title: Cholesterol-dependent pore formation of Clostridium difficile toxin A.
abstract: The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Giesemann
authors.forename: Thomas
authors.initials: T
authors.lastname: Jank
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
authors.forename: Elke
authors.initials: E
authors.lastname: Maier
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
authors.forename: Roland
authors.initials: R
authors.lastname: Benz
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism*; D016360:Clostridioides difficile / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism; D006224:Cricetinae; D003603:Cytotoxins / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012413:Rubidium / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D003603:Cytotoxins; D004768:Enterotoxins; D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008055:Lipids; D010455:Peptides; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C041348:tcdA protein, Clostridium difficile; D002784:Cholesterol; D010738:Type C Phospholipases; D012413:Rubidium