title: Cytoskeletal effects induced by pet, the serine protease enterotoxin of enteroaggregative Escherichia coli.
abstract: We have previously described enteroaggregative Escherichia coli (EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of the pet gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of E. coli HB101 harboring the minimal pet clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C1 cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.
authors.affiliation: Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. fnavarro@servidor.unam.mx
authors.forename: F
authors.initials: F
authors.lastname: Navarro-García
authors.forename: C
authors.initials: C
authors.lastname: Sears
authors.forename: C
authors.initials: C
authors.lastname: Eslava
authors.forename: A
authors.initials: A
authors.lastname: Cravioto
authors.forename: J P
authors.initials: JP
authors.lastname: Nataro
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000633:toxicity*; D001483:Base Sequence; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects*; D017931:DNA Primers / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics / Q000633:toxicity*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D019073:HT29 Cells; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D051381:Rats; D012697:Serine Endopeptidases / Q000235:genetics / Q000633:toxicity*; D013696:Temperature
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D004768:Enterotoxins; D012697:Serine Endopeptidases