New Fks Hot Spot for Acquired Echinocandin Resistance in Saccharomyces cerevisiae and Its Contribution to Intrinsic Resistance of Scedosporium Species

Echinocandins represent a new antifungal group with potent activity against Candida species. These lipopeptides inhibit the synthesis of beta-1,3-glucan, the major cell wall polysaccharide. Acquired resistance or reduced echinocandin susceptibility (RES) is rare and associated with mutations in two hot spot regions of Fks1 or Fks2, the probable beta-1,3-glucan synthases. In contrast, many fungi demonstrate intrinsic RES for reasons that remain unclear. We are using Saccharomyces cerevisiae to understand the basis for RES by modeling echinocandin-Fks interaction. Previously characterized mutations confer cross-RES; we screened for mutations conferring differential RES, implying direct interaction of that Fks residue with a variable echinocandin side chain. One mutant (in an fks1 Delta background) exhibited >= 16-fold micafungin and anidulafungin versus caspofungin RES. Sequencing identified a novel Fks2 mutation, W714L/Y715N. Equivalent W695L/Y696N and related W695L/F/C mutations in Fks1 generated by site-directed mutagenesis and the isolation of a W695L-equivalent mutation in Candida glabrata confirmed the role of the new hot spot 3 in RES. Further mutagenesis expanded hot spot 3 to Fks1 residues 690 to 700, yielding phenotypes ranging from cross-RES to differential hypersusceptibility. Fks1 sequences from intrinsically RES Scedosporium species revealed W695-Fequivalent substitutions; Fks1 hybrids expressing Scedosporium prolificans hot spot 3 confirmed that this substitution imparts RES.