Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity

The beta-toxin gene isolated from Clostridium perfringens type B was expres sed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. T he purified GST-beta-toxin fusion protein from the E. coli transformant cel ls was not lethal. The N-terminal amino acid sequence of the recombinant be ta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin we re indistinguishable from those of native beta-toxin (n toxin) purified fro m C. perfringens type C. Replacement of Cys-265 with alanine or serine by s ite-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of t yrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifica lly modifies histidyl residue resulted in significant decrease in lethal ac tivity, but that of r toxin with the agent did not. These results showed th at replacement of the cysteine residue at position 265 with amino acids wit h large size of side chain or introduction of functional groups in the posi tion resulted in loss of lethal activity of the toxin. Replacement of Tyr-2 66, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simult aneous administration of r toxin and W275A led to a decrease in lethal acti vity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue. (C) 1999 Elsevier Science B. V. All rights reserved.