Temperature-dependent hemolytic activity of membrane pore-forming peptide toxin, tolaasin.

Tolaasin, a pore-forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin-induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 degrees Celsius, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 degrees Celsius and the temperature was decreased, complete hemolysis was observed even at 4 degrees Celsius. These results indicate that tolaasin binding to cell membrane is temperature-sensitive while tolaasin-induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore-forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose-dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin-induced hemolysis can be explained by a multi-hit model.