title: Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.
abstract: Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi alpha-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and alpha-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Papatheodorou
authors.forename: Constantinos
authors.initials: C
authors.lastname: Zamboglou
authors.forename: Selda
authors.initials: S
authors.lastname: Genisyuerek
authors.forename: Gregor
authors.initials: G
authors.lastname: Guttenberg
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism*; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D033922:Clathrin Heavy Chains / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005799:Genes, Dominant / Q000235:genetics; D006031:Glycosylation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D034622:RNA Interference / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D013109:Sphingomyelins; C031815:toxB protein, Clostridium difficile; D033922:Clathrin Heavy Chains; D013108:Sphingomyelin Phosphodiesterase; D034281:Dynamins; D002746:Chlorpromazine