Cytoskeletal effects induced by Pet, the serine protease enterotoxin of enteroaggregative Escherichia coli

We have previously described enteroaggregative Escherichia coli (EAEC) stra ins that induce cytotoxic effects on T84 cells, ligated rat ileal loops, an d human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin), We have also shown previously that the Pe t toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide seq uence 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 C-1 ce lls in culture. The effects were characterized by release of the cellular f ocal contacts from the glass substratum, followed by complete rounding of t he cells and detachment from the glass. Staining of the Pet-treated cells w ith Live/Dead viability stain revealed that >90% of rounded cells were viab le. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled p halloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-alt ering drugs, including colchicine, taxol, cytochalasin D, and phallicidin, The Pet protein induced proteolysis in zymogram gels, and preincubation wit h the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in c omplete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) w as deficient in protease activity and did not produce cytopathic effects, c ytoskeletal damage, or enterotoxic effects in Ussing chambers. These data s uggest that Pet is a cytoskeleton altering toxin and that its protease acti vity is involved in each of the observed phenotypes.