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Infection and Immunity, April 2001, p. 2435-2441, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2435-2441.2001

Clostridium perfringens Iota-Toxin: Mapping of Receptor Binding and Ia Docking Domains on Ib

Jean-Christophe Marvaud,1,2,* Theresa Smith,1 Martha L. Hale,1 Michel R. Popoff,2 Leonard A. Smith,1 and Bradley G. Stiles1,*

Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-5011,1 and Unite des Toxines Microbiennes, Institut Pasteur, Paris, France2

Received 12 October 2000/Returned for modification 21 November 2000/Accepted 19 January 2001

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 >= 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.

* Corresponding author. Mailing address for Bradley G. Stiles: Toxinology Division, USAMRIID, Fort Detrick, MD 21702-5011. Phone: (301) 619-4809. Fax: (301) 619-2348. E-mail: bradley.stiles{at}amedd.army.mil. Mailing address for Jean-Christophe Marvaud: Unite des Toxines Microbiennes, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33-1-45-68-83-07. Fax: 33-1-40-61-31-23. E-mail: jcmarvaud{at}yahoo.com.

Infection and Immunity, April 2001, p. 2435-2441, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2435-2441.2001


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