Role for Fks1 in the intrinsic echinocandin resistance of Fusarium solani as evidenced by hybrid expression in Saccharomyces cerevisiae

Abstract

The opportunistic mold Fusarium solani is intrinsically resistant to cell wall synthesis-inhibiting echinocandins (ECs), including caspofungin and micafungin. Mutations that confer acquired EC resistance in Saccharomyces cerevisiae and other normally susceptible yeast species have been mapped to the Fks1 gene; among these is the mutation of residue 639 from Phe to Tyr (F639Y) within a region designated hot spot 1. Fks1 sequence analysis identified the equivalent of Y639 in F. solani as well as in Scedosporium prolificans, another intrinsically EC-resistant mold. To test its role in intrinsic EC resistance, we constructed Fks1 hybrids in S. cerevisiae that incorporate F. solani hot spot 1 and flanking residues. Hybrid construction was accomplished by a PCR-based method that was validated by studies with Fks1 sequences from EC-susceptible Aspergillus fumigatus and paired EC-susceptible and -resistant Candida glabrata isolates. In support of our hypothesis, hybrid Fks1 incorporating F. solani hot spot 1 conferred significantly reduced EC susceptibility, 4- to 8-fold less than that of wild-type S. cerevisiae and 8- to 32-fold less than that of the same hybrid with an F639 mutation. We propose that Fks1 sequences represent determinants of intrinsic EC resistance in Fusarium and Scedosporium species and, potentially, other fungi.

FIG. 1.

Fks1 and Fks2 hotspots 1 and 2, which are implicated in EC resistance, aligned with the equivalent regions of F. solani and S. prolificans Fks1. Dots represent identity to S. cerevisiae Fks1. Mutated residues associated with EC resistance are underlined, with the mutations indicated underneath (see the text for references). All mutations were spontaneously acquired, except for the A. fumigatus mutation, which was introduced by transformation (38). Arrowheads (▴) indicate the Y639-equivalent residues of F. solani and S. prolificans Fks1, which are proposed here to be the basis for their intrinsic EC resistance. The C. tropicalis and S. prolificans Fks1 sequences are incomplete, and hence are not numbered.

FIG. 2.

(A) Strategy for generating hybrid S. cerevisiae Fks1 incorporating the hot spot 1 region from F. solani Fks1. Step 1 is the construction in S. cerevisiae BY4742 of the internal deletion fks1Δ617-649::URA3. Step 2 is the use of the step 1-generated deletant as the host for the construction of hybrid fks1-Fs617-649, which replaces S. cerevisiae Fks1 codons 617 to 649 with the corresponding region from F. solani. The drawing is not to scale. (B) Alignment of S. cerevisiae Fks1 residues 617 to 649 with the equivalent regions from A. fumigatus and F. solani.