Inactivation of the FCY2 gene encoding purine-cytosine permease promotes cross-resistance to flucytosine and fluconazole in Candida lusitaniae.
In a previous work, we described the possible relationship between a defect of purine-cytosine permease and the acquisition of a cross-resistance to the antifungal combination flucytosine (5FC) and fluconazole (FLC) in Candida lusitaniae (T. Noël, F. François, P. Paumard, C. Chastin, D. Brethes, and J. Villard, Antimicrob. Agents Chemother. 47:1275-1284, 2003). Using degenerate PCR and chromosome walking, we cloned two FCY2-like genes in C. lusitaniae. Northern blot analysis revealed that only one gene was expressed; it was named FCY2. The other one behaved as a pseudogene and was named FCY21. In order to better characterize the possible role of FCY2 in cross-resistance to 5FC-FLC, disruption experiments with auxotrophic strain 6936 ura3(D95V) FCY2 with an integrative vector carrying the URA3 gene and a partial sequence of the C. lusitaniae FCY2 gene were undertaken. Southern blot analysis revealed that homologous recombination events occurred in all transformants analyzed at rates of 50% at resident locus FCY2 and 50% at resident locus URA3, resulting in the genotypes ura3 fcy2::URA3 and ura3::URA3 FCY2, respectively. It was then demonstrated that only transformants harboring a disrupted fcy2 gene were resistant to 5FC, susceptible to FLC, and resistant to the 5FC-FLC combination. Finally, complementation experiments with a functional FCY2 gene restored 5FC and FLC susceptibilities to the wild-type levels. The results of this study provide molecular evidence that inactivation of the sole FCY2 gene promotes cross-resistance to the antifungal association 5FC-FLC in C. lusitaniae.
Chapeland-Leclerc
Florence
F
Laboratoire des Sciences Végétales, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris 5, 4 avenue de l'Observatoire, 75006 Paris, France. thierry.noel@univ-paris5.fr
Bouchoux
Julien
J
Goumar
Abdelhak
A
Chastin
Christiane
C
Villard
Jean
J
Noël
Thierry
T
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D002175:Candida / Q000187:drug effects* / Q000235:genetics; D016386:Chromosome Walking; D003001:Cloning, Molecular; D003596:Cytosine; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics*; D015725:Fluconazole / Q000494:pharmacology*; D005437:Flucytosine / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D033702:Nucleobase Transport Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D000935:Antifungal Agents; D004271:DNA, Fungal; D005656:Fungal Proteins; D033702:Nucleobase Transport Proteins; C022483:purine permease; D003596:Cytosine; D015725:Fluconazole; D005437:Flucytosine