Inhibition of Tolaasin Cytotoxicity Causing Brown Blotch Disease in Cultivated Mushrooms Using Tolaasin Inhibitory Factors

Abstract

Tolaasin, a pore-forming bacterial peptide toxin secreted by Pseudomonas tolaasii, causes brown blotch disease in cultivated mushrooms by forming membrane pores and collapsing the membrane structures. Tolaasin is a lipodepsipeptide, MW 1985, and pore formation by tolaasin molecules is accomplished by hydrophobic interactions and multimerizations. Compounds that inhibit tolaasin toxicity have been isolated from various food additives. Food detergents, sucrose esters of fatty acids, and polyglycerol esters of fatty acids can effectively inhibit tolaasin cytotoxicity. These chemicals, named tolaasin-inhibitory factors (TIF), were effective at concentrations ranging from 10^-4 to 10^-5 M. The most effective compound, TIF 16, inhibited tolaasin-induced hemolysis independent of temperature and pH, while tolaasin toxicity increased at higher temperatures. When TIF 16 was added to tolaasin-pretreated erythrocytes, the cytotoxic activity of tolaasin immediately stopped, and no further hemolysis was observed. In the artificial lipid bilayer, the single-channel activity of the tolaasin channel was completely and irreversibly blocked by TIF 16. When TIF 16 was sprayed onto pathogen-treated oyster mushrooms growing on the shelves of cultivation houses, the development of disease was completely suppressed, and normal growth of oyster mushrooms was observed. Furthermore, the treatment with TIF 16 did not show any adverse effect on the growth of oyster mushrooms. These results indicate that TIF 16 is a good candidate for the biochemical control of brown blotch disease.

Keywords: Pseudomonas tolaasii; artificial lipid bilayer; oyster mushroom (Pleurotus ostreatus); pore-forming peptide toxin; tolaasin.

Figure 1

Effect of TIF 16 at various temperatures and under different pH conditions. TIF 16 was added to the tolaasin solution with a final concentration of 10⁻⁵ M. Filled symbol: tolaasin. Open symbol: tolaasin+TIF 16. (A) Temperature-dependent hemolytic activities of tolaasin at temperatures from 17 to 47 °C. (B) pH-dependence of hemolysis measured at pH levels from 5 to 9.

Figure 2

Effect of TIF 16 at various phases of hemolysis. Symbols: the times of TIF 16 addition during hemolysis. TIF 16 was treated at 1.5 min intervals from 9 min after incubation. The final concentration of TIF 16 was 10⁻⁵ M.

Figure 3

Irreversible binding of TIF 16. Tolaasin was added and washed after 9 min (Con). TIF 16 was added after 9 min of tolaasin addition, and unbound molecules of tolaasin and TIF 16 were washed out after 30 (●) and 60 (▲) s incubations. The washed RBCs were re-suspended with fresh buffer solution.

Figure 4

Inhibitory effects of Zn²⁺ and TIF 16 on the tolaasin channel. (A) Continuous recordings of the tolaasin channel. Additions of Zn²⁺ (B) or TIF 16 (C) in symmetric solution containing 100 mM KCl. Arrows () and H.P. mean closed state and holding potential, respectively.

Figure 5

Suppression of brown blotch by TIF 16 during the shelf cultivation of oyster mushrooms. Disease was observed after 1 day of tolaasin treatment and became serious after 2 days. During the 2nd cycle of harvesting, fungi were spread on the surface of the cultivation shelf, as shown in the circles (P. tolaasii). Con, distilled water; P. tolaasii, P. tolaasii culture supernatant; P. tolaasii + TIF 16, P. tolaasii culture supernatant and TIF 16 at 10⁻⁴ M.