title: Evidence that membrane rafts are not required for the action of Clostridium perfringens enterotoxin.
abstract: The action of bacterial pore-forming toxins typically involves membrane rafts for binding, oligomerization, and/or cytotoxicity. Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with a unique, multistep mechanism of action that involves the formation of complexes containing tight junction proteins that include claudins and, sometimes, occludin. Using sucrose density gradient centrifugation, this study evaluated whether the CPE complexes reside in membrane rafts and what role raft microdomains play in complex formation and CPE-induced cytotoxicity. Western blot analysis revealed that the small CPE complex and the CPE hexamer 1 (CH-1) complex, which is sufficient for CPE-induced cytotoxicity, both localize outside of rafts. The CH-2 complex was also found mainly in nonraft fractions, although a small pool of raft-associated CH-2 complex that was sensitive to cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) was detected. Pretreatment of Caco-2 cells with MbetaCD had no appreciable effect on CPE-induced cytotoxicity. Claudin-4 was localized to Triton X-100-soluble gradient fractions of control or CPE-treated Caco-2 cells, indicating a raft-independent association for this CPE receptor. In contrast, occludin was present in raft fractions of control Caco-2 cells. Treatment with either MbetaCD or CPE caused most occludin molecules to shift out of lipid rafts, possibly due (at least in part) to the association of occludin with the CH-2 complex. Collectively, these results suggest that CPE is a unique pore-forming toxin for which membrane rafts are not required for binding, oligomerization/pore formation, or cytotoxicity.
authors.affiliation: Department of Microbiology and Molecular Genetics, Molecular Virology and Microbiology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Justin A
authors.initials: JA
authors.lastname: Caserta
authors.forename: Martha L
authors.initials: ML
authors.lastname: Hale
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.forename: Bradley G
authors.initials: BG
authors.lastname: Stiles
authors.forename: Bruce A
authors.initials: BA
authors.lastname: McClane
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D002499:Centrifugation, Density Gradient; D062506:Claudin-4; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; D004768:Enterotoxins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C035598:enterotoxin, Clostridium