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-␤-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.

CD101: a novel long‐acting echinocandin

“…The inhibition curves for MCF and CD101 against the C . albicans WT isolate showed the typical pattern of β‐1,3‐D‐glucan synthase echinocandin susceptibility reported with other sensitive Candida species (Park et al, 2005; Garcia‐Effron et al, 2009), with mean IC 50 s of 17.7 and 14.3 ng/ml for MCF and CD101, respectively (supplementary figure and Table 2). Decreased echinocandin susceptibility was observed with the resistant C .…”

“…Reactions were initiated by the addition of product‐entrapped glucan synthase. Sensitivity to MCF and CD101 was measured in a polymerization assay using a 96‐well 0.65 µm multiscreen HTS filtration system (Millipore Corporation, Bedford, MA) in a final volume of 100 µl, as previously described (Park et al, 2005). Serial dilutions of the drugs (0.01–10 000 ng/ml) were used as calibration standards.…”

Development of echinocandin resistance in Candida krusei isolates following exposure to micafungin and caspofungin in a BM transplant unit

“…3,4 Mutations in the Fks1 gene (encoding 1,3-β-D-glucan synthase) are correlated with reduced susceptibility. 5 We report here C. krusei breakthrough fungemia following exposure to prophylactic micafungin in HSC transplanted patients. In two patients, C. krusei with novel mutations in the Fks1 gene and reduced susceptibility to echinocandins emerged in digestive tract surveillance isolates.…”

The activity of echinocandins, amphotericin B and voriconazole against fluconazole-susceptible and fluconazole-resistant Brazilian Candida glabrata isolates

“…Our results demonstrate that the MICs of echinocandins in the FS and the FR groups have increased slightly. Acquired resistance or reduced susceptibility to echinocandins has rarely been reported, with the majority of cases being associated with mutations in the FKS1 gene (Park et al 2005).…”