Effects of Tolaasin Inhibitory Factors on Tolaasin Peptide Channel Evaluated by Competition with Zn2+

Abstract

Tolaasin is a 1.9 kDa bacterial lipodepsipeptide toxin and forms membrane pores causing brown blotch disease on the cultivated mushrooms. Molecular multimerization of tolaasin is required to form membrane channel. Previously, we showed that various chemicals inhibit the multimerization of tolaasins and thus prevent from brown blotch disease. These chemicals named tolaasin-inhibitory factor (TIF) were applicable to control the disease. Various candidates were chosen from different food additives and they were successful for disease control at 1-100 μM. Interestingly, after tolaasin molecules formed channels on the erythrocyte membrane and hemolysis started, further hemolyses were stopped as soon as TIF's were added. The added TIF was not washed by centrifugation and addition of fresh HBS solution. These result imply that TIF's are able to inhibit the preformed tolaasin channels. TIF may inhibit tolaasin-induced hemolysis either by plugging the channel pores or by the dissociation of tolaasin multimers. Zn2+ is a potent tolaasin inhibitor known to bind to tolaasin channel. In order to characterize the TIF-induced inhibition, competition effect of Zn2+ and TIF on tolaasin channel activity was investigated. When Zn2+ and TIF-9 were added simultaneously, no additive effects were observed at various concentration combinations. However, in the combinations of Zn2+ plus either TIF-11 or TIF-16, no additive effect was measured and the inhibitory effect of Zn2+ was reduced in the presence of low concentration of Zn2+. In these experiments, the inhibition of hemolysis was dominated by Zn2+concentration. Based on these results, we suggest that TIF has high affinity to tolaasin channel than Zn2+ although Zn2+ is a strong inhibitor.