Tetrapisispora phaffii killer toxin is a highly specific β-glucanase that disrupts the integrity of the yeast cell wall

BACKGROUND Killer yeasts have been used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and in the medical field, to develop novel antimycotics for the treatment of human and animal fungal infections. Among these killer yeasts, Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii) secretes a glycoprotein known as Kpkt that is lethal to spoilage yeasts under winemaking conditions. In the present study, the mode of action of Kpkt, and the specific damage produced by this toxin on sensitive yeasts is investigated. RESULTS The use of castanospermine, a beta-glucanase inhibitor, demonstrated that beta-glucanase activity is essential for the Kpkt killer activity in vivo. Accordingly, Kpkt has no killer activity on either sensitive yeast spheroplasts or whole sensitive cells in the presence of isosmothic medium (0.8 molar sorbitol). Kpkt induces ultrastructural modifications in the cell wall of sensitive strains, as shown by confocal microscopy, laser-scanning electron microscopy, and atomic force microscopy. The Kpkt killer action is mediated by the glucidic portion of the toxin. This, in turn, appears to be involved both in the stronger cytocidal activity and in the selectivity for the sensitive strain shown by Kpkt compared to laminarinase. CONCLUSION Collectively, these data indicate that the mode of action of Kpkt is directed towards the disruption of cell-wall integrity, and that this is mediated by a highly specific beta-glucanase activity. In this, Kpkt differs from other microbial beta-glucanases that do not show killer activities.