Pet toxin from enteroaggregative Escherichia coli produces cellular damageassociated with fodrin disruption

Pet toxin is a serine protease from enteroaggregative Escherichia coli whic h 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, w e 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 b reakdown product. Spectrin degradation and production of the 120-kDa subpro duct were confirmed using specific antibodies against the alpha- and beta-s pectrin chains. The same degradation effect was observed in alpha-fodrin fr om 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 confir med using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif, Fluorescence an d light microscopy of HEp-2 Pet-treated cells showed morphological alterati ons, which were associated with irregular distribution of fodrin in situ. S pectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet anti bodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, wh ich had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectr in or fodrin in epithelial cell assays. This is a new system of cellular da mage identified in bacterial toxins which includes the internalization of t he protease, induction of some unknown intermediate signaling steps, and fi nally the fodrin degradation to destroy the cell.