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

Clostridium difficile toxin A displays both cytotoxic and enterotoxic activ ities. It has recently been demonstrated that toxin A exerts its cytotoxic effect by the glucosylation of the small GTP-binding proteins of the Rho fa mily. Diethyl pyrocarbonate, at pH 7.0, was used to chemically modify expos ed histidine residues on toxin A. Modification of toxin A with diethyl pyro carbonate abolished both its cytotoxic activity and the ability of the toxi n to bind Zn-Sepharose gel. Treatment of toxin A with [C-14]-diethyl pyroca rbonate revealed concentration dependent labelling of histidine residues on the toxin molecules. The effects of diethyl pyrocarbonate could be reverse d by hydroxylamine treatment. These data suggest the modified histidine res idues on toxin A are critical to its cytotoxic activity. Histidine modifica tion had no effect on the glucosyl transferase enzyme activity of toxin A. However, modification abolished the 'cold' binding of toxin to bovine thyro globulin in an ELISA and reduced ligand binding activity in a rabbit erythr ocyte haemagglutination assay. The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A. Exposed histid ines on toxin A are available for zinc chelation, and these have been explo ited in the development of a novel purification protocol for toxin A using zinc-chelating chromatography. (C) 2000 Elsevier Science Ltd. All rights re served.