Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-beta-D-glucan synthase: implication for the existing susceptibility breakpoint

Abstract

Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-beta-D-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-beta-D-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 microg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 microg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of > or = 2 microg/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.

FIG. 1.

Summary of echinocandin MIC (A) and IC₅₀ (B) values for the C. glabrata strains harboring various Fks1p and Fks2p amino acid substitutions. Lines represent average MICs and IC₅₀s for ANF, CSF, and MCF for the group of mutants harboring amino acid substitutions in each Fks protein. MICs were obtained using the CLSI document M27-A3 guidelines, and IC₅₀s were obtained using product entrapped partially purified 1,3-β-d-glucan synthase enzyme complexes. WT, wild type.

FIG. 2.

Echinocandin inhibition profiles for product-entrapped 1,3-β-d-glucan synthase enzyme complexes assessed by the incorporation of [³H]glucose into radiolabeled product. (A) CSF titration curves for enzymes isolated from strains ATCC 90030 (wild type), 5847 (Fks1p-S629P), and 3830 (Fks2p-S663P). (B) ANF inhibition curves for 1,3-β-d-glucan synthase enzyme complexes extracted from strains ATCC 90030 (wild type), 42997 (Fks1p-S625S), 234 (Fks2p-S659V), and 41026 (Fks2p-F659S).

FIG. 3.

Lineweaver-Burke double-reciprocal plots used to determine the maximum velocity (V_max) and the Michaelis-Menten constant (K_m) for product-entrapped 1,3-β-d-glucan synthase complexes by varying the amount of [³H]UDPG. (A) Comparison of the kinetic properties of the 1,3-β-d-glucan synthase enzymes isolated from strains ATCC 90030 (wild type [WT]), 5847 (Fks1p-S629P), and 3830 (Fks2p-S663P). (B) Evaluation of the kinetic properties of the 1,3-β-d-glucan synthase enzyme obtained from the isogenic strains 3168 (WT) and 3169 (Fks1p-D632G).

FIG. 4.

MIC/IC₅₀ correlation as a function of Fksp amino acid substitutions. The echinocandin drug ANF (A), CSF (B), or MCF (C) was used to obtain IC₅₀ values and MICs in the presence of RPMI 1640 medium (CLSI document M27-A3) (4) or RPMI 1640 medium with 50% serum. Vertical dashed lines represent the CLSI susceptibility breakpoint (2 μg/ml). WT, wild type.

FIG. 4.

MIC/IC₅₀ correlation as a function of Fksp amino acid substitutions. The echinocandin drug ANF (A), CSF (B), or MCF (C) was used to obtain IC₅₀ values and MICs in the presence of RPMI 1640 medium (CLSI document M27-A3) (4) or RPMI 1640 medium with 50% serum. Vertical dashed lines represent the CLSI susceptibility breakpoint (2 μg/ml). WT, wild type.