Hypermutation inCryptococcusreveals a novel pathway to 5-fluorocytosine (5FC) resistance

AbstractDrug resistance is a critical challenge in treating infectious disease. For fungal infections, this issue is exacerbated by the limited number of available and effective antifungal agents. Patients infected with the fungal pathogenCryptococcusare most effectively treated with a combination of amphotericin B and 5-fluorocytosine (5FC). Isolates causing infections frequently develop resistance to 5FC although the mechanism of this resistance is poorly understood. Here we show that resistance is acquired more frequently in isolates with defects in DNA mismatch repair that confer an elevated mutation rate. Natural isolates ofCryptococcuswith mismatch repair defects have recently been described and defective mismatch repair has been reported in other pathogenic fungi. In addition, whole genome sequencing was utilized to identify mutations associated with 5FC resistancein vitro. Using a combination of candidate-based Sanger and whole genome Illumina sequencing, the presumptive genetic basis of resistance in 16 independent isolates was identified, including mutations in the known resistance genesFUR1andFCY2, as well as a novel gene,UXS1. Mutations inUXS1lead to accumulation of a metabolic intermediate that appears to suppress toxicity of both 5FC and its toxic derivative 5FU. Interestingly, while aUXS1ortholog has not been identified in other fungi likeSaccharomyces cerevisiae, where the mechanisms underlying 5FC and 5FU resistance were elucidated, aUXS1ortholog is found in humans, suggesting that mutations inUXS1in cancer cells may also play a role in resistance to 5FU when used during cancer chemotherapy in humans.