A naturally occurring proline-to-alanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility

Abstract

Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced V(max) values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

FIG. 1.

Sequence alignments of Fks1p and Fks2p hot spot regions from diverse fungal species. The aligned sequences are as follows: C. parapsilosis (Cp; GenBank accession no. ABX80511); C. metapsilosis (Cm; ABY67254); C. orthopsilosis (Co; ABY67253); C. albicans wild-type strain 5314 (Ca; XP_721429); C. albicans mutant strain 122, FKS1p P649H (Ca*); S. cerevisiae (Sc; AAC48981); C. glabrata Fks2p (Cg; XP_448401); C. glabrata mutant strain 916, Fks2p P633T (Cg*); C. krusei (Ck; AAY40291); Aspergillus fumigatus (Af; AAB58492); Debaryomyces hansenii (Dh; XP_457762); Yarrowia lipolytica (Yl; XP_504213); Kluyveromyces lactis (Kl; CAH02189); Schizosaccharomyces pombe (Sp; NP_588501); and Coccidioides immitis (Ci; EAS36399).

FIG. 2.

Kinetic properties of GS inhibition by echinocandin drugs. (A) CSF inhibition profiles of trapped GS complexes from wild-type C. albicans SC5314 (gray squares), C. glabrata ATCC 90030 (gray diamonds), C. parapsilosis ATCC 22019 (triangles), C. metapsilosis 960161 (inverted triangles), and C. orthopsilosis H10 (circles). (B) ANF titration of the GS complexes isolated from C. albicans Sc5314 (gray squares), C. albicans mutant strain 122 (black squares), C. parapsilosis ATCC 22019 (triangles), C. metapsilosis 960161 (inverted triangles), and C. orthopsilosis H10 (circles). (C) Average IC₅₀ values comparison between the following groups: C. parapsilosis (Cp), C. metapsilosis (Cm), C. orthopsilosis (Co), and S. cerevisiae wild type (Sc WT) and P647A mutant (Sc P647A). (D) ANF inhibition profiles of partially purified GS complexes from S. cerevisiae wild type (circles) and FKS1 P647A (inverted triangles).