Jm. Villaseca et al., Pet toxin from enteroaggregative Escherichia coli produces cellular damageassociated with fodrin disruption, INFEC IMMUN, 68(10), 2000, pp. 5920-5927
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.