Oligomerization of Vibrio cholerae cytolysin yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in the target membrane

Vibrio cholerae cytolysin permeabilizes animal cell membranes Upon binding to the target lipid bilayer, the protein assembles into homo oligomeric por es of an as yet unknown stoichiometry Pore formation has been observed with model liposomes consisting of phosphatidylcholine and cholesterol, but the latter were much less susceptible to the cytolysin than were erythrocytes or intestinal epithelial cells. We here show that liposome permeabilization is strongly promoted if cholesterol is combined with sphingolipids, whereb y the most pronounced effects are observed with monohexosylceramides and fr ee ceramide. These two lipid species are prevalent in mammalian intestinal brush border membranes. We therefore propose that, on its natural target me mbranes, the cytolysin has a dual specificity for both cholesterol and cera mides. To assess the stoichiometry of the pore, we generated hybrid oligome rs of two naturally occurring variants of the toxin that differ in molecula r weight. On SDS-polyacrylamide gel electrophoresis, the mixed oligomers fo rmed a pattern of six distinct bands. Ordered by decreasing electrophoretic mobility, the six oligomer species must comprise 0 to 5 subunits of the la rger form; the pore thus is a pentamer, Due to both lipid specificity and p ore stoichiometry, V. cholerae cytolysin represents a novel prototype in th e class of bacterial pore-forming toxins.