• Journal of Applied Biological Chemistry
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• v.52 no.1
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• pp.28-32
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• 2009

The bacterial toxin, tolaasin, causes brown blotch disease on the cultivated mushrooms by collapsing fungal and fruiting body structure of mushroom. Cytotoxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasins form membrane pores on the red blood cells and destroy cell structure. While we investigated the inhibitions of hemolytic activity of tolaasin by $Zn^{2+}$ and $Cd^{2+}$, we found that $Ni^{2+}$ is another antagonist to block the toxicity of tolaasin. $Ni^{2+}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its Ki value was $\sim10$ mM, implying that the inhibitory effect of $Ni^{2+}$ is stronger than that of $Cd^{2+}$. The hemolytic activity was completely inhibited by $Ni^{2+}$ at the concentration higher than 50 mM. The effect of $Ni^{2+}$ was reversible since it was removed by the addition of EDTA. When the tolaasin-induced hemolysis was suppressed by the addition of 20 mM $Ni^{2+}$, the subsequent addition of EDIA immediately initiated the hemolysis. Although the mechanism of $Ni^{2+}$ -induced inhibition on tolaasin toxicity is not known, $Ni^{2+}$ could inhibit any of fallowing processes of tolaasin action, membrane binding, molecular multimerization, pore formation, and massive ion transport through the membrane pore. Our results indicate that $Ni^{2+}$ inhibits the pore activity of tolaasin, the last step of the toxic process.

• Journal of Applied Biological Chemistry
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• v.61 no.3
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• pp.213-217
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• 2018

Tolaasin secreted by Pseudomonas tolaasii is a peptide toxin and causes brown blotch disease on the cultivated mushrooms by collapsing cellular and fruiting body structure. Toxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasin molecules form membrane pores on the red blood cells and destroy cell membrane structure. In the previous studies, we found that tolaasin cytotoxicity was suppressed by $Zn^{2+}$ and $Ni^{2+}$. $Ni^{2+}$ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its $K_i$ value was 1.8 mM. The hemolytic activity was completely inhibited at the concentration higher than 10 mM. The inhibitory effect of $Zn^{2+}$ on tolaasin-induced hemolysis was increased in alkaline pH, while that of $Ni^{2+}$was not much dependent on pH. When the pH of buffer solution was increased from pH 7 to pH 9, the time for 50% hemolysis ($T_{50}$) was increased greatly by $100{\mu}M$ $Zn^{2+}$; however, it was slightly increased by 1 mM $Ni^{2+}$ at all pH values. When the synergistic effect of $Zn^{2+}$ and $Ni^{2+}$ on tolaasin-induced hemolysis was measured, it was not dependent on the pH of buffer solution. Molecular elucidation of the difference in pH-dependence of these two metal ions may contribute to understand the mechanism of tolaasin pore formation and cytotoxicity.

• Journal of Applied Biological Chemistry
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• v.60 no.2
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• pp.179-184
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• 2017

Tolaasin, a 1.9 kDa peptide toxin, is produced by Pseudomonas tolaasii and causes the brown blotch disease of cultivated oyster mushroom. It forms pores on the membrane and thus destroys cellular membrane structure, seriously reducing the productivity of mushroom cultivation. The mechanism of tolaasin-induced cytotoxicity is not known in detail. However, it has been reported to form a pore structure in the cytoplasmic membrane through the molecular multimerization. Therefore, food additives which can interact with tolaasin molecules may inhibit the pore formation by hydrophobic interactions with tolaasin molecules. In this study, various food additive materials have been identified as inhibitors of the tolaasin activity and named tolaasin-inhibitory factors (TIF). Most of TIFs are emulsifying agents for food processing procedures. Among various TIFs, polyglycerol and sucrose esters of fatty acids blocked effectively the cytotoxicity of tolaasins at the concentrations $10^{-4}-10^{-5}M$. These TIFs also successfully suppressed the blotch disease development in the shelf cultivation of oyster mushroom.

• Journal of Applied Biological Chemistry
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• v.64 no.4
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• pp.369-374
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• 2021

Brown blotch disease of oyster mushrooms is caused by tolaasin and its analog peptide toxins which are produced by Pseudomonas tolaasii. Tolaasin peptides form pores in the plasma membrane and destroy the fruiting body structure of mushroom. Lysis of red blood cells, hemolysis, can be occurred by cytotoxic activity of tolaasin. The hemolytic activity of tolaasin is inhibited by metal ions, such as Zn²⁺ and Ni²⁺. When Gadolinium ion was added, a biphasic effect was observed on tolaasin-induced hemolysis, an increase in hemolysis at submillimolar concentrations and an inhibition at millimolar concentrations. The mechanism of gadolinium ion-induced inhibition of tolaasin activity may not be similar to those of the inhibitions by other metal ions. Since gadolinium ion has been reported to change a lateral pressure of lipid membrane by binding to the negative charges of membrane lipids, it may not directly work on the tolaasin channel gating, but rather decrease the stability of tolaasin channel by increasing firmness of membrane.

• Journal of Applied Biological Chemistry
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• v.60 no.4
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• pp.351-355
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• 2017

Tolaasin, peptide toxin produced by Pseudomonas tolaasii, causes a brown blotch disease on the cultivated mushrooms. Tolaasin peptides form membrane pores and disrupt cellular membrane structure. Molecular actions of tolaasin consist of the aggregation of peptide molecules, binding to the cell membrane, and formation of membrane pores. Therefore, the inhibitions of any of these actions are able to suppress the blotch disease. We have isolated and identified several tolaasin inhibitors (named tolaasin inhibitory factors, TIF) from food additives. TIFs were able to suppress the blotch-formation by the pathogen inoculated to the mushrooms. In this study, TIFs were incubated under various conditions and their activities for the inhibition of tolaasin-induced hemolytic activity were investigated. Since TIFs are unsaturated carbon compounds, they were sensitive to the air exposure and light irradiation. In the anaerobic conditions, TIFs were stable and their activities were decreased by 10% for three months. However, near 90% of TIF activity was suppressed by two weeks in the presence of air and sun light. Temperature did not show any significant effects on the activity of TIF, since storages at 5, 25, $45^{\circ}C$ did not show any difference. Therefore, for the stable storage of TIF compounds, container should be designed to be dark and air-tight.