Evidence for the involvement of the surface active properties of the extracellular toxin tolaasin in the manifestation of brown blotch disease symptoms by Pseudomonas tolaasii on Agaricus bisporus

Abstract

Pseudomonas tolaasii produces an ion channel forming haemolytic lipodepsipeptide toxin tolaasin which has been shown to be a potent biosurfactant. Following purification by high performance liquid chromatography, the critical micellar concentration and the surface tension at the critical micellar concentration of purified toxin were determined in distilled water as 0·46 mg ml −1 and 41 mN m −1 respectively by the drop weight method and as 0·42 mg ml −1 and 38 mN m −1 respectively by the contact angle method. The critical micellar concentration of tolaasin varied as a function of pH, with a minimum critical micellar concentration of 1·2 mg ml −1 in 0·1 m Na borate buffer at pH 6·3. However the surface tension at the critical micellar concentration of tolaasin remained almost constant at 40–42 mN m −1 over the pH range 3·0–10·0. The surfactant properties of a mutant truncated form of the toxin (tolaasin 144), which is unable to lyse erythrocytes at low concentrations but which reproduces disease symptoms, were also studied. Tolaasin 144 was also shown to be a biosurfactant with a critical micellar concentration in water of 1·2 mg ml −1 , a surface tension at the critical micellar concentration of 34 mN m −1 and was capable of reducing the surface tension of distilled water to 29 mN m −1 . Both tolaasin and tolaasin 144 (which is unable to form ion channels in artificial lipid bilayers) were able to induce rapid erythrocyte lysis in the presence or absence of the ion channel blocking agent Zn 2+ at concentrations above their critical micellar concentration. Since tolaasin 144 is only able to cause lysis of intact mushroom caps, whereas tolaasin can lyse both mushroom caps and cut mushroom tissue, these results suggest that the ion channel forming activity of tolaasin plays a role in lysis of cut mushroom tissue, whereas the surface active properties of tolaasin and tolaasin 144 are required for lysis of mushroom caps. A second role for tolaasin in brown blotch disease is suggested by the observation that the ability to synthesize tolaasin or tolaasin 144 increased the rate of spread of P. tolaasii colonies in vitro . These results are discussed in relation to the biology of brown blotch disease.