Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1995 May;95(5):2004–2011. doi: 10.1172/JCI117885

Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro.

M Riegler 1, R Sedivy 1, C Pothoulakis 1, G Hamilton 1, J Zacherl 1, G Bischof 1, E Cosentini 1, W Feil 1, R Schiessel 1, J T LaMont 1, et al.
PMCID: PMC295778  PMID: 7738167

Abstract

Toxin A but not toxin B, appears to mediate intestinal damage in animal models of Clostridium difficile enteritis. The purpose of this study was to investigate the electrophysiologic and morphologic effects of purified C. difficile toxins A and B on human colonic mucosa in Ussing chambers. Luminal exposure of tissues to 16-65 nM of toxin A and 0.2-29 nM of toxin B for 5 h caused dose-dependent epithelial damage. Potential difference, short-circuit current and resistance decreased by 76, 58, and 46%, respectively, with 32 nM of toxin A and by 76, 55, and 47%, respectively, with 3 nM of toxin B, when compared with baseline (P < 0.05). 3 nM of toxin A did not cause electrophysiologic changes. Permeability to [3H]mannitol increased 16-fold after exposure to 32 nM of toxin A and to 3 nM of toxin B when compared with controls (P < 0.05). Light and scanning electron microscopy after exposure to either toxin revealed patchy damage and exfoliation of superficial epithelial cells, while crypt epithelium remained intact. Fluorescent microscopy of phalloidin-stained sections showed that both toxins caused disruption and condensation of cellular F-actin. Our results demonstrate that the human colon is approximately 10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important than toxin A in the pathogenesis of C. difficile colitis in man.

Full text

PDF
2004

Images in this article

2007Image
on p.2007
2008Image
on p.2008
2008Image
on p.2008
2008Image
on p.2008
2008Image
on p.2008
2009Image
on p.2009

Selected References

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

  1. Abrams G. D., Allo M., Rifkin G. D., Fekety R., Silva J., Jr Mucosal damage mediated by clostridial toxin in experimental clindamycin-associated colitis. Gut. 1980 Jun;21(6):493–499. doi: 10.1136/gut.21.6.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barroso L. A., Wang S. Z., Phelps C. J., Johnson J. L., Wilkins T. D. Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res. 1990 Jul 11;18(13):4004–4004. doi: 10.1093/nar/18.13.4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartlett J. G., Chang T. W., Gurwith M., Gorbach S. L., Onderdonk A. B. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med. 1978 Mar 9;298(10):531–534. doi: 10.1056/NEJM197803092981003. [DOI] [PubMed] [Google Scholar]
  4. Bartlett J. G., Moon N., Chang T. W., Taylor N., Onderdonk A. B. Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis. Gastroenterology. 1978 Nov;75(5):778–782. [PubMed] [Google Scholar]
  5. Bartlett J. G., Onderdonk A. B., Cisneros R. L., Kasper D. L. Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. J Infect Dis. 1977 Nov;136(5):701–705. doi: 10.1093/infdis/136.5.701. [DOI] [PubMed] [Google Scholar]
  6. Donta S. T., Sullivan N., Wilkins T. D. Differential effects of Clostridium difficile toxins on tissue-cultured cells. J Clin Microbiol. 1982 Jun;15(6):1157–1158. doi: 10.1128/jcm.15.6.1157-1158.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Ehrich M. Biochemical and pathological effects of Clostridium difficile toxins in mice. Toxicon. 1982;20(6):983–989. doi: 10.1016/0041-0101(82)90100-3. [DOI] [PubMed] [Google Scholar]
  9. Feil W., Lacy E. R., Wong Y. M., Burger D., Wenzl E., Starlinger M., Schiessel R. Rapid epithelial restitution of human and rabbit colonic mucosa. Gastroenterology. 1989 Sep;97(3):685–701. doi: 10.1016/0016-5085(89)90640-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Flegel W. A., Müller F., Däubener W., Fischer H. G., Hadding U., Northoff H. Cytokine response by human monocytes to Clostridium difficile toxin A and toxin B. Infect Immun. 1991 Oct;59(10):3659–3666. doi: 10.1128/iai.59.10.3659-3666.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hecht G., Koutsouris A., Pothoulakis C., LaMont J. T., Madara J. L. Clostridium difficile toxin B disrupts the barrier function of T84 monolayers. Gastroenterology. 1992 Feb;102(2):416–423. doi: 10.1016/0016-5085(92)90085-d. [DOI] [PubMed] [Google Scholar]
  13. Hecht G., Pothoulakis C., LaMont J. T., Madara J. L. Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers. J Clin Invest. 1988 Nov;82(5):1516–1524. doi: 10.1172/JCI113760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Just I., Fritz G., Aktories K., Giry M., Popoff M. R., Boquet P., Hegenbarth S., von Eichel-Streiber C. Clostridium difficile toxin B acts on the GTP-binding protein Rho. J Biol Chem. 1994 Apr 8;269(14):10706–10712. [PubMed] [Google Scholar]
  15. Kelly C. P., Becker S., Linevsky J. K., Joshi M. A., O'Keane J. C., Dickey B. F., LaMont J. T., Pothoulakis C. Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit. J Clin Invest. 1994 Mar;93(3):1257–1265. doi: 10.1172/JCI117080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim P. H., Iaconis J. P., Rolfe R. D. Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters. Infect Immun. 1987 Dec;55(12):2984–2992. doi: 10.1128/iai.55.12.2984-2992.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Meltzer S. J., Ahnen D. J., Battifora H., Yokota J., Cline M. J. Protooncogene abnormalities in colon cancers and adenomatous polyps. Gastroenterology. 1987 May;92(5 Pt 1):1174–1180. doi: 10.1016/s0016-5085(87)91074-2. [DOI] [PubMed] [Google Scholar]
  20. Miller P. D., Pothoulakis C., Baeker T. R., LaMont J. T., Rothstein T. L. Macrophage-dependent stimulation of T cell-depleted spleen cells by Clostridium difficile toxin A and calcium ionophore. Cell Immunol. 1990 Mar;126(1):155–163. doi: 10.1016/0008-8749(90)90308-e. [DOI] [PubMed] [Google Scholar]
  21. Mitchell T. J., Ketley J. M., Haslam S. C., Stephen J., Burdon D. W., Candy D. C., Daniel R. Effect of toxin A and B of Clostridium difficile on rabbit ileum and colon. Gut. 1986 Jan;27(1):78–85. doi: 10.1136/gut.27.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moore R., Pothoulakis C., LaMont J. T., Carlson S., Madara J. L. C. difficile toxin A increases intestinal permeability and induces Cl- secretion. Am J Physiol. 1990 Aug;259(2 Pt 1):G165–G172. doi: 10.1152/ajpgi.1990.259.2.G165. [DOI] [PubMed] [Google Scholar]
  23. Pothoulakis C., Barone L. M., Ely R., Faris B., Clark M. E., Franzblau C., LaMont J. T. Purification and properties of Clostridium difficile cytotoxin B. J Biol Chem. 1986 Jan 25;261(3):1316–1321. [PubMed] [Google Scholar]
  24. Pothoulakis C., Castagliuolo I., LaMont J. T., Jaffer A., O'Keane J. C., Snider R. M., Leeman S. E. CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):947–951. doi: 10.1073/pnas.91.3.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pothoulakis C., Karmeli F., Kelly C. P., Eliakim R., Joshi M. A., O'Keane C. J., Castagliuolo I., LaMont J. T., Rachmilewitz D. Ketotifen inhibits Clostridium difficile toxin A-induced enteritis in rat ileum. Gastroenterology. 1993 Sep;105(3):701–707. doi: 10.1016/0016-5085(93)90886-h. [DOI] [PubMed] [Google Scholar]
  26. Pothoulakis C., Kelly C. P., Joshi M. A., Gao N., O'Keane C. J., Castagliuolo I., Lamont J. T. Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum. Gastroenterology. 1993 Apr;104(4):1108–1115. doi: 10.1016/0016-5085(93)90280-p. [DOI] [PubMed] [Google Scholar]
  27. Pothoulakis C., LaMont J. T., Eglow R., Gao N., Rubins J. B., Theoharides T. C., Dickey B. F. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein. J Clin Invest. 1991 Jul;88(1):119–125. doi: 10.1172/JCI115267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pothoulakis C., Triadafilopoulos G., Clark M., Franzblau C., LaMont J. T. Clostridium difficile cytotoxin inhibits protein synthesis in fibroblasts and intestinal mucosa. Gastroenterology. 1986 Nov;91(5):1147–1153. doi: 10.1016/s0016-5085(86)80010-5. [DOI] [PubMed] [Google Scholar]
  29. Rolfe R. D. Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters. Infect Immun. 1991 Apr;59(4):1223–1230. doi: 10.1128/iai.59.4.1223-1230.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Torres J., Jennische E., Lange S., Lönnroth I. Enterotoxins from Clostridium difficile; diarrhoeogenic potency and morphological effects in the rat intestine. Gut. 1990 Jul;31(7):781–785. doi: 10.1136/gut.31.7.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Triadafilopoulos G., Pothoulakis C., O'Brien M. J., LaMont J. T. Differential effects of Clostridium difficile toxins A and B on rabbit ileum. Gastroenterology. 1987 Aug;93(2):273–279. doi: 10.1016/0016-5085(87)91014-6. [DOI] [PubMed] [Google Scholar]
  33. Wedel N., Toselli P., Pothoulakis C., Faris B., Oliver P., Franzblau C., LaMont T. Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts. Exp Cell Res. 1983 Oct 15;148(2):413–422. doi: 10.1016/0014-4827(83)90163-5. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES