Clostridium perfringens iota-toxin requires activation of both binding andenzymatic components for cytopathic activity

Iota-toxin is produced by Clostridium perfringens type E strains and consis ts of two independent components, the enzymatic and binding components, ref erred to as Ia and Ib, respectively. A recombinant C, perfringens strain, s train 667/pMRP147, produced processed Ia and partially processed Ib, while a recombinant C, perfringens type A strain, strain TS133/pMRP147, in which the VirR-VirS two-component system is inactivated, produced only precursor forms of Ia and Ib, This suggests that iota-toxin is processed by a VirR-Vi rS-responsive protease, although not completely in the recombinant type A s train. The precursor forms of Ia and Ib were purified from cultures of the latter strain, and their proteolytic activation was examined. Treatment wit h proteases cleaved off small peptides (9 to 13 amino acid residues) and a 20-kDa peptide from the N termini of the Ia and Ib precursors, respectively , leading to their active forms. They were activated efficiently by alpha-c hymotrypsin, pepsin, proteinase K, subtilisin, and thermolysin but only wea kly by trypsin, as demonstrated by the cell-rounding assay. lambda-Protease from the C, perfringens type E strain, which was found to be a zinc-depend ent protease related to thermolysin, activated iota-toxin as eWciently as d id alpha-chymotrypsin. These results suggest that lambda-protease is most r esponsible for the activation of iota-toxin in type E strains.