MECHANISM OF MEMBRANE DAMAGE BY EL-TOR HEMOLYSIN OF VIBRIO-CHOLERAE O1

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cho lerae O1 spontaneously assembled oligomeric aggregates on the membrane s of rabbit erythrocyte ghosts and liposomes, Membrane-associated olig omers were resolved by sodium dodecyl sulfate-polyacrylamide gel elect rophoresis and immunoblotting into two to nine bands with apparent mol ecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposoma l membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could he repla ced with diosgenin or ergosterol but not with 5 alpha-cholestane-3-one , suggesting that sterol is essential for the oligomerization. The tre atment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 2 0 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysi s, this hemolysis is likely to be caused by pore formation on the memb rane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte m embrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemol ysin oligomers and pore formation on the membrane.