Clostridium perfringens iota-toxin: mapping of receptor binding and Ia docking domains on Ib.
Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing > or = 200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice.
Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-5011, USA. jcmarvaud@yahoo.com
J C
JC
Marvaud
T
T
Smith
M L
ML
Hale
M R
MR
Popoff
L A
LA
Smith
B G
BG
Stiles
D036002:ADP Ribose Transferases; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D001665:Binding Sites; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000472:pathogenicity*; D004768:Enterotoxins / Q000737:chemistry*; D018604:Epitope Mapping; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D011994:Recombinant Proteins; C050737:iota toxin, Clostridium perfringens; D036002:ADP Ribose Transferases