Inactivation of the FCY2 Gene Encoding Purine-Cytosine Permease Promotes Cross-Resistance to Flucytosine and Fluconazole in Candida lusitaniae

Abstract

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.Treatment of fungal infections represents a crucial problem for clinicians because of the emergence of resistance to a small number of antifungal drugs available for systemic use (18). In order to minimize the risk of therapeutic failure, the use of bitherapy, which relies on antifungal combinations, is more frequent and is strongly recommended when the treatment includes flucytosine (5FC). Indeed, resistance to 5FC can easily develop as a result of mutations mainly in genes involved in the uptake of 5FC (the FCY2 gene, which encodes purinecytosine permease [PCP]), in the conversion of intracellular 5FC to 5-fluorouracil (5FU; the FCY1 gene, which encodes cytosine deaminase), or in the transformation of 5FU to 5-fluorouridine monophosphate (the FUR1 gene, which encodes uracil phosphoribosyltransferase) (9, 16, 34).However, combinations of antifungals may be challenged by the occurrence of cross-resistance, particularly in the commonly used class of azole antifungals, where it can derive from enhanced expression of multidrug resistance genes, such as CDR1 and CDR2 of the ATP-binding cassette transporter family (26, 28, 30), or from point mutations of the genes that encode the lanosterol demethylase and the ⌬ 5,6 -sterol desaturase of the ergosterol biosynthesis pathway (17,29).Because of the increasing use of antifungal bitherapy, we investigated the possible occurrence of cross-resistance in Candida lusitaniae. Despite its low prevalence ...

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

“…It was then hypothesized that extracellular 5FC would behave as a competitive inhibitor of FLC uptake (23). We recently provided molecular evidence that inactivation of the C. lusitaniae FCY2 and FCY1 genes promoted FLC resistance in the presence of subinhibitory 5FC concentrations (7,24).The present work explores the molecular events supporting 5FC resistance and 5FC/FLC cross-resistance in 11 genetically unrelated C. lusitaniae clinical isolates from our collection, including the 4 previously described and characterized at the biochemical level (23). In particular, we analyzed the se-…”

“…Yeast suspensions were diluted with RPMI 1640 medium (Sigma) to a final cell count of approximately 10 3 cells/ml, as previously described (7). The 96-well microtiter plates were incubated for 48 h at 35°C.…”

“…It was then hypothesized that extracellular 5FC would behave as a competitive inhibitor of FLC uptake (23). We recently provided molecular evidence that inactivation of the C. lusitaniae FCY2 and FCY1 genes promoted FLC resistance in the presence of subinhibitory 5FC concentrations (7,24).…”

Future therapies targeted towards eliminatingCandidabiofilms and associated infections

“… Mutations of cytosine permease coded by FCY2 may also lower the uptake of the antifungal in to the cell (Figure 2) [238,240].…”

Differential Involvement of Histidine Kinase Receptors in Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast Candida lusitaniae

“…It is also an emerging pathogen which is characterized by its propensity to develop resistance to antifungal agents during treatment (16,24,31,32,47,52,58,60). The recent achievement of C. lusitaniae whole genome sequencing clearly reflects a growing interest of the scientific community in this opportunistic yeast.…”