Intracellular expression of the plasmid-encoded toxin from enteroaggregative Escherichia coli.
Intracellular expression of the plasmid-encoded toxin from enteroaggregative Escherichia coli.
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来自肠聚集性大肠杆菌的质粒编码毒素的细胞内表达。
DOI：
10.1128/iai.71.9.5364-5370.2003
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发表时间：
2003
影响因子：
3.1
通讯作者：
Nataro,JamesP
中科院分区：
文献类型：
--
作者：
Sui,BaoQuan;Dutta,PinakiR;Nataro,JamesP
The plasmid-encoded toxin (Pet) from enteroaggregativeEscherichia coliis 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-petin 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.
