Caracterização e purificação da toxina killer produzida pela levedura Kodamaea ohmeri ES92

Abstract

Killer toxins or mycocins are extracellular molecules produced by yeaststhat have a deleterious effect on organisms taxonomically related or not to its producers, without cell to cell contact. The yeast Kodamaea ohmeri, target of this study, was isolated from the cactus Opuntia sp. and was previously characterized as a killer yeast. Its killer toxin has a deleterious effect on Candida species, relevant opportunistic agents, whose medical importance is growing with the increasing number of immunocompromised patients or presenting other predisposing conditions. The search for new antifungal drugs becomes necessary, since these microorganisms are associated with high morbidity and mortality and some strains are less susceptible to traditionalantifungal drugs. This motivates the study of the killer toxin of K. ohmeri, which has not yet been characterized. The aims of this study were to determine the spectrum of action of the killer toxin as well as to characterize, purify and identify it. In order to determine its spectrum of action strains belonging to different Candida species, Saccharomyces cerevisiae, Kodamaea ohmeri and different species of dermatophytes were used. Along the experiments it became necessary to confirm the identity of some of the isolates used, previously identified by Candida fermentation API20AUX kit (Biomerieux). The confirmation was performed by growth on CHROMagar and ribosomal DNA sequencing. Theidentification of Candida by CRHOMagar was more accurate than that made by the APIAUX20, even considering its preferential application to C. albicans, C. krusei and C. tropicalis, since it was highly similar to that provided by sequencing. The characterization of the killer toxin was based on an evaluation of its resistance to temperature, pH, presence of different ions, definition of its nature, determination of its mass and investigation of its genetic determinant. For toxin purification and identification, liquid chromatography (HPLC), electrophoresis (SDS-PAGE) and mass spectrometry (MS) were used. The strains of C. albicans (86.2%) and C. glabrata (60%) were more sensitive to killer toxin followed by samples of S. cerevisiae (50%) and other strains of K.ohmeri (50%). The dermatophytes tested were resistant. The results indicate that the sensitivity of these samples to the killer toxin may be of taxonomic significance, since the species C. albicans is significantly more sensitive than other species. The results indicate that the killer toxin of K. ohmeri might be a glycoprotein having mass above 30kDa, sensitive to temperatures greater than or equal to 37°C, more active between pH 3.5 and 4.5, with antifungal activity increased in the presence of calcium ions and possibly encoded by a plasmidial gene. The tests performed until now did not indicate that cell death caused by the killer toxin would be by forming pores in cell membranes.