title: Intoxication of epithelial cells by plasmid-encoded toxin requires clathrin-mediated endocytosis.
abstract: It has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target alpha-fodrin (alphaII spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl-beta-cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm.
authors.affiliation: Department of Cell Biology, Centro de Investigación y de Estudios Avanzados (Cinvestav-Zacatenco), Ap. Postal 14-740, 07000 México, DF, Mexico.
authors.forename: Fernando
authors.initials: F
authors.lastname: Navarro-García
authors.affiliation: Department of Cell Biology, Centro de Investigación y de Estudios Avanzados (Cinvestav-Zacatenco), Ap. Postal 14-740, 07000 México, DF, Mexico.
authors.forename: Adrián
authors.initials: A
authors.lastname: Canizalez-Roman
authors.affiliation: Department of Cell Biology, Centro de Investigación y de Estudios Avanzados (Cinvestav-Zacatenco), Ap. Postal 14-740, 07000 México, DF, Mexico.
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Vidal
authors.affiliation: Department of Cell Biology, Centro de Investigación y de Estudios Avanzados (Cinvestav-Zacatenco), Ap. Postal 14-740, 07000 México, DF, Mexico.
authors.forename: Ma Isabel
authors.initials: MI
authors.lastname: Salazar
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000494:pharmacology*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D004847:Epithelial Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases
chemical_list: D001427:Bacterial Toxins; D002966:Clathrin; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D034741:RNA, Small Interfering; C027538:heat-labile enterotoxin, E coli; C530786:Pet protein, E coli; D012697:Serine Endopeptidases