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Infection and Immunity, February 2001, p. 1053-1060, Vol. 69, No. 2
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.2.1053-1060.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Plasmid-Encoded Toxin of Enteroaggregative Escherichia coli is Internalized by Epithelial Cells

Fernando Navarro-García,1,* Adrián Canizalez-Roman,1 José Luna,2 Cynthia Sears,3 and James P. Nataro4

Departments of Cell Biology1 and Physiology, Biophysics, and Neuroscience,2 CINVESTAV-IPN, 07000 México, DF, Mexico; Divisions of Infectious Diseases and Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland 212053; and Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 212014

Received 12 June 2000/Returned for modification 2 August 2000/Accepted 7 October 2000

We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative Escherichia coli (EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet (J. M. Villaseca, F. Navarro-García, G. Mendoza-Hernández, J. P. Nataro, A. Cravioto, and C. Eslava, Infect. Immun. 68:5920-5927, 2000).

* Corresponding author. Mailing address: Department of Cell Biology, CINVESTAV-IPN, Ap. Postal 14-740, 07000 Mexico, DF, Mexico. Phone: (525) 747-7000, ext. 5527. Fax: (525) 747-7081. E-mail: fnavarro{at}cell.cinvestav.mx.

Infection and Immunity, February 2001, p. 1053-1060, Vol. 69, No. 2
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.2.1053-1060.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.