title: Pet toxin from enteroaggregative Escherichia coli produces cellular damage associated with fodrin disruption.
abstract: 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.
authors.affiliation: Department of Public Health, Faculty of Medicine, UNAM, 04510 Mexico DF, Mexico.
authors.forename: J M
authors.initials: JM
authors.lastname: Villaseca
authors.forename: F
authors.initials: F
authors.lastname: Navarro-García
authors.forename: G
authors.initials: G
authors.lastname: Mendoza-Hernández
authors.forename: J P
authors.initials: JP
authors.lastname: Nataro
authors.forename: A
authors.initials: A
authors.lastname: Cravioto
authors.forename: C
authors.initials: C
authors.lastname: Eslava
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects*; D004768:Enterotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000472:pathogenicity*; D004927:Escherichia coli Infections / Q000503:physiopathology; D029968:Escherichia coli Proteins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011817:Rabbits; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D013049:Spectrin / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D002352:Carrier Proteins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008840:Microfilament Proteins; D012697:Serine Endopeptidases; D013049:Spectrin; C027538:heat-labile enterotoxin, E coli; C530786:Pet protein, E coli