title: Cholesterol- and sphingolipid-rich microdomains are essential for microtubule-based membrane protrusions induced by Clostridium difficile transferase (CDT).
abstract: Clostridium difficile toxin (CDT) is a binary actin-ADP-ribosylating toxin that causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions, which are suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains. Here, we studied the involvement of membrane lipid components of human colon adenocarcinoma (Caco-2) cells in formation of membrane protrusions. Depletion of cholesterol by methyl-β-cyclodextrin inhibited protrusion formation in a concentration-dependent manner but had no major effect on the toxin-catalyzed modification of actin in target cells. Repletion of cholesterol reconstituted formation of protrusions and increased velocity and total amount of protrusion formation. Methyl-β-cyclodextrin had no effect on the CDT-induced changes in the dynamics of microtubules. Formation of membrane protrusions was also inhibited by the cholesterol-binding polyene antibiotic nystatin. Degradation or inhibition of synthesis of sphingolipids by sphingomyelinase and myriocin, respectively, blocked CDT-induced protrusion formation. Benzyl alcohol, which increases membrane fluidity, prevented protrusion formation. CDT-induced membrane protrusions were stained by flotillin-2 and by the fluorescent-labeled lipid raft marker cholera toxin subunit B, which selectively interacts with GM1 ganglioside mainly located in lipid microdomains. The data suggest that formation and especially the initiation of CDT-induced microtubule-based membrane protrusions depend on cholesterol- and sphingolipid-rich lipid microdomains.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schwan
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Nölke
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Kruppke
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Schubert
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Lang
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany. Electronic address: Klaus.Aktories@pharmakol.uni-freiburg.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000187:drug effects / Q000502:physiology*; D001427:Bacterial Toxins / Q000378:metabolism*; D018938:Caco-2 Cells; D002784:Cholesterol / Q000378:metabolism*; D016360:Clostridioides difficile / Q000201:enzymology* / Q000472:pathogenicity; D004305:Dose-Response Relationship, Drug; D004761:Enterocolitis, Pseudomembranous / Q000201:enzymology / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D009761:Nystatin / Q000494:pharmacology; D013107:Sphingolipids / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D001427:Bacterial Toxins; D005229:Fatty Acids, Monounsaturated; D013107:Sphingolipids; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009761:Nystatin; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase; C001996:thermozymocidin