F. Navarro-garcia et al., Cytoskeletal effects induced by Pet, the serine protease enterotoxin of enteroaggregative Escherichia coli, INFEC IMMUN, 67(5), 1999, pp. 2184-2192
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.