Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex.
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.
Stiles
Bradley G
BG
Toxinology Division, Department of Immunology and Molecular Biology, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702-5011, USA. bradley.stiles@amedd.army.mil
Hale
Martha L
ML
Marvaud
Jean Christophe
JC
Popoff
Michel R
MR
D036002:ADP Ribose Transferases / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D001704:Biopolymers / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003016:Clostridium perfringens / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D013696:Temperature; D014709:Vero Cells
D000906:Antibodies; D001427:Bacterial Toxins; D001704:Biopolymers; D004768:Enterotoxins; C050737:iota toxin, Clostridium perfringens; D036002:ADP Ribose Transferases