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-␤-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-␤-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 CDTinduced microtubule-based membrane protrusions depend on cholesterol-and sphingolipid-rich lipid microdomains.Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis (1). Both diseases depend on the production of toxins. Major virulence factors are the glycosylating C. difficile toxins A and B, which inactivate Rho GTPases (2-5). Especially hypervirulent strains additionally produce C. difficile transferase (CDT) 3 (6). CDT belongs to the family of actin-ADP-ribosylating toxins like Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin (7-9). These toxins are binary in structure and consist of an enzymatic component, which possesses ADP-ribosyltransferase activity, and a separated binding/translocation component. The binding component is proteolytically activated and forms heptamers, which interact with the enzymatic component (8, 10). After binding of the toxin to a cell surface receptor, the toxin complex is endocytosed (11,12). At low pH of endosomal compartments, the binding component inserts into the membrane of endosomes and forms pores, which allow the translocation of the enzymatic component into the cytosol (10, 13). Here, the toxin ADP-ribosylates actin at arginine 177 (14). Modification at this site inhibits actin polymerization and causes destruction of the actin cytoskeleton.Recently, we reported that following ADP-ribosylation of actin, CDT induces the formation of microtubule-based protrusions in epithelial cells (15). These protrusions are in general Ͻ1 m in diameter ...

Identification of the Cellular Receptor of Clostridium spiroforme Toxin

“…The methodology was adapted from protocols that were previously established for the recombinant production of various other clostridial toxins (11,17,20,22). The B. megaterium expression system often offers an advantage over conventional bacterial expression systems (e.g., Escherichia coli) in producing clostridial toxins, obviously due to a similar codon usage of the related genera Bacillus and Clostridium.…”

“…The components of C. botulinum C2 toxin (C2I and C2II) and C. difficile transferase (CDTa and CDTb) were purified from Escherichia coli and Bacillus megaterium, respectively, as described elsewhere, and binding components (C2II and CDTb) were activated by protease treatment (3,5,20).…”

“…This system was extremely instrumental for expression of members of the family of large glucosylating clostridial toxins, which possess a mass of 250 to 308 kDa and are notoriously poorly expressed in Escherichia coli (22). We previously employed this procedure for recombinant production of the clostridial toxins C. sordellii lethal toxin, C. novyi alpha-toxin, and C. difficile transferase (11,17,20). The current study describes the usefulness of the B. megaterium expression system for recombinant production of CSTa and CSTb.…”

The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes

“…As a result, the plasma membrane would favor more planar-like conformations, explaining the smoothing of the cell surface and the retraction of disconnected ICNs (Figure 2 and Figure 5A). Since it has been recently shown that cholesterol-sphingomyelin nanodomains were essential for microtubule-based protrusion growth, 44 it could be also possible that the stability of the ICN depends on the binding of microtubules to the ICN membrane through cholesterol-based membrane nanodomains.…”

Clostridium difficile Infection

“…Experimental data have suggested that CDT results in the formation of microtubule-based protrusions on epithelial cells that might increase the adherence and colonization of C. difficile 142 .…”