title: Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis.
abstract: Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.
authors.affiliation: Dept. of Medicine, Division of Gastroenterology, 918 Ross Research Bldg., 720 Rutland Ave., Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Malyukova
authors.forename: Karen F
authors.initials: KF
authors.lastname: Murray
authors.forename: Chengru
authors.initials: C
authors.lastname: Zhu
authors.forename: Edgar
authors.initials: E
authors.lastname: Boedeker
authors.forename: Anne
authors.initials: A
authors.lastname: Kane
authors.forename: Kathleen
authors.initials: K
authors.lastname: Patterson
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Peterson
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
authors.forename: Olga
authors.initials: O
authors.lastname: Kovbasnjuk
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D004195:Disease Models, Animal; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D004927:Escherichia coli Infections / Q000378:metabolism* / Q000382:microbiology; D019453:Escherichia coli O157 / Q000378:metabolism* / Q000472:pathogenicity; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects / Q000382:microbiology; D008297:Male; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism; D010873:Pinocytosis / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014281:Trihexosylceramides / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D002966:Clathrin; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C018549:globotriaosylceramide; D019061:src-Family Kinases; D052066:N-Ethylmaleimide-Sensitive Proteins; D020764:cdc42 GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide; D002746:Chlorpromazine