Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Nov 1;367(Pt 3):801–808. doi: 10.1042/BJ20020566

Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex.

Bradley G Stiles 1, Martha L Hale 1, Jean Christophe Marvaud 1, Michel R Popoff 1
PMCID: PMC1222948  PMID: 12175336

Abstract

Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass approximately 47 kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass approximately 81 kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37 degrees C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220 kDa) within 15 s, which was still evident 110 min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5 min at 37 degrees C, cell-bound Ib generated Na(+)/K(+) permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4 degrees C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632-655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28-66. The Ib protoxin (molecular mass approximately 98 kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.

Full Text

The Full Text of this article is available as a PDF (198.1 KB).

Selected References

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

  1. Arora N., Klimpel K. R., Singh Y., Leppla S. H. Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells. J Biol Chem. 1992 Aug 5;267(22):15542–15548. [PubMed] [Google Scholar]
  2. Ballard J. D., Collier R. J., Starnbach M. N. Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12531–12534. doi: 10.1073/pnas.93.22.12531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barth H., Blocker D., Behlke J., Bergsma-Schutter W., Brisson A., Benz R., Aktories K. Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification. J Biol Chem. 2000 Jun 23;275(25):18704–18711. doi: 10.1074/jbc.M000596200. [DOI] [PubMed] [Google Scholar]
  4. Barth H., Hofmann F., Olenik C., Just I., Aktories K. The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion toxin. Infect Immun. 1998 Apr;66(4):1364–1369. doi: 10.1128/iai.66.4.1364-1369.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barth Holger, Roebling Robert, Fritz Michaela, Aktories Klaus. The binary Clostridium botulinum C2 toxin as a protein delivery system: identification of the minimal protein region necessary for interaction of toxin components. J Biol Chem. 2001 Dec 6;277(7):5074–5081. doi: 10.1074/jbc.M109167200. [DOI] [PubMed] [Google Scholar]
  6. Blöcker D., Barth H., Maier E., Benz R., Barbieri J. T., Aktories K. The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding. Infect Immun. 2000 Aug;68(8):4566–4573. doi: 10.1128/iai.68.8.4566-4573.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blöcker D., Behlke J., Aktories K., Barth H. Cellular uptake of the Clostridium perfringens binary iota-toxin. Infect Immun. 2001 May;69(5):2980–2987. doi: 10.1128/IAI.69.5.2980-2987.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradley K. A., Mogridge J., Mourez M., Collier R. J., Young J. A. Identification of the cellular receptor for anthrax toxin. Nature. 2001 Nov 8;414(6860):225–229. doi: 10.1038/n35101999. [DOI] [PubMed] [Google Scholar]
  9. Eckhardt M., Barth H., Blöcker D., Aktories K. Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates. J Biol Chem. 2000 Jan 28;275(4):2328–2334. doi: 10.1074/jbc.275.4.2328. [DOI] [PubMed] [Google Scholar]
  10. Escuyer V., Collier R. J. Anthrax protective antigen interacts with a specific receptor on the surface of CHO-K1 cells. Infect Immun. 1991 Oct;59(10):3381–3386. doi: 10.1128/iai.59.10.3381-3386.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Falnes P. O., Sandvig K. Penetration of protein toxins into cells. Curr Opin Cell Biol. 2000 Aug;12(4):407–413. doi: 10.1016/s0955-0674(00)00109-5. [DOI] [PubMed] [Google Scholar]
  12. Gibert M., Petit L., Raffestin S., Okabe A., Popoff M. R. Clostridium perfringens iota-toxin requires activation of both binding and enzymatic components for cytopathic activity. Infect Immun. 2000 Jul;68(7):3848–3853. doi: 10.1128/iai.68.7.3848-3853.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gill D. M. The arrangement of subunits in cholera toxin. Biochemistry. 1976 Mar 23;15(6):1242–1248. doi: 10.1021/bi00651a011. [DOI] [PubMed] [Google Scholar]
  14. Han S., Craig J. A., Putnam C. D., Carozzi N. B., Tainer J. A. Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex. Nat Struct Biol. 1999 Oct;6(10):932–936. doi: 10.1038/13300. [DOI] [PubMed] [Google Scholar]
  15. Iwasaki M., Ohishi I., Sakaguchi G. Evidence that botulinum C2 toxin has two dissimilar components. Infect Immun. 1980 Aug;29(2):390–394. doi: 10.1128/iai.29.2.390-394.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kimura K., Kubota T., Ohishi I., Isogai E., Isogai H., Fujii N. The gene for component-II of botulinum C2 toxin. Vet Microbiol. 1998 Apr 30;62(1):27–34. doi: 10.1016/s0378-1135(98)00195-3. [DOI] [PubMed] [Google Scholar]
  17. Knapp Oliver, Benz Roland, Gibert Maryse, Marvaud Jean C., Popoff Michel R. Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia. J Biol Chem. 2001 Dec 10;277(8):6143–6152. doi: 10.1074/jbc.M103939200. [DOI] [PubMed] [Google Scholar]
  18. Krasilnikov O. V., Merzlyak P. G., Yuldasheva L. N., Rodrigues C. G., Bhakdi S., Valeva A. Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha-toxin in planar lipid bilayers. Mol Microbiol. 2000 Sep;37(6):1372–1378. doi: 10.1046/j.1365-2958.2000.02080.x. [DOI] [PubMed] [Google Scholar]
  19. Marvaud J. C., Smith T., Hale M. L., Popoff M. R., Smith L. A., Stiles B. G. Clostridium perfringens iota-toxin: mapping of receptor binding and Ia docking domains on Ib. Infect Immun. 2001 Apr;69(4):2435–2441. doi: 10.1128/IAI.69.4.2435-2441.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Milne J. C., Furlong D., Hanna P. C., Wall J. S., Collier R. J. Anthrax protective antigen forms oligomers during intoxication of mammalian cells. J Biol Chem. 1994 Aug 12;269(32):20607–20612. [PubMed] [Google Scholar]
  21. Mogridge Jeremy, Cunningham Kristina, Collier R. John. Stoichiometry of anthrax toxin complexes. Biochemistry. 2002 Jan 22;41(3):1079–1082. doi: 10.1021/bi015860m. [DOI] [PubMed] [Google Scholar]
  22. Nagahama Masahiro, Nagayasu Koichi, Kobayashi Keiko, Sakurai Jun. Binding component of Clostridium perfringens iota-toxin induces endocytosis in Vero cells. Infect Immun. 2002 Apr;70(4):1909–1914. doi: 10.1128/IAI.70.4.1909-1914.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olsnes S., Pihl A. Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry. 1973 Jul 31;12(16):3121–3126. doi: 10.1021/bi00740a028. [DOI] [PubMed] [Google Scholar]
  24. Perelle S., Gibert M., Boquet P., Popoff M. R. Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli. Infect Immun. 1993 Dec;61(12):5147–5156. doi: 10.1128/iai.61.12.5147-5156.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Petit L., Maier E., Gibert M., Popoff M. R., Benz R. Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers. J Biol Chem. 2001 Feb 7;276(19):15736–15740. doi: 10.1074/jbc.M010412200. [DOI] [PubMed] [Google Scholar]
  26. Petosa C., Collier R. J., Klimpel K. R., Leppla S. H., Liddington R. C. Crystal structure of the anthrax toxin protective antigen. Nature. 1997 Feb 27;385(6619):833–838. doi: 10.1038/385833a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Popoff M. R., Milward F. W., Bancillon B., Boquet P. Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells. Infect Immun. 1989 Aug;57(8):2462–2469. doi: 10.1128/iai.57.8.2462-2469.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sakurai J., Kobayashi K. Lethal and dermonecrotic activities of Clostridium perfringens lota toxin: biological activities induced by cooperation of two nonlinked components. Microbiol Immunol. 1995;39(4):249–253. doi: 10.1111/j.1348-0421.1995.tb02197.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Schmid A., Benz R., Just I., Aktories K. Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine. J Biol Chem. 1994 Jun 17;269(24):16706–16711. [PubMed] [Google Scholar]
  32. Singh U., Van Itallie C. M., Mitic L. L., Anderson J. M., McClane B. A. CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junction protein occludin. J Biol Chem. 2000 Jun 16;275(24):18407–18417. doi: 10.1074/jbc.M001530200. [DOI] [PubMed] [Google Scholar]
  33. Singh Y., Khanna H., Chopra A. P., Mehra V. A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo. J Biol Chem. 2001 Mar 16;276(25):22090–22094. doi: 10.1074/jbc.M010222200. [DOI] [PubMed] [Google Scholar]
  34. Singh Y., Klimpel K. R., Goel S., Swain P. K., Leppla S. H. Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells. Infect Immun. 1999 Apr;67(4):1853–1859. doi: 10.1128/iai.67.4.1853-1859.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Singh Y., Klimpel K. R., Quinn C. P., Chaudhary V. K., Leppla S. H. The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity. J Biol Chem. 1991 Aug 15;266(23):15493–15497. [PubMed] [Google Scholar]
  36. Steinthorsdottir V., Halldórsson H., Andrésson O. S. Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells. Microb Pathog. 2000 Jan;28(1):45–50. doi: 10.1006/mpat.1999.0323. [DOI] [PubMed] [Google Scholar]
  37. Stiles B. G., Hale M. L., Marvaud J. C., Popoff M. R. Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry. Infect Immun. 2000 Jun;68(6):3475–3484. doi: 10.1128/iai.68.6.3475-3484.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. van der Goot F. G., Harder T. Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack. Semin Immunol. 2001 Apr;13(2):89–97. doi: 10.1006/smim.2000.0300. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES