Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates

Abstract

An association between reduced susceptibility to echinocandins and changes in the 1,3-beta-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

FIG. 1.

Integration of plasmid pGSC8 at CaFKS1. (A) Schematic map of the Cafks1 locus of strain T1FOA, showing the Cafks1h::hisG and CaFKS1b alleles. Abbreviations for restriction enzyme cleavage sites are as follows: P, PvuII; H, HindIII; K, KpnI; Sa, SalI; B, BamHI; Sp, SpeI. The K, Sa, and B sites are included for reference only and should not be considered unique within the regions shown. The open box represents the hisG sequence remaining after the Ura blaster method (16) was used to construct a Ura⁻ strain with a deletion of CaFKS1. Rectangles with stippling below the alleles indicate regions encoding either CaFks1p or Cafks1p. (B). Map of plasmid pGSC8. The pBR322 vector sequences (thin line), the 1.2-kb URA3 gene (stippled line), and DNA at the Cafks1h-1 locus (hatched line) are indicated. Arrows within the circle illustrate the direction of transcription of the URA3, CaFKS1, and E. coli bla genes. The plasmid was linearized by digestion with SpeI (thick line intersecting the circle) prior to transformation. (C and D) Chromosomal maps following pGSC8 integration at CaFKS1. The maps depict pGSC8 integration at the Cafks1h::hisG allele (C) or the CaFKS1b allele (D).

FIG. 2.

Alignment of Fks protein sequences important for echinocandin susceptibility in S. cerevisiae and C. albicans. The Fks proteins from wild-type S. cerevisiae (ScFks1p and ScFks2p; GenBank accession numbers U12893 and U16783, respectively) and S. cerevisiae mutants R560-1C (ScFks1-2p), MS10 (ScFks1-3p), and YFK978 (ScFks2-1p), as well as wild-type C. albicans (CaFks1p; accession number D88815) and C. albicans mutants CAI4-R1 (CaFks1h-1p) and NR3 (CaFks1h-3p), were aligned. Subscripts for the first and last residue of each entry identify amino acid positions in the Fksp sequence.

FIG. 3.

Schematic diagram of CaFks1 loci associated with reduced susceptibility to caspofungin. The locations of regions linked to reduced caspofungin susceptibility are shown superimposed on a linear profile of CaFks1p (numbers refer to amino acid positions). The gray bar depicts highly conserved regions of the Fks protein, and arrows provide the approximate hybridization positions of primer sets (HS1F, etc.) used to amplify the two regions associated with reduced susceptibility.

FIG. 4.

Caspofungin inhibition profiles of enriched GS complexes from wild-type and mutant C. albicans strains. Relative GS activity was assessed by the incorporation of [³H]glucose into radiolabeled product. (A) Caspofungin titration curves for the C. albicans parental strain CAI4 (squares), heterozygous S645P/WT strain NR4 (triangles), and homozygotic pseudo-haploid S645P/null strain T25 (circles). Percent incorporation values for GS from strain T25 did not fit a simple sigmoidal curve. (B) Caspofungin titration curves for the parental strain CAI4 (squares) and the homozygous S645Y/S645Y strain NR3 (triangles).

FIG. 5.

Dendrogram and splits tree analysis of genetically related isolates. Dendrogram showing the genetic relatedness among 21 C. albicans isolates, per Robles et al. (36). The splits tree demonstrates the genetic relatedness among isolates. Sixteen strains with a CLY number from protocol 026 (including those described in Table 5) and five laboratory reference strains were evaluated.