Nonsense and Missense Mutations in FCY2 and FCY1 Genes Are Responsible for Flucytosine Resistance and Flucytosine-Fluconazole Cross-Resistance in Clinical Isolates of Candida lusitaniae

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Abstract
The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae . We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2 ( C505T ) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC- and 5FC/FLC-resistant fcy1 Δ strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.

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