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Infection and Immunity, May 1999, p. 2184-2192, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cytoskeletal Effects Induced by Pet, the Serine Protease Enterotoxin of Enteroaggregative Escherichia coli

Fernando Navarro-García,1,2,* Cynthia Sears,3 Carlos Eslava,2 Alejandro Cravioto,2 and James P. Nataro1

Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 212011; Department of Public Health, Faculty of Medicine, UNAM, 04510 Mexico DF, Mexico2; and Divisions of Infectious Diseases and Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland 212053

Received 20 November 1998/Returned for modification 21 January 1999/Accepted 11 February 1999

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.

* Corresponding author. Mailing address: Department of Public Health, Faculty of Medicine, UNAM, Ap. Postal 70-443, 04510 Mexico DF, Mexico. Phone: (525) 622-0822. Fax: (525) 622-0827. E-mail: fnavarro{at}servidor.unam.mx.

Infection and Immunity, May 1999, p. 2184-2192, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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