Increase in Cytotoxic Effect of Tolaasin by Phospholipids Composed of Medium-Chain Fatty Acids

Abstract

Tolaasin produced by Pseudomonas tolaasii disrupts membrane structures of cultivated mushrooms, such as Agaricus bisporus and Pleurotus ostreatus (oyster mushroom), and causes brown blotch disease. It consists of 18 amino acids, its molecular mass is 1,985 Da, and it forms a left-handed α-helix. The mechanism of membrane-pore formation of tolaasin molecule has not known in detail. When tolaasin molecule is inserted into the membrane, N-terminus of tolaasin forms 4 turns of α-helix and the length of tolaasin channel corresponds to near 20 A, a little shorter than the thickness of membrane. Tolaasin channels are unstable in the artificial lipid bilayer and this may be explained by the comparison between the length of tolaasin channel and the thickness of lipid bilayer membrane. In control condition, bilayer was made with phosphatidyl ethanolamine (PE18:1/16:0) and phosphatidyl serine (PS18:0/18:1). Since this membrane is thicker than the estimated length of tolaasin channel, mismatch in thickness may make the channel unstable. Phospholipids composed of medium or short-chain fatty acids may be helpful to the stability of tolaasin channel by making the membrane thinner. When phosphatidyl ethanolamines made with decanoic acids (capric acid, DDPE), myristic acids (DMPE), and stearic acids (DSPE) were added, DDPE (200 nM) facilitated tolaasin-induced hemolysis. When the concentration of DDPE was adjusted from 0.2-200 nM, the hemolysis was stimulated at the concentrations above 2 nM. Ks value of DDPE effect was obtained at 6 μM DDPE. When the preincubation effect of tolaasin and DDPE was measured, binding of tolaasin to DDPE was completed within 5 min. In the lipid bilayer recording, the addition of DDPE increased tolaasin channel activity by increasing open probability. Therefore, tolaasin molecules make more stable channels with phospholipids composed of medium-chain fatty acids.