Characterization of a Novel FKS1 Mutation in Candida lusitaniae Shows a Potential Critical Role for MKC1 in Echinocandin Resistance

Caspofungin is an echinocandin antifungal that inhibits glucan synthesis in the fungal cell wall. A Candida parapsilosis bloodstream isolate resistant to echinocandins was recovered from a patient who had undergone allogeneic hematopoietic stem cell transplantation. The FKS1 gene, encoding the target glucan synthase, contained a heterozygous mutation resulting in an I1380T amino acid change, in addition to the naturally occurring P660A polymorphism. When expressed at the equivalent position in the Fks1p protein of C. lusitaniae, P642A and I1359T, alone and in combination, led to 6-, 12-, and [≥]256-fold increases in the minimal inhibitory concentration (MIC) of caspofungin, respectively. The caspofungin concentration needed to inhibit 50% of glucan synthase activity was increased 3-, 37-, and 270-fold, respectively. At high drug concentrations, and also in drug-free medium, infrared spectroscopy revealed a decrease in {beta}-glucan content and an increase in chitin in the cell wall of the I1359T Fks1p mutants. Atomic force microscopy showed cell wall damage and cell swelling in both susceptible and resistant strains under caspofungin exposure. Analysis of susceptibility to cell-wall stressors and key factors in cell wall integrity (CWI) and high-osmolarity glycerol (HOG) pathways showed that all strains activated these pathways under caspofungin stress. In the I1359T Fks1p mutants, Mkc1p was constitutively activated even without caspofungin. Deletion of MKC1 restored caspofungin susceptibility, indicating that activation of the CWI pathway is a key molecular determinant of resistance in vitro to caspofungin in these mutants.