Elevated chitin content reduces the susceptibility of Candida species to caspofungin

Abstract

The echinocandin antifungal drugs inhibit synthesis of the major fungal cell wall polysaccharide β(1,3)-glucan. Echinocandins have good efficacy against Candida albicans but reduced activity against other Candida species, in particular Candida parapsilosis and Candida guilliermondii. Treatment of Candida albicans with a sub-MIC level of caspofungin has been reported to cause a compensatory increase in chitin content and to select for sporadic echinocandin-resistant FKS1 point mutants that also have elevated cell wall chitin. Here we show that elevated chitin in response to caspofungin is a common response in various Candida species. Activation of chitin synthesis was observed in isolates of C. albicans, Candida tropicalis, C. parapsilosis, and C. guilliermondii and in some isolates of Candida krusei in response to caspofungin treatment. However, Candida glabrata isolates demonstrated no exposure-induced change in chitin content. Furthermore, isolates of C. albicans, C. krusei, C. parapsilosis, and C. guilliermondii which were stimulated to have higher chitin levels via activation of the calcineurin and protein kinase C (PKC) signaling pathways had reduced susceptibility to caspofungin. Isolates containing point mutations in the FKS1 gene generally had higher chitin levels and did not demonstrate a further compensatory increase in chitin content in response to caspofungin treatment. These results highlight the potential of increased chitin synthesis as a potential mechanism of tolerance to caspofungin for the major pathogenic Candida species.

Fig 1

Chitin distribution in Candida clinical isolates. DIC microscopy images (top panels) and CFW fluorescence images (bottom panels). Isolates were grown in YPD alone to establish basal chitin levels (A), treated with a sub-MIC level of CSF (B), grown in YPD with 200 mM CaCl₂ and 100 μg ml⁻¹ CFW (C), or pregrown with CaCl₂ and CFW and then exposed to caspofungin at the IC₅₀ (D). Bars, 2 μm.

Fig 2

Pregrowth of Candida isolates with CaCl₂ and CFW reduces susceptibility to caspofungin. Plate dilution sensitivity tests were performed on Candida clinical isolates on YPD agar containing caspofungin at the IC₅₀ for each isolate. The following CSF concentrations were used: for C. albicans isolate 1, 0.064 μg ml⁻¹; for C. glabrata isolates 1 and 4, 0.032 μg ml⁻¹; for C. tropicalis isolate 5, 0.032 μg ml⁻¹; for C. krusei isolate 1, 0.13 μg ml⁻¹; for C. krusei isolate 4, 0.032 μg ml⁻¹; for C. krusei isolate 6, 16 μg ml⁻¹; for C. parapsilosis isolates 1 and 4, 0.13 μg ml⁻¹; for C. guilliermondii isolates 1 and 2, 16 μg ml⁻¹; and for C. guilliermondii isolate 3, 0.064 μg ml⁻¹. Rows marked with asterisks indicate pregrowth of the inoculum in YPD containing both 200 mM CaCl₂ and 100 μg ml⁻¹ CFW. Cell numbers per spot were 5,000, 500, 50, and 5 cells, from left to right.

Fig 3

Sequencing of the FKS1 hot spot region in Candida species. For each species, the sequenced control strain was used as a template to determine whether any of the clinical isolates contained point mutations in FKS1. All isolates of C. parapsilosis contained a P660A substitution, and isolate 4 contained an additional S645P point mutation (highlighted in bold). C. guilliermondii isolate 3 contained an L646I point mutation (highlighted in bold).