Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1

Abstract

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5␣-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. 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 membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

Haemolysins in Vibrio species

“…The pro-region of HlyA was reported to function as a molecular chaperone, suggesting that it exerts a role in the correct folding of the mature HlyA haemolysin (Nagamune et al 1997). It is considered likely that HlyA haemolysin acts as a poreforming toxin through oligomer formation (Ikigai et al 1996), with the oligomers having molecular masses of 170-350 kDa on SDS-PAGE (Ikigai et al 1996). In comparison, the monomer has a molecular mass of 60 kDa (Ikigai et al 1996(Ikigai et al , 1999.…”

“…It is considered likely that HlyA haemolysin acts as a poreforming toxin through oligomer formation (Ikigai et al 1996), with the oligomers having molecular masses of 170-350 kDa on SDS-PAGE (Ikigai et al 1996). In comparison, the monomer has a molecular mass of 60 kDa (Ikigai et al 1996(Ikigai et al , 1999. Overall, it was estimated that the pores were of 1AE2-1AE6 nm in size (Ikigai et al 1996).…”

“…In comparison, the monomer has a molecular mass of 60 kDa (Ikigai et al 1996(Ikigai et al , 1999. Overall, it was estimated that the pores were of 1AE2-1AE6 nm in size (Ikigai et al 1996). Cholesterol in the target cell membrane was considered to have an important role in the assembly of the toxin oligomers needed for pore formation (Ikigai et al 1996;Menzl et al 1996).…”

“…Overall, it was estimated that the pores were of 1AE2-1AE6 nm in size (Ikigai et al 1996). Cholesterol in the target cell membrane was considered to have an important role in the assembly of the toxin oligomers needed for pore formation (Ikigai et al 1996;Menzl et al 1996). The conclusion is that HlyA interacts with cholesterol and forms oligomers at the interface of cholesterol microcrystals and water (Harris et al 2002).…”

Cholesterol and the activity of bacterial toxins

“…The tryptophan-rich motif involved in membrane binding is highlighted. cytolysin [35], and Streptococcus agalactiae CAMP factor [36]. These toxins have no significant sequence similarity to each other or to the thiol-activated toxins; no high-resolution structural information is available for either of them.…”