Modification of surface histidine residues abolishes the cytotoxic activity of Clostridium difficile toxin A

April K. Roberts, Clifford Shone

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


Clostridium difficile toxin A displays both cytotoxic and enterotoxic activities. It has recently been demonstrated that toxin A exerts its cytotoxic effect by the glucosylation of the small GTP-binding proteins of the Rho family. Diethyl pyrocarbonate, at pH 7.0, was used to chemically modify exposed histidine residues on toxin A. Modification of toxin A with diethyl pyrocarbonate abolished both its cytotoxic activity and the ability of the toxin to bind Zn-Sepharose gel. Treatment of toxin A with [14C]-diethyl pyrocarbonate revealed concentration dependent labelling of histidine residues on the toxin molecules. The effects of diethyl pyrocarbonate could be reversed by hydroxylamine treatment. These data suggest the modified histidine residues on toxin A are critical to its cytotoxic activity. Histidine modification had no effect on the glucosyl transferase enzyme activity of toxin A. However, modification abolished the 'cold' binding of toxin to bovine thyroglobulin in an ELISA and reduced ligand binding activity in a rabbit erythrocyte haemagglutination assay. The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A. Exposed histidines on toxin A are available for zinc chelation, and these have been exploited in the development of a novel purification protocol for toxin A using zinc-chelating chromatography. Copyright (C) 2000 Elsevier Science Ltd.

Original languageEnglish
Pages (from-to)325-333
Number of pages9
Issue number2-3
Publication statusPublished - 1 Feb 2001

Bibliographical note

Funding Information:
The authors wish to thank Mr. Glyn Choules for excellent technical assistance. This work was supported by the Department of Health, UK.

Copyright 2019 Elsevier B.V., All rights reserved.


  • Cytotoxicity
  • Enterotoxin
  • Glucosy transferase
  • Histidine residue
  • Receptor-binding
  • Toxin A


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