Published Ahead of Print 27 May 2014. 10.1128/AAC.00009-14.

2014, 58(8):4476. DOI:Antimicrob. Agents Chemother. Dementhon, Isabelle Accoceberry and Thierry NoëlSophie Pujol, Valérie Fitton-Ouhabi, Daniel Brèthes, Karine Frédéric Gabriel, Ayman Sabra, Sofiane El-Kirat-Chatel, Fluorinated Nucleotides and FluconazoleHighlights Antagonistic Interaction betweenand Azoles in Candida lusitaniae and

5-FluorouracilConfers Cross-Resistance to Deletion of the Uracil Permease Gene

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Deletion of the Uracil Permease Gene Confers Cross-Resistance to5-Fluorouracil and Azoles in Candida lusitaniae and HighlightsAntagonistic Interaction between Fluorinated Nucleotides andFluconazole

Frédéric Gabriel,a,b,c Ayman Sabra,a,b Sofiane El-Kirat-Chatel,a,b* Sophie Pujol,c Valérie Fitton-Ouhabi,a,b Daniel Brèthes,a,d

Karine Dementhon,a,b Isabelle Accoceberry,a,b,c Thierry Noëla,b

University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, Francea; CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234,Bordeaux, Franceb; Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Parasitologie-Mycologie, Bordeaux, Francec; Institut de Biochimie et Génétique Cellulaires,UMR 5095, Bordeaux, Franced

We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) familyinvolved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae.Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter wascharacterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured us-ing radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transportsallantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazolesand imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are notinvolved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance toazoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtainedled us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resis-tance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest thatintracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.

Candida lusitaniae is an opportunistic pathogenic yeast that canbe responsible for deep and invasive fungal infections, partic-

ularly in neonatology and in hematology and oncology units (1,2). Although much less frequent than Candida albicans, C. lusita-niae is feared because of its ability to acquire antifungal resistanceduring treatment, noticeably to polyene (3–5) and azole antifun-gals (1, 6), and it is considered to have decreased susceptibility toechinocandins in vitro (7).

In a preceding work, we demonstrated cross-resistance be-tween flucytosine (5FC) and fluconazole (FLC) in several geneti-cally unrelated clinical isolates of C. lusitaniae (8). These isolateswere resistant to flucytosine, susceptible to fluconazole, and resis-tant to both antifungals, but only when they were used simultane-ously. Such cross-resistance between flucytosine and fluconazoleis intriguing in several regards, first because the two moleculeshave very different targets in the cell. Flucytosine is a prodrug thatuses the salvage pathway of pyrimidine to exert its toxic activity(Fig. 1). The molecule enters the fungal cell using the specifictransporter cytosine permease, then it is deaminated by a cytosinedeaminase to 5-fluorouracil (5FU), which is finally converted totoxic fluorinated UMP (5FUMP) through the activity of uracilphosphoribosyl transferase (UPRTase). The diverse fluorinatedderivatives that are generated further inhibit either the enzymethymidylate synthetase and affect the synthesis of DNA or areincorporated into the mRNA and block the translation process(9). It is noteworthy that fluorinated UMP also can be generatedthrough the de novo pathway of pyrimidine synthesis when fedwith 5-fluoroorotic acid (Fig. 1), a toxic analog of orotate that is

widely used for the selection of ura3 auxotrophs. Fluconazole isthought to enter the cell via facilitated transport, which has notbeen characterized so far (10), and then reach the endoplasmicreticulum, where its target is located (11), the lanosterol 14-alpha-demethylase (Erg11p), a key enzyme of the ergosterol biosyntheticpathway (12). Our ongoing goal is to decipher the underlyingmechanisms of the flucytosine-fluconazole cross-resistance phe-notype. We already know that a single genetic event supports bothresistance to flucytosine and cross-resistance to fluconazole, ei-ther a mutation in the FCY2 gene encoding purine-cytosine per-mease (13, 14) or a mutation in the FCY1 gene encoding cytosinedeaminase (14, 15). The mutation of FUR1, encoding UPRTase,confers cross-resistance to flucytosine and 5-fluorouracil but, sur-prisingly, does not support cross-resistance to fluconazole (15).Because of the structural similitude between flucytosine and 5-flu-

Received 6 January 2014 Returned for modification 9 March 2014Accepted 17 May 2014

Published ahead of print 27 May 2014

Address correspondence to Thierry Noël, thierry.noel@u-bordeaux2.fr.

* Present address: Sofiane El-Kirat-Chatel, Université Catholique de Louvain,Institute of Life Sciences, Louvain-la-Neuve, Belgium.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00009-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.00009-14

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orouracil and of their different properties to support or not sup-port cross-resistance to fluconazole, we decided in this study tocomplete the characterization in C. lusitaniae of the membranetransporters of the NCS1 (nucleobase cation symporter 1) family

that are involved in the uptake transport of pyrimidine nucleo-bases, focusing on the ortholog of Fur4p, the plasma membranepermease which specifically transports uracil in Saccharomycescerevisiae (16), and to determine its possible contribution to thefluoropyrimidine-fluconazole cross-resistant phenotype in C. lus-itaniae. The results obtained allow us to conclude that fluorinatednucleotides derived from 5-fluorouracil are the key molecules re-sponsible for resistance to triazole and imidazole antifungalsthrough a mechanism that remains to be elucidated, either bypreventing azoles from reaching the target, Erg11p, at the intra-cellular level or by activating efflux pumps, which leads to de-creased azole concentrations at the enzyme target within the yeastcell. We also show that this mechanism is not restricted to C.lusitaniae but that it is widespread in the genus Candida.

MATERIALS AND METHODSCandida lusitaniae strains and media. The yeast strains used and con-structed in this study are listed in Table 1. The Candida lusitaniae wild-type 6936 MATa strain (Centraalbureau voor Schimmelcultures, Utrecht,The Netherlands) was used for gene cloning and as a susceptible referencestrain for antifungal agents susceptibility tests. The clinical isolate CL38MAT� fcy2, which has a defective purine-cytosine permease resultingfrom a C505T nonsense mutation in FCY2, is resistant to flucytosine,susceptible to 5-fluorouracil and to fluconazole, and cross-resistant to theflucytosine-fluconazole association (8). This strain was used for geneticcrosses. The 6936 MATa fur1�::URA3 strain, defective for uracil phos-phoribosyl transferase, and the 6936 MATa fcy2�::URA3 strain, defectivefor purine-cytosine permease, were used as control strains for antifungaltesting (15). The auxotrophic 6936 MATa ura3� (bearing a deletion of360 bp in the orotidine 5=-phosphate decarboxylase gene) and 6936 MATatrp1� (bearing a deletion of 798 bp in the phosphoribosyl anthranilateisomerase gene) strains, along with the double auxotrophic 6936 MATaura3� trp1� strain, were used for transformation experiments. Thesestrains were described or constructed as already described elsewhere (17).

The yeast cells were cultivated in liquid YPD medium (1% yeast ex-tract, 2% peptone, 2% glucose) at 35°C under agitation (250 rpm). Aftertransformation, selection was accomplished on synthetic YNB medium

FIG 1 Salvage pathway and de novo biosynthesis pathway of pyrimidines inCandida lusitaniae. 5FC, flucytosine; 5FU, 5-fluorouracil; 5FUMP, 5-fluorou-ridine monophosphate; 5-FOMP, 5-fluoroorotidine monophosphate; 5FOA,5-fluoroorotic acid. The nomenclature of the genes used is the same as that forSaccharomyces cerevisiae: URA1, dihydroorotate dehydrogenase; URA2, car-bamoylphosphate synthetase-aspartate transcarbamylase; URA3, orotidine-5=-phosphate decarboxylase; URA4, dihydroorotase; URA5, orotate phospho-ribosyltransferase; FCY1, cytosine deaminase; FCY2, purine-cytosinepermease; FCY21, pseudogene paralog of FCY2; FUR1, uracil phosphoribosyl-transferase; FUR4, uracil permease; DAL4, allantoin permease. The genes withasterisks have been characterized in C. lusitaniae.

TABLE 1 Candida yeast strains used and constructed in this study

Species and strain Genotype Origin Reference or source

Candida lusitaniaeCL38 fcy2 MAT� fcy2[C505T] Clinical isolate, 5FCr 842720 MAT� Clinical isolate, ATCC 46936 MATa Wild type, CBS6936 ura3� MATa ura3�360 6936 176936 trp1� MATa trp1�798 6936 This study6936 ura3� trp1� MATa ura3�360 trp1�798 6936 This study6936 fur1� MATa fur1�::URA3 ura3[D95V] 6936 ura3[D95V] 156936 fcy2� MATa fcy2�::URA3 ura3[D95V] 6936 ura3[D95V] 136936 dal4� MATa dal4�::URA3 ura3�360 6936 ura3� This study6936 fur4� MATa fur4�::TRP1 trp1�798 6936 trp1� This study6936 dal4� fur4� MATa dal4�::URA3 fur4�::TRP1 ura3�360 trp1�798 6936 ura3� trp1� This study6936 dal4� fur4� fcy2 MATa dal4�::URA3 fur4�::TRP1 fcy2[C505T] Progeny of CL38 fcy2 � 6936 dal4� fur4� This study6936 dal4�::DAL4 MATa dal4�::DAL4 ura3�360::URA3 6936 dal4� This study6936 fur4�::FUR4 MATa fur4�::FUR4 trp1�798::TRP1 6936 fur4� This study

Other Candida speciesC. albicans SC5314 Clinical isolate, ATCC 37C. tropicalis IP1275 Institut Pasteur, Paris, FranceC. parapsilosis 22019 ATCCC. guilliermondii 6260 ATCCC. glabrata 90030 ATCCC. krusei 6258 ATCC

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(0.67% yeast nitrogen base without amino acids [Difco Laboratories] and2% glucose) supplemented with 1 M sorbitol and, when needed, 25 �g/mluracil, 0.5 mg/ml 5-fluorootic acid (5FOA), or 0.5 mg/ml 5-fluoroanthra-nilate. Other media used in this study were thiamine-free YNB medium(US Biological) for phenotypic characterization of the mutants, YNB me-dium without amino acids and without ammonium sulfate (0.17%; DifcoLaboratories), YCB medium (1.17% yeast carbon base; Difco Laborato-ries) for mating and genetic crosses, and RPMI 1640 (Sigma) for antifun-gal testing. Solid media were obtained with 2% agar (Sigma).

Genetic crosses and ascospore isolation. The dal4�::URA3 fur4�::TRP1 fcy2 triple mutant strain was obtained in the progeny of a geneticcross involving the 6936 MATa dal4�::URA3 fur4�::TRP1 double mutantstrain and the CL38 MAT� fcy2 sexually compatible clinical isolate (8).Genetic crosses were performed by mixing cells of opposite mating typeson solid YCB as described previously (18). Ascospores were isolated afterspheroplast formation and osmotic lysis of the parental yeast cells andwere germinated on solid RPMI medium supplemented with 4 �g/ml5-fluorouracil and 8 �g/ml 5-fluorocytosine at 35°C for 3 to 4 days. Thegenotype of the selected progeny then was verified using PCR assay andSouthern blot analysis. Nucleotide sequencing was used to verify the pres-ence of the C505T nonsense mutation in the fcy2 mutant allele inheritedfrom the clinical isolate CL38 (14).

Antifungal agents and susceptibility tests. Stock solutions of 2 mg/mlfluconazole (ICN Biomedicals Inc.), 2 mg/ml flucytosine (Sigma), 2mg/ml 5-fluorouracil (Sigma), and 2 mg/ml 5-fluorouridine (5FUI) wereprepared in sterile distilled water. A solution of 5-fluoroorotic acid wasprepared at 100 mg/ml in dimethylsulfoxide (DMSO). Microdilution as-says were performed according to CLSI standards (19) in RPMI 1640, pH7.0, buffered with 0.165 M morpholinepropanesulfonic acid (MOPS).Yeast suspensions were diluted with RPMI 1640 medium at a final cellcount of 103 cells/ml. The 96-well plates were incubated for 48 h at 35°C,and growth was measured with an automated microtiter reader at 450 nm.All experiments were done at least in triplicate, and growth variations didnot exceed 10%. Cross-resistance between fluoropyrimidines and azoleantifungals were determined using Etest (bioMérieux, France) with con-centration ranges of 0.002 to 32 �g/ml for itraconazole (ITC), voricona-zole (VRC), posaconazole (POS), and ketoconazole (KTC) and 0.016 to256 �g/ml for fluconazole. Etest strips were deposited onto solid RPMImedium left unsupplemented or supplemented with different concentra-tions of flucytosine or 5-fluorouracil (e.g., 8, 16, or 32 �g/ml) and inocu-lated with the yeast strains at the cellular density recommended by thesupplier. The checkerboard method used for drug combination studieswas also based on microdilution CLSI standards (19) using RPMI me-dium. Fluconazole concentrations ranging from 0.06 to 64 �g/ml werecombined with flucytosine, 5-fluorouracil, or 5-fluorouridine concentra-tions ranging from 0.0075 to 0.5 �g/ml. The fractional inhibitory concen-trations (FICs) of the drugs used in combination were calculated to obtainthe FIC indices (�FIC) as described previously (20) and are interpreted asstated in Instructions to Authors of Antimicrobial Agents and Chemotherapy(synergy for �FIC, �0.5; indifferent for �FIC, �0.5 and �4; antagonismfor �FIC, �4) (21).

DNA extraction, PCR amplifications, and nucleotide sequencing.Genomic DNA was extracted by a glass bead method for yeast cell disrup-tion as previously described (17). The high-fidelity DNA polymerase PfuTurbo (Stratagene) was used to amplify DNA fragments by PCR for clon-ing steps and for overlapping PCR. Routine PCRs were performed withHot-Start Taq DNA polymerase (Qiagen) as recommended by the sup-plier. All primers used in this study (see Table S1 in the supplementalmaterial) were synthesized by Eurofins MWG Operon (Europe). Whenrequired, PCR products were sequenced using the BigDye Terminatorv1.1 kit (Applied Biosystems) at the Genotyping–Sequencing Pole of theFunctional Genomic Platform of Bordeaux.

Yeast transformations. Auxotrophic strains were transformed by anelectroporation procedure previously described (22), slightly modified byadding 1 M sorbitol to the lithium acetate buffer (17), using 1 to 2 �g of

transforming DNA per experiment. Prototrophic transformants were se-lected on YNB selective medium after 3 days of incubation at 35°C.

Identification of the C. lusitaniae FUR4 and DAL4 genes. The openreading frames (ORFs) of the C. lusitaniae FUR4 and DAL4 genes wereretrieved from the C. lusitaniae genome database of the Broad Institute(http://www.broad.mit.edu/annotation/fungi/candida_lusitaniae/index.html) and were identified as CLUG_05588.1 on supercontig 1.7 andCLUG_04321 on supercontig 1.5, respectively. Similarity searches in thedatabase were performed with the basic local alignment search tool(BLAST) algorithm (23) using the amino acid sequence encoded by thegenes FUR4 and DAL4 of Saccharomyces cerevisiae available at the Saccha-romyces Genome Database (http://www.yeastgenome.org/).

Genetic constructions and gene deletions. The genes DAL4 andFUR4 were amplified from the C. lusitaniae wild-type strain 6936 with theprimer pairs F1.5/R1.5 and F1.7/R1.7 (see Table S1 in the supplementalmaterial), respectively, and were cloned into the vector pGEM-T (Pro-mega) to give the plasmids pGDAL4 and pGFUR4. Because of the occur-rence of synthetic lethality between the ura3� and fur4� mutations, theFUR4 gene was deleted with the TRP1 marker and DAL4 was deleted withthe URA3 marker. A deletion cassette was obtained for DAL4 by replacingan 800-bp Mfe1/Ale1 restriction fragment located within the ORF ofDAL4 cloned in pGDAL4 with the 2.1-kbp GUN fragment made of theURA3 gene of C. lusitaniae flanked by two 300-bp noncoding repeatedsequences, called NPT, derived from the bacterial neomycin phospho-transferase NPT1 gene, as described previously (15). Likewise, a deletioncassette for FUR4 was obtained by replacing a 997-bp Mfe1/NheI restric-tion fragment located within the ORF of FUR4 cloned in pGFUR4 with the2.0-kbp GTN fragment made of the TRP1 gene of C. lusitaniae flanked bythe two repeated NPT sequences. The deletion cassettes then were ampli-fied from the plasmids and used in yeast cell transformation experimentsto obtain, by homologous recombination, the simple mutant strains 6936dal4�::URA3 and 6936 fur4�::TRP1 and the 6936 dal4�::URA3, fur4�::TRP1 double mutant strain. Reintegrant strains were obtained directly bytransformation of the dal4�::URA3 and fur4�::TRP1 strains with a linearDNA fragment carrying the wild allele DAL4 or FUR4, respectively. Thedal4�::DAL4 and fur4�::FUR4 reintegrant strains that had lost the geneticmarkers URA3 and TRP1 were selected onto YNB minimal medium sup-plemented with either 500 �g/ml of 5-fluorooroctic acid plus 25 �g/mluracil or 500 �g/ml of 5-fluoroanthranilic acid plus 25 �g/ml tryptophan,respectively. Subsequently, the wild alleles URA3 and TRP1 were reintro-duced to their own locus in the reintegrant strains. The genotype of allstrains was verified by PCR and Southern blot hybridizations (see Table S1and Fig. S1 in supplemental material).

Southern blotting. Approximately 10 �g of C. lusitaniae DNA wasdigested with the appropriate restriction enzyme, separated by electro-phoresis in a 1% agarose gel, and transferred onto nylon membranes(Hybond N�; Roche Molecular Biochemicals). Hybridization was car-ried out with digoxigenin (DIG)-labeled probes synthesized with a PCRDIG probe synthesis kit (Roche Molecular Biochemicals), as recom-mended by the supplier.

Uptake measurements. Uptake measurements were performed as de-scribed previously (8, 24) by using [2-14C]uracil (3.81 GBq/mmol) fromMoravek Biochemicals (Brea, CA, USA). Yeast cells were grown in YPDmedium at 35°C under agitation, harvested by centrifugation at mid-logphase (5 � 107 to 7 � 107 cells/ml), and resuspended at a density of 4 �106 to 5 � 106 cells/ml in 50 mM sodium citrate (pH 5.0) containing 2%glucose at 30°C. The reaction was started by adding [2-14C]uracil at con-centrations ranging from 5 to 150 �M. After 4, 8, and 12 s of incubation,aliquots (0.3 ml) were filtered through 0.8-�m-pore-size cellulose acetatefilters (Sartorius, Germany), washed twice with 3 ml of cold water, dried,and assayed for radioactivity. As already reported (24), we verified that theinitial rate of uptake was constant over the first 10 s of base accumulation.The maximal rate of uptake (Vmax) and the apparent Michaelis constant oftransport (Ktapp) were calculated by nonlinear regression analysis of the

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initial rates of uptake versus the solute concentrations using the Michae-lis-Menten equation.

Nucleotide sequence accession numbers. The FUR4 and DAL4 se-quences of the C. lusitaniae strain 6936 have been deposited in theGenBank database under accession numbers KC832301 and KC832300,respectively.

RESULTSIn silico identification of the FUR4 and DAL4 nucleobase trans-porters of the NCS1 family in the genome of C. lusitaniae. InSaccharomyces cerevisiae, the nucleobase cation symporter 1(NCS1) family is constituted by several uptake transporters of thesalvage pathway for purines and pyrimidines and related mole-cules. The purine-cytosine permease gene FCY2 has been charac-terized already in C. lusitaniae (8, 13), and its paralog, FCY21, wasshown to be unexpressed (13). Our aim was to characterize othergenes of the NCS1 family, such as the uracil permease Fur4p (16),and to define its possible role in azole resistance. In S. cerevisiae,Fur4p has similarities with five other transporters (85% and 70%similarity with allantoin permease Dal4p and uridine permeaseFui1p, respectively, and 50% similarity with the nicotinamidetransporter Nrt1p and the thiamine transporters Thi7p andThi72p). A tblastn analysis of the C. lusitaniae genome database(http://www.broadinstitute.org), using S. cerevisiae Fur4p as thequery, allowed us to identify only two very similar paralogs in C.lusitaniae, CLUG_05588.1 and CLUG_04321.1, with the samepercentages of amino acid identity and similarity to Fur4p (48%and 68%, respectively). The two ORFs are highly similar, exhibit-ing 49% identity and 80% similarity. Bioinformatics comparisonto each member of the NCS1 transporters of S. cerevisiae did notallow us to predict a specific function for either one of the trans-porters. This could be achieved only from the phenotypic charac-terization of the null mutants, whose results for this study areprovided below. For further clarity in this report, we use the fol-lowing correct names for the ORFs: CLUG_05588.1 correspondsto FUR4, an intronless gene of 1,746 nucleotides (nt), encoding apredicted protein of 581 amino acids, and CLUG_04321.1 corre-sponds to DAL4, an intronless gene of 1,809 nt, encoding a pre-dicted protein of 602 amino acids.

Characterization of NCS1 transporter-defective mutantstrains of C. lusitaniae. In S. cerevisiae, the Fur4p protein familymainly includes transporters of uracil, uridine, allantoin, and thi-

amine. C. lusitaniae is auxotrophic for thiamine and exhibitsgrowth failure for thiamine concentrations as low as 1 � 10�3

�g/ml. The growth ability of the different fur4 and dal4 mutantstrains of C. lusitaniae constructed in this study was tested in thi-amine-free YNB, in both liquid and solid medium, supplementedwith thiamine concentrations ranging from 2 � 10�5 to 0.012�g/ml. Growth of the mutant strains was no more impaired in thepresence of limiting concentrations of thiamine than that of thewild strain, indicating that Fur4p and Dal4p were not involved inthiamine uptake (Fig. 2). Similar experiments were performedwith allantoin, a nitrogen-rich product of purine catabolism, in aconcentration range from 1 � 10�3 mg/ml to 10 mg/ml. All of thestrains which bore a dal4� deletion were unable to grow normallyon YNB supplemented with 0.1 mg/ml allantoin (Fig. 2), indicat-ing that Dal4p was involved in allantoin uptake, as confirmed bythe wild-type phenotype restored in the dal4�::DAL4 reintegrantstrain.

The ability of Fur4p and Dal4p to transport uracil and/or uri-dine then was investigated. First, we observed that growth of theura3� auxotrophic strain on YNB medium could be restored with25 �g/ml of uracil only and not with 25 �g/ml of uridine. We thenmeasured the susceptibility of the different strains of C. lusitaniaeto 5-fluorouracil, which is known to be transported by uracil per-mease in S. cerevisiae (25). The previously characterized 5-fluo-rouracil-resistant fur1� strain was used as a positive control (15).The results showed that the wild-type strain, the dal4� mutant,and the fur4�::FUR4 reintegrant strain all were susceptible to5-fluorouracil, even at the lowest concentration used (Fig. 3). Allof the mutant strains harboring the fur4� mutation were resistantto 5-fluorouracil, which suggested that Fur4p was responsible for5-fluorouracil uptake and that it very likely was involved in uraciluptake in C. lusitaniae.

Uptake measurement of [14C]uracil in C. lusitaniae. Uracilpermease activity was assayed by the use of [14C]uracil uptaketransport measurements in the wild-type and different mutantstrains, and the kinetic parameters were determined (Table 2). Allof the strains susceptible to 5-fluorouracil (MIC, �0.25 �g/ml)showed detectable transport activity, with Vmax and Ktapp valuesfor uracil not being statistically different, even for the strains de-fective for purine-cytosine permease (CL38 fcy2 strain) or defec-

FIG 2 Drop tests showing the growth of wild-type and mutant strains of C.lusitaniae on thiamine-free YNB supplemented with thiamine and YNB sup-plemented with allantoin as the sole source of nitrogen.

FIG 3 Growth of wild-type and mutant strains of C. lusitaniae in RPMI sup-plemented with increasing concentrations of 5FU. Growth is expressed as apercentage of growth observed in RPMI without 5-fluorouracil. The wild-typestrain 6936, the dal4� strain, and the fur4�::FUR4 reintegrant strain all aresusceptible to 5-fluorouracil.

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tive for uracil phosphoribosyl transferase (6936 fur1� strain).These assays confirmed that only Fur4p, and not Dal4p, was in-volved in uracil uptake, as also shown by the restoration of both5-fluorouracil susceptibility and uracil transport in the reinte-grant strain 6936 fur4�::FUR4.

Cross-resistance to azole antifungals. In previous studies (8,13, 15), we reported that mutant strains of C. lusitaniae, whichwere affected in different genes of the pyrimidine salvage path-way, had specific resistance patterns with regard to the fluoro-pyrimidines flucytosine and 5-fluorouracil and to the triazoleantifungal fluconazole. For testing the ability of the fur4� mu-tant to support cross-resistance to 5-fluorouracil and flucona-zole, we developed a new assay based on the use of fluconazoleEtest strips and RPMI medium left unsupplemented or supple-mented with increasing concentrations of 5-fluorouracil (Fig.4). Using unsupplemented RPMI, the fluconazole MIC of thefur4� strain was 0.25 �g/ml. Using RPMI supplemented with 8,16, and 32 �g/ml 5-fluorouracil, the fluconazole MIC increasedin a dose-dependent manner to reach 12, 64, and 256 �g/ml,respectively. As a control strain, we used the UPRTase-defi-cient 6936 fur1� mutant strain, which was also 5-fluorouracilresistant (MIC, �256 �g/ml) but remained susceptible to flu-conazole, whatever the 5-fluorouracil concentration used. Wetook advantage of the simplicity of the test to demonstrate theoccurrence of the cross-resistance phenotype toward all otherazole antifungals, i.e., the imidazole ketoconazole as well as thetriazoles itraconazole, voriconazole, and posaconazole (Table3), for the fur4� strain and the fcy2 clinical and laboratorymutant strains. The increase of MIC was much more markedfor fluconazole, to which the mutant strains became fully resis-tant (with MICs ranging from 64 to 256 �g/ml).

Pyrimidine transporters are not involved in fluconazole up-take. In our previous works, we suggested that cross-resistancebetween flucytosine and fluconazole was due to the competitiveinhibition of fluconazole uptake by extracellular flucytosine (8,13, 15). The data obtained with 5-fluorouracil and the fur4� mu-tant in the present work are consistent with this hypothesis, show-ing, notably, that the MIC of fluconazole increases as the 5-fluo-

rouracil concentration increases. To determine whether thepyrimidine transporters could mediate fluconazole import, triplemutants were selected in the progeny of a genetic cross involvingan fcy2 parental strain having a nonfunctional purine-cytosinepermease and the fur4� dal4� double mutant constructed in thisstudy. None of the triple mutants exhibited an increase of MIC forthe different azole antifungals compared to the parental strains,indicating that these transporters were not involved in azole up-take.

Attempts to induce fluconazole resistance with other chem-ical compounds. In another set of experiments, RPMI mediumcontaining 8, 16, or 32 �g/ml fluconazole was supplemented withdifferent concentrations of the pyrimidine nucleobase cytosine oruracil, with the amino acid histidine, which contains an imidazolering, or with thiamine and thiamine pyrophosphate, both harbor-ing an aminopyrimidine and a thiazole ring. None of these mole-cules was able to promote resistance to fluconazole like fluoropy-rimidines did in the fur4� strain (data not shown). This resultpointed out the particular importance of the fluorine atom inazole resistance. Nevertheless, we failed to trigger azole resistanceby the use of NaF (concentration range, 0.7 to 200 mM; the MIC ofNaF measured in this work for the fur4� and wild-type 6936strains was 400 mM for both), 2-amino-5-fluorobenzoic acid(concentration range, 0.2 to 50 mM; the MIC of 5-fluorobenzoatemeasured in this work was 100 mM for fur4� and wild-type 6936strains), and 5-fluorodeoxyuridine (concentration range, 0.5 to256 �g/ml; the MIC measured in this work was �256 �g/ml forthe fur4� and wild-type 6936 strains).

TABLE 2 MICs of 5FU and kinetic parameters of [14C]uracil uptake forthe C. lusitaniae strainsa

Strain5FU MIC(�g/ml)

Uracil uptake parameterb

Ktapp (�M)Vmax (nmol/min/107 cells)

6936 wild type �0.25 23.04 5.85 0.34 0.03CL38 fcy2 �0.25 30.75 16.5 0.17 0.056936 fur1� �256 33.30 4.7 0.16 0.026936 dal4� �0.25 22.63 15.35 0.27 0.056936 fur4� 128 NM NM6936 dal4� fur4� 128 NM NM6936 fur4�::FUR4 �0.25 49.01 10.9 0.23 0.07a Results shown are MICs of 5FU and kinetic parameters of [14C]uracil uptake for theC. lusitaniae wild-type strain, the fur1� mutant, and mutants defective for nucleobasetransporters in RPMI medium.b Uracil uptake was measured over a concentration range from 5 to 150 �M. Iftransport could not be detected by using the highest concentration of uracil, it wasnoted as nonmeasurable (NM). Ktapp and Vmax were calculated by nonlinear regressionof the saturation curves obtained with at least five different solute concentrations, andall experiments were done in triplicate. The kinetic parameters of uracil uptake for thewild-type strain 6936 in YPD were a Ktapp of 21.64 4.81 �M and a Vmax of 1.92 0.18 nmol/min/107 cells.

FIG 4 Etest determination of the fluconazole MIC of the C. lusitaniae fur4�and fur1� mutant strains on RPMI medium supplemented with increasingconcentrations of 5FU or left unsupplemented.

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Subinhibitory doses of fluorinated pyrimidines mediate re-sistance to fluconazole in a susceptible wild-type strain. We pre-viously reported that a fur1 mutant strain, which had lost theability to convert 5-fluorouracil to phosphoribosylated deriva-tives, was resistant to both flucytosine and 5-fluorouracil but notcross-resistant to fluconazole (15), indicating that the fluorinatednucleobase 5-fluorouracil, even when it accumulated into the fun-gal cell, was unable to promote fluconazole resistance by itself.Only mutants of the pyrimidine salvage pathway, which keptfunctional uracil phosphoribosyl transferase activity, such as fcy1,fcy2, and fur4 mutant strains, exhibited cross-resistance to azole inthe presence of certain concentrations of fluoropyrimidines. Wehypothesized that small amounts of 5-fluorouracil, present in thecytoplasm of the mutant fungal cells, could be converted by Fur1pinto fluorinated nucleotides that then would be responsible for thetriggering of azole resistance.

To test this hypothesis, we studied the effect of the supplemen-tation of RPMI medium with subinhibitory doses of 5-fluoroura-cil or flucytosine (from 0.19 � 10�4 �g/ml to 1 �g/ml) on thegrowth of the fluconazole-susceptible wild-type strain 6936 (flu-conazole MIC, 1 �g/ml) in the presence of either 8 �g/ml or 16�g/ml fluconazole. Under these conditions, the growth of wild-type yeast cells was inhibited by fluconazole, except in the pres-ence of 5-fluorouracil concentrations ranging from 0.06 to 0.25�g/ml (i.e., from 0.48 to 1.92 �M) or flucytosine from 0.015 to0.06 �g/ml (i.e., from 0.12 to 0.48 �M) (Fig. 5). The difference ofactive concentrations between flucytosine and 5-fluorouracil fortriggering azole resistance can be explained by the difference ofaffinity of the respective flucytosine and 5-fluorouracil permeases,with the purine-cytosine permease having about 4 times moreaffinity for cytosine (6.2 �M) (8) than uracil permease for uracil(21.6 �M) in the 6936 wild-type cells of C. lusitaniae.

The de novo pathway of pyrimidine synthesis can be used togenerate fluorinated pyrimidine molecules and to trigger flu-conazole resistance. 5-Fluoroorotic acid is a fluorinated analogthat can enter the de novo pathway of pyrimidine biosynthesis togenerate the toxic 5-fluoro-UMP through two enzymatic activi-ties, orotate phosphoribosyl transferase (URA5) and orotidine 5=-phosphate decarboxylase (URA3) (Fig. 1). We observed that thewild-type strain of C. lusitaniae cultivated in the presence of uracil,5-fluoroorotic acid, and inhibitory concentrations of fluconazolecould exhibit, at certain concentrations of 5-fluoroorotic acid,growth corresponding to 40% of that observed in inhibitor-freeRPMI medium (Fig. 6). Growth of a ura3� mutant strain underthe same conditions was much lower, indicating that the fluori-nated orotidine phosphate, which is probably accumulated in the

TABLE 3 MICs of azole antifungals for wild-type and mutant strains of C. lusitaniaeb

Antifungal

MIC by strain and culture mediuma

6936 inRPMI-0

CL38 fcy2 6936 fcy2� 6936 fur4�

RPMI-0 RPMI � 5FC RPMI-0 RPMI � 5FC RPMI-0 RPMI � 5FU

Fluconazole 0.5 0.75 96 (�128) 0.5 64 (�128) 0.5 256 (�512)Itraconazole 0.006 0.023 1.5 (�65) 0.004 1.0 (�25) 0.008 0.38 (�48)Voriconazole 0.012 0.016 0.38 (�24) 0.012 0.38 (�32) 0.012 0.5 (�42)Posaconazole 0.008 0.012 0.19 (�16) 0.008 0.19 (�24) 0.008 0.125 (�16)Ketoconazole 0.032 0.032 0.19 (�6) 0.032 0.25 (�8) 0.032 0.19 (�6)a Solid RPMI medium (RPMI-0) was supplemented with 32 �g/ml 5FC or 5FU according to the genotype of the strain. Numbers in parentheses indicate the increase in MICobserved on RPMI supplemented with 5-fluoropyrimidine compared to the level with unsupplemented RPMI.b MICs were determined with Etest strips on RMPI solid medium supplemented with 5-fluoropyrimidines or left unsupplemented.

FIG 5 Growth of the antifungal-susceptible wild-type strain 6936 in RPMIsupplemented with inhibitory concentrations of fluconazole along withvarious low concentrations of 5-fluorouracil (A) or flucytosine (B).Growth is given as a percentage of the growth of the wild-type strain inantifungal-free RPMI. The lack of bars indicates a complete inhibition ofgrowth.

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mutant strain, was less efficient at triggering azole resistance thanfluorinated uridylic acid.

Resistance to fluconazole can be induced in other species ofCandida using flucytosine, 5-fluorouracil, and 5-fluorouridine.We then investigated whether the interaction between fluoropy-rimidines and fluconazole was specific to C. lusitaniae or if it couldbe observed in other strains of different Candida species. First, theMICs of fluoropyrimidines and fluconazole were determined for arepresentative panel of strains of different species, including thestrain ATCC 42720 of C. lusitaniae, used as a control (Table 4). Allof the strains were susceptible to flucytosine except C. krusei,which was susceptible dose dependent (SDD), indicating thatthe metabolic pathway involved in the uptake and the intracel-lular conversion of flucytosine to toxic derivatives of fluorou-racil was functional in each strain of each species. Species of theCTG clade of Candida are divided in two groups, those whichwere susceptible to 5-fluorouracil and resistant to 5-fluorouridine(C. tropicalis and C. lusitaniae) and those which were resistant to5-fluorouracil and susceptible to 5-fluorouridine (C. albicans, C.parapsilosis, and C. guilliermondii), but all were susceptible to flu-conazole. The two other species, C. glabrata and C. krusei, wereSDD for 5-fluorouracil, 5-fluorouridine, and fluconazole.

Each strain then was cultivated in liquid RPMI containing flu-conazole at a concentration corresponding to 4 times the MIC(e.g., from 8 �g/ml for C. tropicalis to 128 �g/ml for C. krusei) anda fluoropyrimidine (flucytosine, 5-fluorouracil, or 5-fluorouri-dine) at various concentrations, from 0.002 �g/ml to 1 �g/ml orfrom 0.25 �g/ml to 128 �g/ml, depending on the susceptibility orresistance of the strain to the relevant fluoropyrimidine. Resultsfor three species (C. guilliermondii, C. glabrata, and C. tropicalis)are shown in Fig. 7, and results for the others are shown in Fig. S2in the supplemental material (C. parapsilosis, C. krusei, and C.lusitaniae). With the exception of C. albicans SC5314 (data notshown), antagonism between fluoropyrimidines at certain con-centrations and fluconazole could be observed for each strain ofeach species, allowing growth in the medium with inhibiting con-centrations of fluconazole up to 70% of the growth of the controlwithout inhibitor. The results of an RPMI microdilution checker-board analysis confirmed strong antagonism (�FIC, �4) betweenfluconazole and flucytosine and between fluconazole and 5-fluo-rouracil or 5-fluorouridine for strains of C. lusitaniae and forstrains of C. guilliermondii, C. tropicalis, and C. glabrata (Table 5).

Antagonism results specifically from an elevation of the MIC offluconazole in the presence of fluoropyrimidine, whereas the MICof fluoropyrimidine is invariable and indifferent to the presence offluconazole.

DISCUSSION

This work completes the characterization of uptake transportersof the nucleobase cation symporter 1 family, which are involved inthe pyrimidine nucleobases salvage pathway, and of related mol-ecules in the opportunistic yeast C. lusitaniae. After having char-acterized the purine-cytosine permease in clinical isolates and inlaboratory mutants of C. lusitaniae (8, 13), we were interested inthe uptake transport of uracil. In S. cerevisiae, members of theNCS1 family generally possess 12 transmembrane �-helical span-ners and are thought to function in uptake by a substrate-H�

symport mechanism. More specifically, uracil uptake is mediatedby the protein Fur4p (26), a permease that has similarity to agroup of five other transporters specialized in the uptake of uri-dine, allantoin, nicotinamide, and thiamine. In contrast, theFur4p family in C. lusitaniae, as well as in the CTG clade of Can-dida species, consists of only two very similar transporters. Usinggene deletion and loss-of-function analysis, we showed that one ofthem, named Dal4p, was involved in the uptake transport of al-lantoin, a nitrogen-rich molecule generated from purine catabo-lism, that can be used as a nitrogen source by yeast cells (27). Thesecond one was called Fur4p and was shown to be the permease foruracil. [14C]uracil uptake transport was abolished in the fur4�mutant but not in the dal4� mutant, indicating that Fur4p specif-ically transported uracil. In wild-type strains of S. cerevisiae, thekinetic parameters of the transport of cytosine by Fcy2p and uracilby Fur4p are very similar, with Ktapp values around 2 �M and Vmax

values in the range of 8 to 10 nmol/min/107 cells (16, 28). In the C.lusitaniae wild-type strain, Fcy2p shows a greater affinity for cyto-sine [Ktapp, 6 �M] (8) than Fur4p for uracil [Ktapp, 20 to 24 �M],which itself has 10 times less affinity for uracil than Fur4p of S.cerevisiae (2.5 �M) (16). These differences can be interpreted as again of substrate specificity of the transporters during evolution ofS. cerevisiae following whole-genome duplication, while thenucleobase transporters of the more ancient clade of Candidawould have retained certain plasticity for different substrates. Thiscan be illustrated by comparing C. lusitaniae to C. albicans.Growth of a ura3� strain of C. lusitaniae is restored with uracil but

FIG 6 Growth of wild-type and ura3� strains of C. lusitaniae in RMPI sup-plemented with 8 �g/ml fluconazole and different concentrations of 5FOA.Growth is expressed as the percentage of growth observed in inhibitor-freeRPMI. The lack of bars indicates the complete inhibition of the growth.

TABLE 4 MICs of 5FC, 5FU, 5FUI, and FLC for strains of differentCandida species determined by the CLSI method

Strain

MICa (�g/ml)

5FC 5FU 5FUI FLC

C. albicans SC5314 �0.12 64 �0.12 �0.25b

C. tropicalis IP1275 �0.12 �0.12 �64 2C. parapsilosis 22019 0.25 �64 �0.12 2C. lusitaniae 42720 �0.12 �0.12 �64 2C. guilliermondii 6260 0.12 �64 0.25 8C. glabrata 90030 0.25 2 16 16C. krusei 6258 16 8 2 32a The value corresponding to the IC50 (concentration inhibiting at least 50% of thegrowth of the control) is given only for fluconazole and for C. albicans. Other MICs aregiven for 100% growth inhibition.b Value corresponding to IC50 (concentration inhibiting at least 50% of the growth ofthe control) is given only for fluconazole and for C. albicans. Other MICs are given for100% growth inhibition.

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not with uridine, suggesting that Fur4p is unable to transport thenucleoside form, which is consistent with the resistance of C. lus-itaniae to 5-fluorouridine. Inversely, it is known that growth of aura3 mutant strain of C. albicans can be restored with uridine butnot with uracil. In parallel, we observed that C. albicans SC5314was susceptible to 5-fluorouridine (MIC, �0.12 �g/ml) and had ahigh MIC for 5-fluorouracil (64 �g/ml), although the wild-typestrain possesses a functional uracil phosphoribosyl transferase(29), suggesting that resistance to 5-fluorouracil resulted from adefect in uptake transport (30). Like C. lusitaniae, C. albicans pos-sesses only two transporters that are very similar to Fur4p andDal4p. It can be inferred that Fur4p transports uracil and noturidine in C. lusitaniae, while it transports uridine and not uracilin C. albicans. This shows that an ortholog protein like Fur4p mayhave evolved differently with regard to its substrate specificity,even in two species belonging to the same evolutionary clade.

Interestingly, the fur4� null mutant of C. lusitaniae is resistantto 5-fluorouracil and cross-resistant to 5-fluorouracil and flu-conazole, but also to all other azole antifungals used in this study,when both the fluoropyrimidine and the azole are used simulta-

FIG 7 Growth of C. guilliermondii ATCC 6260, C. glabrata ATCC 90030, and C. tropicalis IP1275 in RPMI medium supplemented with inhibitory concentrationsof fluconazole (4� the MIC for each strain) in the presence of different concentrations of the fluoropyrimidines 5FC, 5FU, or 5FUI. Growth is expressed as apercentage of the growth observed in inhibitor-free medium. The lack of bars indicates the complete inhibition of growth.

TABLE 5 FIC index determined by checkerboard analysis of fluconazoleand fluoropyrimidine combinations in different Candida species

StrainAntifungalcombinationa

FICvariation �FIC Interpretationb

C. lusitaniae 6936 FLC � 5FC 1–128.5 28 ANTC. lusitaniae 6936 FLC � 5FU 1–64.5 15 ANTC. lusitaniae 42720 FLC � 5FC 1–64.5 14 ANTC. lusitaniae 42720 FLC � 5FU 1–64.5 12.8 ANTC. tropicalis IP1275 FLC � 5FC 1–32.5 8.6 ANTC. tropicalis IP1275 FLC � 5FU 1–32.5 10 ANTC. guilliermondii 6260 FLC � 5FC 1–16.5 5.6 ANTC. guilliermondii 6260 FLC � 5FUI 1–16.5 6.3 ANTC. glabrata 90030 FLC � 5FC 1–16.5 4.4 ANTC. glabrata 90030 FLC � 5FU 1–16.5 4.4 ANTC. krusei 6258 FLC � 5FC 1–�4 2 INDC. krusei 6258 FLC � 5FU 1–�4 1.5 INDC. parapsilosis 22019 FLC � 5FC 1–4.5 1.5 INDC. parapsilosis 22019 FLC � 5FUI 1–4.5 1.8 INDa The fluoropyrimidines selected for this analysis were 5FC and either 5FU or 5FUIaccording to the susceptibility of the Candida strain to one or the other of these twomolecules. The higher starting antifungal concentrations were 64 �g/ml for FLC and0.5 �g/ml for fluoropyrimidines.b ANT, antagonism; IND, indifferent.

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neously. This phenotype contrasts completely with that of thefur1� mutant, which is also resistant to 5-fluorouracil but has nocross-resistant phenotype to fluconazole (15). Indeed, in previousworks with C. lusitaniae, we demonstrated that mutations affect-ing the purine-cytosine permease Fcy2p and the cytosine deami-nase Fcy1p, but not uracil phosphoribosyl transferase Fur1p,conferred cross-resistance to flucytosine and fluconazole in adose-dependent manner. It was speculated that flucytosine, butnot 5-fluorouracil, was responsible for competitive inhibition ofthe uptake transport of fluconazole (8, 13–15). Although it wasshown more recently that flucytosine itself did not compete for[3H]fluconazole import (10), it cannot be completely excludedthat the nucleobase transporters in C. lusitaniae could transportboth fluoropyrimidines and azole antifungals. It has long beenknown that the nucleoside transporter of human cells can trans-locate a large variety of toxic anticancer and antiviral nucleosideanalogs and also structurally unrelated pharmacologically activemolecules (for a review, see reference 31). Closer to the field ofmicrobiology, an adenosine transporter of Trypanosoma bruceiwas shown to be implicated in the uptake transport of two struc-turally unrelated antiparasitic molecules, melarsoprol and pent-amidine, which are used to treat sleeping sickness in humans andNagana disease in animals (32), with a null mutant of this trans-porter being cross-resistant to both drugs (for a review, see refer-ence 33). Little is known about the uptake transport of azole an-tifungals in Candida yeasts, except that it is mediated by facilitateddiffusion with a very low Vmax in C. albicans through transportersthat remain to be identified (10). Unfortunately, our resultsindicate that the uptake transport of fluconazole and otherazoles probably is not mediated, or not exclusively mediated,by nucleobase transporters in C. lusitaniae, because a triple mu-tant harboring the fur4� and dal4� null mutations, associatedwith the clinical nonsense mutation fcy2[C505T], showed no re-duced susceptibility to fluconazole.

From previous and present data, we observed that only somemutations leading to the accumulation of the fluorinated nucleo-bases flucytosine and 5-fluorouracil, either at the intracellular(fcy1� mutant) or at the extracellular (fcy2� and fur4� mutants)level, were able to confer cross-resistance to fluoropyrimidinesand azole antifungals. All of these mutants possessed a functionalFUR1 gene. On the other hand, mutation of the FUR1 gene itself isthe only one that results in resistance to flucytosine and 5-fluo-rouracil but not in cross-resistance to azole antifungals. Thus, wehypothesized that some intracellular 5-fluorouracil, derived fromspontaneous deamination of flucytosine in the fcy1� mutant andfrom the entry by diffusion of fluoropyrimidines in the fcy2� andfur4� mutants, can be converted by Fur1p to fluorinated nucleo-tides, which in turn are responsible for cross-resistance to azoles.This hypothesis was verified by showing that subinhibitory dosesof fluorinated pyrimidines were able to trigger cross-resistance tofluconazole in susceptible wild-type strains of C. lusitaniae by boththe salvage and the de novo pathways of pyrimidine synthesis. Thedemonstration was enlarged to other Candida species by showingthat fluconazole resistance could be induced by flucytosine, 5-flu-orouracil, and 5-fluorouridine in a panel of strains of C. parapsi-losis, C. tropicalis, C. guilliermondii, C. krusei, and C. glabrata butnot in C. albicans SC5314, maybe because of the strong trailinggrowth of that strain in the presence of fluconazole alone. Theexact mechanism by which the fluorinated nucleotides do triggercross-resistance is not known. A competition for transport at the

intracellular level between fluorinated nucleotides and azole anti-fungals cannot be ruled out, because Erg11p, the target of azoles, islocated at the membrane of the endoplasmic reticulum, and accessfor fluconazole could require a membrane transporter. Anotherpossibility is that fluorinated nucleotides block the entry or thefixation of azole antifungals into the catalytic pocket of Erg11p. Athird possibility relies on the activation of an efflux system. In C.glabrata, the recently reported antagonism between flucytosineand azole antifungals was explained in part as the result of aPdr1p-dependent overexpression of the Cdr1p efflux pump (34).Alternatively, it was recently described in bacteria that a fluoride-responsive riboswitch class of noncoding RNA (35) could be in-volved in fluoride export or detoxification. As riboswitches alsowere described in eukaryotes (36), and even though we failed toinduce cross-resistance to azoles in C. lusitaniae with NaF or5-fluorobenzoate, it would be interesting to investigate if a fluo-ride export mechanism does exist in Candida yeasts and if it isinvolved in the export of fluorinated azole antifungals.

ACKNOWLEDGMENTS

We thank Benoît Pinson (IBGC, Bordeaux) for helpful discussion andadvice on nucleobase, nucleoside, and nucleotide transport in yeast.

We acknowledge the Genotyping and Sequencing Facility of Bordeauxfor participating in the sequencing of the FUR4 and DAL4 genes.

This work was supported by grants from the University of Bordeauxand the Centre National de la Recherche Scientifique (CNRS).

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