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Infection and Immunity, October 2000, p. 5920-5927, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Pet Toxin from Enteroaggregative Escherichia coli Produces Cellular Damage Associated with Fodrin Disruption

Jorge M. Villaseca,1 Fernando Navarro-García,1,2 Guillermo Mendoza-Hernández,3 James P. Nataro,4 Alejandro Cravioto,1 and Carlos Eslava1,*

Department of Public Health1 and Department of Biochemistry,3 Faculty of Medicine, UNAM, 04510 Mexico DF, and Department of Cell Biology, CINVESTAV-IPN, 07000 Mexico DF,2 Mexico, and Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 212014

Received 7 April 2000/Returned for modification 1 June 2000/Accepted 26 July 2000

Pet toxin is a serine protease from enteroaggregative Escherichia coli which has been described as causing enterotoxic and cytotoxic effects. In this paper we show that Pet produces spectrin and fodrin (nonerythroid spectrin) disruption. Using purified erythrocyte membranes treated with Pet toxin, we observed degradation of alpha - and beta -spectrin chains; this effect was dose and time dependent, and a 120-kDa protein fraction was observed as a breakdown product. Spectrin degradation and production of the 120-kDa subproduct were confirmed using specific antibodies against the alpha - and beta -spectrin chains. The same degradation effect was observed in alpha -fodrin from epithelial HEp-2 cells, both in purified cell membranes and in cultured cells which had been held in suspension for 36 h; these effects were confirmed using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif. Fluorescence and light microscopy of HEp-2 Pet-treated cells showed morphological alterations, which were associated with irregular distribution of fodrin in situ. Spectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet antibodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, which had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectrin or fodrin in epithelial cell assays. This is a new system of cellular damage identified in bacterial toxins which includes the internalization of the protease, induction of some unknown intermediate signaling steps, and finally the fodrin degradation to destroy the cell.

* Corresponding author. Mailing address: Department of Public Health, Faculty of Medicine, UNAM, Ap. Postal 70-443, 04510 Mexico DF, Mexico. Phone and fax: (525) 622-0822. E-mail: eslava{at}servidor.unam.mx.

Infection and Immunity, October 2000, p. 5920-5927, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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