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.
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 fcy1Delta 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.
Florent
Martine
M
EA209 Eucaryotes Pathogènes, Transports Membranaires et Chimiorésistances, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 Avenue de l'Observatoire, Paris, France.
Noël
Thierry
T
Ruprich-Robert
Gwenaël
G
Da Silva
Bruno
B
Fitton-Ouhabi
Valérie
V
Chastin
Christiane
C
Papon
Nicolas
N
Chapeland-Leclerc
Florence
F
D000935:Antifungal Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015139:Blotting, Southern; D002175:Candida / Q000187:drug effects / Q000235:genetics; D002177:Candidiasis / Q000382:microbiology*; D018389:Codon, Nonsense / Q000235:genetics*; D025141:Drug Resistance, Fungal / Q000235:genetics; D015725:Fluconazole / Q000494:pharmacology*; D005437:Flucytosine / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D006801:Humans; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense / Q000235:genetics*; D016133:Polymerase Chain Reaction
D000935:Antifungal Agents; D018389:Codon, Nonsense; D005656:Fungal Proteins; D015725:Fluconazole; D005437:Flucytosine