Hypermutation in Cryptococcus reveals anovel pathway to 5-fluorocytosine (5FC) resistance

Drug 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 pathogen Cryptococcus are 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 of Cryptococcus with 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 resistance in 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 genes FUR1 and FCY2, as well as a novel gene, UXS1. Mutations in UXS1 lead to accumulation of a metabolic intermediate that appears to suppress toxicity of both 5FC and its toxic derivative 5FU. Interestingly, while a UXS1 ortholog has not been identified in other fungi like Saccharomyces cerevisiae, where the mechanisms underlying 5FC and 5FU resistance were elucidated, a UXS1 ortholog is found in humans, suggesting that mutations in UXS1 in cancer cells may also play a role in resistance to 5FU when used during cancer chemotherapy in humans.