Mechanisms of antifungal drug resistance in Candida species

Shriya Raj

doi:10.17918/00008478

Candida albicans and the recently emerged Candida glabrata are the most frequent agents of opportunistic fungal infection. Antifungals commonly used to treat these infections inhibit synthesis of the cell wall or plasma membrane. These agents are, respectively, the [beta]-1,3-glucan synthesis-inhibiting echinocandins (e.g., caspofungin) and the ergosterol biosynthesis-inhibiting azoles (e.g., fluconazole). In C. albicans, [beta]-1,3-glucan synthesis is linked to essential gene GSC1, the site of mutations conferring high-level echinocandin resistance. In mutants exhibiting low-level resistance, expression of GSC1 paralog GSL2 was upregulated. To further study its role, the UAU1 technique was employed to generate homozygous gsl2 disruptants. One of four disruptants obtained, D4, was caspofungin-hypersensitive. Furthermore, D4 demonstrated an osmostabilizer-suppressed growth defect, hypersensitivity to cell wall-binding agents calcofluor white and congo red, and temperature sensitivity, consistent with a role for GSL2 in cell wall integrity. Intriguingly, GSL2 expression was highly growth-phase dependent - abundant in early to mid-log and declining in late log - suggesting a role during rapid cell wall growth. Intrinsic and acquired azole resistance in C. glabrata is mediated by transcription factor Pdr1-dependent upregulation of azole/multidrug transporter gene CDR1. The CDR1 promoter contains six candidate Pdr1 response elements (PDRE) located 133 to 1200 bp upstream of the start codon, followed by a GGAYYT repeat and a candidate TATA box. To analyze the role of these elements, a PCDR1-TRP1 plasmid reporter system was developed in conjunction with trp1[delta] strains encoding either wild type or constitutively active Pdr1 from azole-resistant mutant F15. TRP1 expression was monitored by evaluating growth on or in trp-minus medium. Truncation analysis revealed that five PDRE are required for full TRP1 expression in Pdr1WT and three in Pdr1F15 cells. In a 4 PDRE construct, TRP1 expression in Pdr1F15 cells was progressively reduced when 1 to 3 PDRE were mutated. Mutational inactivation of the TATA box only modestly reduced TRP1 expression. Interestingly, mutation of the GGAYYT repeat eliminated expression in Pdr1WT but had no effect in Pdr1F15 cells. In summary, these studies characterized new factors that play a role in echinocandin and azole activity, and suggest new ways in which this activity may be enhanced.

Mechanisms of antifungal drug resistance in Candida species

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Mechanisms of antifungal drug resistance in Candida species

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