A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase

Abstract

A novel, potent, semisynthetic pneumocandin, L-733,560, was Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-p-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etgl-l (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etgl-l mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble (B-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-I-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etgl-l mutant gene encodes a subunit of the 1,3-0-D-glucan synthase complex.

Identification of two cell cycle regulated genes affecting the β1,3‐glucan content of cell walls in Saccharomyces cerevisiae

“…comm.). The etgl-1 mutant was isolated as a mutant resistant to L-733,560, a semisynthetic echinocandin [37], which inhibits fll,3-glucan synthase in Candida albicans [38]. In vitro, the ICso offl-glucan synthase activity to L-733,560 ofetgl-1 was found to be 50-fold higher compared to wild type, explaining the resistancy to L-733,560 in vivo, and indicating that C WH53/ETG1 is involved in the formation offl 1,3-glucan.…”

Resistance to echinocandin-class antifungal drugs

“…Early genetic studies by Myra Kurtz and Cameron Douglas (Merck Research Labs) with caspofungin in S. cerevisiae (Douglas et al, 1994a;Douglas et al, 1994b) and C. albicans indicated that Fks1, the major subunit of glucan synthase, is the presumed target of the echinocandins. These genetic studies suggested that target-site modification was a likely cause of reduced susceptibility.…”

Role of Small G Proteins in Yeast Cell Polarization and Wall Biosynthesis

“…However, glucan synthase has been purified extensively from solubilized membranes by the product entrapment procedure (125). These preparations were enriched in the product of the FKS1 gene, which, together with its homologue, FKS2 (126), has been implicated in the activity of glucan synthase (127,128). Mutants in FKS1 were first isolated in a screen for hypersensitivity to immunosuppressants (129), and again, under the name etg1, in a screen for strains resistant to glucan synthase inhibitors (127).…”

“…These preparations were enriched in the product of the FKS1 gene, which, together with its homologue, FKS2 (126), has been implicated in the activity of glucan synthase (127,128). Mutants in FKS1 were first isolated in a screen for hypersensitivity to immunosuppressants (129), and again, under the name etg1, in a screen for strains resistant to glucan synthase inhibitors (127). Null ( f ks1 ) mutants show a decrease in glucan synthase activity (128) and in incorporation of glucose into β (1 → 3)glucan in vivo (130); double null ( f ks1 f ks2 ) mutants are inviable (126).…”