc. Infect Immun. 1986 Sep;53(3):573–581. doi: 10.1128/iai.53.3.573-581.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Libby J. M., Wilkins T. D. Production of antitoxins to two toxins of Clostridium difficile and immunological comparison of the toxins by cross-neutralization studies. Infect Immun. 1982 Jan;35(1):374–376. doi: 10.1128/iai.35.1.374-376.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lyerly D. M., Barroso L. A., Wilkins T. D., Depitre C., Corthier G. Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. Infect Immun. 1992 Nov;60(11):4633–4639. doi: 10.1128/iai.60.11.4633-4639.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lyerly D. M., Krivan H. C., Wilkins T. D. Clostridium difficile: its disease and toxins. Clin Microbiol Rev. 1988 Jan;1(1):1–18. doi: 10.1128/cmr.1.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lyerly D. M., Lockwood D. E., Richardson S. H., Wilkins T. D. Biological activities of toxins A and B of Clostridium difficile. Infect Immun. 1982 Mar;35(3):1147–1150. doi: 10.1128/iai.35.3.1147-1150.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lyerly D. M., Saum K. E., MacDonald D. K., Wilkins T. D. Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun. 1985 Feb;47(2):349–352. doi: 10.1128/iai.47.2.349-352.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lyerly D. M., Sullivan N. M., Wilkins T. D. Enzyme-linked immunosorbent assay for Clostridium difficile toxin A. J Clin Microbiol. 1983 Jan;17(1):72–78. doi: 10.1128/jcm.17.1.72-78.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Martinez R. D., Wilkins T. D. Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C. difficile. J Med Microbiol. 1992 Jan;36(1):30–36. doi: 10.1099/00222615-36-1-30. [DOI] [PubMed] [Google Scholar]
  15. Martinez R. D., Wilkins T. D. Purification and characterization of Clostridium sordellii hemorrhagic toxin and cross-reactivity with Clostridium difficile toxin A (enterotoxin). Infect Immun. 1988 May;56(5):1215–1221. doi: 10.1128/iai.56.5.1215-1221.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mitchell M. J., Laughon B. E., Lin S. Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro. Infect Immun. 1987 Jul;55(7):1610–1615. doi: 10.1128/iai.55.7.1610-1615.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Müller H., von Eichel-Streiber C., Habermann E. Morphological changes of cultured endothelial cells after microinjection of toxins that act on the cytoskeleton. Infect Immun. 1992 Jul;60(7):3007–3010. doi: 10.1128/iai.60.7.3007-3010.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. NISHIDA S., NAKAGAWARA G. ISOLATION OF TOXIGENIC STRAINS OF CLOSTRIDIUM NOVYI FROM SOIL. J Bacteriol. 1964 Dec;88:1636–1640. doi: 10.1128/jb.88.6.1636-1640.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Navin T. R., Krug E. C., Pearson R. D. Effect of immunoglobulin M from normal human serum on Leishmania donovani promastigote agglutination, complement-mediated killing, and phagocytosis by human monocytes. Infect Immun. 1989 Apr;57(4):1343–1346. doi: 10.1128/iai.57.4.1343-1346.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Oksche A., Nakov R., Habermann E. Morphological and biochemical study of cytoskeletal changes in cultured cells after extracellular application of Clostridium novyi alpha-toxin. Infect Immun. 1992 Jul;60(7):3002–3006. doi: 10.1128/iai.60.7.3002-3006.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Popoff M. R., Boquet P. Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin. Biochem Biophys Res Commun. 1988 May 16;152(3):1361–1368. doi: 10.1016/s0006-291x(88)80435-2. [DOI] [PubMed] [Google Scholar]
  22. Popoff M. R. Purification and characterization of Clostridium sordellii lethal toxin and cross-reactivity with Clostridium difficile cytotoxin. Infect Immun. 1987 Jan;55(1):35–43. doi: 10.1128/iai.55.1.35-43.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roff C. F., Hall C. W., Robbins A. R. Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification. J Cell Biol. 1990 Apr;110(4):1023–1032. doi: 10.1083/jcb.110.4.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schering B., Bärmann M., Chhatwal G. S., Geipel U., Aktories K. ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin. Eur J Biochem. 1988 Jan 15;171(1-2):225–229. doi: 10.1111/j.1432-1033.1988.tb13780.x. [DOI] [PubMed] [Google Scholar]
  25. Schranner I., Erhard M. H., Kaltner H., Lösch U. Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B. Toxicon. 1992 May-Jun;30(5-6):653–668. doi: 10.1016/0041-0101(92)90859-4. [DOI] [PubMed] [Google Scholar]
  26. Simpson L. L., Stiles B. G., Zepeda H. H., Wilkins T. D. Molecular basis for the pathological actions of Clostridium perfringens iota toxin. Infect Immun. 1987 Jan;55(1):118–122. doi: 10.1128/iai.55.1.118-122.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stiles B. G., Wilkins T. D. Clostridium perfringens iota toxin: synergism between two proteins. Toxicon. 1986;24(8):767–773. doi: 10.1016/0041-0101(86)90101-7. [DOI] [PubMed] [Google Scholar]
  28. Stiles B. G., Wilkins T. D. Purification and characterization of Clostridium perfringens iota toxin: dependence on two nonlinked proteins for biological activity. Infect Immun. 1986 Dec;54(3):683–688. doi: 10.1128/iai.54.3.683-688.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sullivan N. M., Pellett S., Wilkins T. D. Purification and characterization of toxins A and B of Clostridium difficile. Infect Immun. 1982 Mar;35(3):1032–1040. doi: 10.1128/iai.35.3.1032-1040.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Taylor N. S., Thorne G. M., Bartlett J. G. Comparison of two toxins produced by Clostridium difficile. Infect Immun. 1981 Dec;34(3):1036–1043. doi: 10.1128/iai.34.3.1036-1043.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tucker K. D., Carrig P. E., Wilkins T. D. Toxin A of Clostridium difficile is a potent cytotoxin. J Clin Microbiol. 1990 May;28(5):869–871. doi: 10.1128/jcm.28.5.869-871.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vandekerckhove J., Schering B., Bärmann M., Aktories K. Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177. FEBS Lett. 1987 Dec 10;225(1-2):48–52. doi: 10.1016/0014-5793(87)81129-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES