title: Intracellular expression of the plasmid-encoded toxin from enteroaggregative Escherichia coli.
abstract: The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a serine protease autotransporter that acts as an enterotoxin and cytotoxin. When applied to epithelial cells in culture, purified toxin induces cell elongation and rounding, followed by exfoliation of cells from the substratum. These effects are accompanied by loss of actin stress fibers and electrophysiologic changes. Although it has been hypothesized that Pet has an intracellular site of action, evidence for this is indirect. In addition, Pet has recently been shown to cleave spectrin in vitro and in vivo. If Pet requires intracellular localization to execute its toxic effects, then intracellular expression of the protein could induce cytopathic effects similar to those observed when the toxin is applied to the cell surface. To test this hypothesis, we expressed the mature Pet toxin (comprising only the passenger domain of the Pet precursor) in the cytoplasm of HEp-2 cells by using mammalian expression vectors. Separately, we expressed the Pet passenger domain mutated at the catalytic serine (PetS260A), a construct that has been reported to lack toxic effects. Forty-eight hours after transient transfection of pcDNA3.1-pet in HEp-2 cells, we observed cell elongation and other morphological changes similar to those induced by applied toxin. Cells transfected with pcDNA3.1 vector alone appeared normal, while cells expressing the PetS260A mutant displayed similar (though less pronounced) changes compared with those in cells expressing pcDNA3.1-pet. Notably, intracellular expression of Pet was accompanied by condensation of the spectrin cytoskeleton. These studies corroborate an intracellular site of action for the Pet toxin, further implicate a role for spectrin in Pet intoxication, and provide a powerful tool for Pet structure and function analyses.
authors.affiliation: Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Bao Quan
authors.initials: BQ
authors.lastname: Sui
authors.forename: Pinaki R
authors.initials: PR
authors.lastname: Dutta
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity; D001483:Base Sequence; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000633:toxicity; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000633:toxicity; D013049:Spectrin / Q000378:metabolism; D014162:Transfection
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D013049:Spectrin