Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)

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...

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Veröffentlicht in:The Journal of biological chemistry 2011-08, Vol.286 (33), p.29356-29365
Hauptverfasser: Schwan, Carsten, Nölke, Thilo, Kruppke, Anna S., Schubert, Daniel M., Lang, Alexander E., Aktories, Klaus
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container_end_page 29365
container_issue 33
container_start_page 29356
container_title The Journal of biological chemistry
container_volume 286
creator Schwan, Carsten
Nölke, Thilo
Kruppke, Anna S.
Schubert, Daniel M.
Lang, Alexander E.
Aktories, Klaus
description 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.
doi_str_mv 10.1074/jbc.M111.261925
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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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>21705797</pmid><doi>10.1074/jbc.M111.261925</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record>
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subjects Actin
Anti-Bacterial Agents - pharmacology
Antifungal Agents - pharmacology
Bacterial Adhesion - drug effects
Bacterial Adhesion - physiology
Bacterial Toxins
Bacterial Toxins - metabolism
beta-Cyclodextrins - pharmacology
Caco-2 Cells
Cell Biology
Cholesterol - metabolism
Clostridioides difficile - enzymology
Clostridioides difficile - pathogenicity
Dose-Response Relationship, Drug
Enterocolitis, Pseudomembranous - enzymology
Enterocolitis, Pseudomembranous - metabolism
Fatty Acids, Monounsaturated - pharmacology
Humans
Lipid Raft
Membrane
Membrane Microdomains - metabolism
Microtubules
Microtubules - metabolism
Nystatin - pharmacology
Sphingolipids - metabolism
Sphingomyelin Phosphodiesterase - pharmacology
title Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)
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