The AUR1 gene in Saccharomyces cerevisiae encodes dominant resistance to the antifungal agent aureobasidin A (LY295337)

Abstract

Aureobasidin A (LY295337) is a cyclic depsipeptide antifungal agent with activity against Candida spp. The mechanism of action of LY295337 remains unknown. LY295337 also shows activity against the yeast Saccharomyces cerevisiae. Generation of a mutant of S. cerevisiae resistant to LY295337 is reported. Resistance was found to reside in a dominant mutation of a single gene which has been named AUR1 (for aureobasidin resistance). This gene was cloned and sequenced. A search for homologous sequences in GenBank and by BLAST did not elucidate the function of this gene, although sequence homology to an open reading frame from the Saccharomyces genome sequencing project and several other adjacent loci was noted. Deletion of aur1 was accomplished in a diploid S. cerevisiae strain. Subsequent sporulation and dissection of the aur1/aur1⌬ diploid resulted in tetrads demonstrating 2:2 segregation of viable and nonviable spores, indicating that deletion of aur1 is lethal. As LY295337 is fungicidal and deletion of aur1 is lethal, aur1 represents a potential candidate for the target of LY295337.Aureobasidins are antifungal cyclic depsipeptides isolated from Aureobasidium pullulans R106 (15, 25). Aureobasidin A (LY295337) is the major factor isolated from the fermentation broth of A. pullulans R106 (25). This compound has potent in vitro antifungal activity against Candida albicans, other species of Candida, and Cryptococcus neoformans (9,26).The model yeast Saccharomyces cerevisiae is susceptible to LY295337. The study of mutants of S. cerevisiae resistant to the action of LY295337 could lead to an understanding of its mechanism of action. In this study, generation of a dominant mutant of S. cerevisiae which is resistant to LY295337 has allowed the cloning of a gene, ABR1, which encodes resistance to LY295337 (12, 13). Hashida-Okado and coworkers and Okado et al. have recently isolated the same gene as ABR1 through resistance to aureobasidin A (named AUR1) from S. cerevisiae (11,16). The gene encoding resistance to aureobasidin A will hereafter be referred to as AUR1. AUR1 is unique and essential for viability of S. cerevisiae. AUR1 may encode the target for LY295337. MATERIALS AND METHODSStrains, media, plasmids, and transformations. S. cerevisiae strains used in this study are presented in Table 1. Cultivation, storage, and genetic manipulation of S. cerevisiae were carried out as previously described (19). The yeast shuttle plasmid pRS416 was used for construction of the yeast library, and pRS406 was used for construction of a deletion plasmid (24). Transformation of yeast strains was carried out by the method of Reddy and Maley (18). Transformants bearing the URA3-marked plasmids were cured by the use of 5-fluoroorotic acid (5-FOA) (4).Selective medium for drug resistance was standard yeast-peptone-dextrose (YPD) agar with 5 g of LY295337 per ml and 1% ethanol (19).Library construction and propagation of plasmids were performed with Escherichia coli DH5␣, grown and selected for by standard methods (21).Determination of ant...

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