Abstract
Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass approximately 47 kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass approximately 81 kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37 degrees C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220 kDa) within 15 s, which was still evident 110 min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5 min at 37 degrees C, cell-bound Ib generated Na(+)/K(+) permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4 degrees C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632-655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28-66. The Ib protoxin (molecular mass approximately 98 kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.