title: Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes.
abstract: Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.
authors.affiliation: Institutional Program in Molecular Biomedicine, National School of Homeopathy and Medicine, Instituto Politecnico Nacional, Mexico City, Mexico. rcg@hp.fciencias.unam.mx
authors.forename: Renato E
authors.initials: RE
authors.lastname: Cappello
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Estrada-Gutierrez
authors.forename: Claudine
authors.initials: C
authors.lastname: Irles
authors.forename: Silvia
authors.initials: S
authors.lastname: Giono-Cerezo
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bloch
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
mesh_terms: D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D002460:Cell Line; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D005434:Flow Cytometry; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051419:Paxillin / Q000378:metabolism; D010957:Plasmids; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D013049:Spectrin / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C496061:PXN protein, human; D051419:Paxillin; D000185:Actinin; D016596:Vinculin; D013049:Spectrin; D051416:Focal Adhesion Protein-Tyrosine Kinases; C530786:Pet protein, E coli; D012697:Serine Endopeptidases