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Infection and Immunity, June 2000, p. 3475-3484, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Clostridium perfringens Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry

Bradley G. Stiles,1,* Martha L. Hale,1 Jean-Christophe Marvaud,2 and Michel R. Popoff2

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

Received 10 December 1999/Returned for modification 3 February 2000/Accepted 15 March 2000

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

* Corresponding author. Mailing address: Department of Immunology and Molecular Biology, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702-5011. Phone: (301) 619-4809. Fax: (301) 619-2348. E-mail: bradley.stiles{at}det.amedd.army.mil.

Infection and Immunity, June 2000, p. 3475-3484, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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