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Antimicrobial Agents and Chemotherapy, January 2007, p. 369-371, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00824-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Molecular Mechanism of Flucytosine Resistance in Candida lusitaniae: Contribution of the FCY2, FCY1, and FUR1 Genes to 5-Fluorouracil and Fluconazole Cross-Resistance{triangledown}

Nicolas Papon,1 Thierry Noël,2 Martine Florent,1 Stéphanie Gibot-Leclerc,1 Dorothée Jean,1 Christiane Chastin,1 Jean Villard,1 and Florence Chapeland-Leclerc1*

Laboratoire des Sciences Végétales, EA209, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris 5, 4 avenue de l'Observatoire, 75006 Paris, France,1 Laboratoire de Mycologie Moléculaire, UMR 5162 CNRS-Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France2

Received 7 July 2006/ Returned for modification 4 August 2006/ Accepted 16 October 2006


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ABSTRACT
 
Inactivation of the FCY2 (cytosine permease), FCY1 (cytosine deaminase), and FUR1 (uracil phosphoribosyltransferase) genes in Candida lusitaniae produced two patterns of resistance to flucytosine. Mutant fur1 demonstrated resistance to 5-fluorouracil, whereas mutants fcy1 and fcy2 demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.


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TEXT
 
Flucytosine (5FC) is one of the oldest antifungal agents. When this drug is taken up and converted to 5-fluorouracil (5FU) by fungal cells, it inhibits DNA replication and protein synthesis (11). However, it should be administered in combination with amphotericin B or azole antifungal agents, such as fluconazole (FLC), because the frequency of resistance development precludes its use as a single agent (10). In a previous work, we demonstrated that 5FC resistance in four Candida lusitaniae clinical isolates was due to a defect of purine-cytosine permease and that these isolates were specifically cross-resistant to FLC when both the antifungals 5FC and FLC were used in combination (8). More recently, we provided molecular evidence that inactivation of the FCY2 gene in C. lusitaniae promotes cross-resistance to the antifungal combination 5FC-FLC (3). The goal of this study was to determine the precise contribution of the two other main genes possibly involved in 5FC resistance, FCY1 and FUR1, to the 5FC-FLC cross-resistance phenotype.

Cloning and disruption of FCY1, FUR1, and FCY2 genes. BLAST analysis of the C. lusitaniae database (http://www.broad.mit.edu/annotation/fungi/candida_lusitaniae/) allowed identification of a 507-bp gene encoding a predicted protein of 153 amino acids (16.8 kDa) that bore a significant identity with other cytosine deaminases from Candida albicans (69%), Candida glabrata (65%), and Saccharomyces cerevisiae (64%). The C. lusitaniae FCY1 gene was located on supercontig 1.8 from positions 326868 to 326357 and contained a predicted intron located at nucleotides 56 to 105 from the ATG codon. In the same way, we identified one gene putatively encoding a uracil phosphoribosyltransferase (UPRTase) protein of 216 amino acid residues (24.3 kDa) that exhibited strong identity with the Fur1p proteins of C. albicans (90%), C. glabrata (75%), and S. cerevisiae (74%). The 651-bp intronless C. lusitaniae FUR1 gene was located on supercontig 1.6 from positions 699508 to 700158. The complete FCY1 and FUR1 genes with their 5' and 3' UTR were isolated by PCR amplification and cloned into pGEM-T (Promega). Cloning of the C. lusitaniae FCY2 gene (GenBank accession no. AY506668) has been described in a previous work (3).

Null mutants were constructed for the FCY2, FCY1, and FUR1 genes by using an improved integrative transformation system based upon the "URA3-blaster" strategy. For that, the central part of the coding region of each cloned gene was deleted by digestion with adequate restriction enzymes and was replaced by the GUN fragment; this fragment consisted of the C. lusitaniae URA3 gene flanked on both sides by a noncoding 327-bp repeat (fragment REP) obtained by amplification from the prokaryotic NPTI gene encoding neomycin phosphotransferase. The resulting disruption cassettes (FCY1-GUN, FCY2-GUN, FUR1-GUN) were excised from the cloning vector with restriction enzymes and were separately used to transform strain 6936 ura3[D95V] to prototrophy, as described previously (4). Correct targeting to each locus was verified by Southern analysis of the genomic DNA of Ura+ transformants (results not shown). Gene replacement resulted in genotypes ura3[D95V], fcy2{Delta}::REP-URA3-REP (abbreviated fcy2{Delta}::URA3), ura3[D95V], fcy1{Delta}::REP-URA3-REP (abbreviated fcy1{Delta}::URA3), and ura3[D95V], fur1{Delta}::REP-URA3-REP (abbreviated fur1{Delta}::URA3).

Antifungal susceptibilities of the transformants. Testing of the susceptibilities of reference strain 6936 and the null mutants to 5FC, 5FU, FLC, and the association 5FC-FLC was performed (Table 1). Strain 6936 was susceptible to all antifungals tested and to the 5FC-FLC association. Null mutants were all resistant to 5FC, with MICs varying according to the strain genotype. Mutants fcy2{Delta}::URA3 and fcy1{Delta}::URA3 displayed the lowest MIC of 5FC (64 to 128 µg/ml), whereas mutant fur1{Delta}::URA3 had the highest MIC (≥512 µg/ml). Only mutant fur1{Delta}::URA3 was strongly resistant to 5FU (MIC, ≥512 µg/ml), whereas mutants fcy2{Delta}::URA3 and fcy1{Delta}::URA3 were as susceptible as strain 6936. All the strains tested were susceptible to FLC. However, when 5FC and FLC (at 16 µg/ml, i.e., 8x MIC) were used in association, fcy2{Delta}::URA3 and fcy1{Delta}::URA3 developed cross-resistance to FLC, allowing up to 50% of the growth observed for the drug-free control (results not shown) over a range of 5FC subinhibitory concentrations that ranged from 4 to 32 µg/ml 5FC.


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  		TABLE 1. Susceptibilities to 5FC, 5FU, and FLC of C. lusitaniae wild-type strain 6936, null mutants, and the revertant strains constructed for this studya

Complementation of fcy2 and fcy1 null mutant alleles. Ura– clones resistant to 5-fluoro-orotic acid (5FOA) were selected from the fcy2{Delta}::URA3 and fcy1{Delta}::URA3 mutants; and their genetic organization, i.e., loss of the URA3 gene and of one of the flanking REP fragments, was confirmed by Southern blot analysis (data not shown). The genotypes ura3[D95V] fcy2{Delta}::REP (abbreviated fcy2{Delta}) and ura3[D95V] fcy1{Delta}::REP (abbreviated and fcy1{Delta}) were assigned to the 5FOA-resistant clones. Attempts to select a ura3[D95V] fur1{Delta}::REP mutant failed recurrently, even when UMP or uridine was used as supplementation. Complementation plasmids containing the URA3 and FCY2 genes or the URA3 and FCY1 genes were used to transform the fcy2{Delta} and fcy1{Delta} mutants to prototrophy, respectively. Southern blotting (not shown) was used to demonstrate the occurrence of the relevant genotypes ura3[D95V] fcy2{Delta}::[REP-URA3-FCY2] and ura3[D95V] fcy1{Delta}::[REP-URA3-FCY1]. The antifungal susceptibilities of these genetically engineered revertants were identical to that of susceptible reference strain 6936 (Table 1). We concluded that reintroduction of functional FCY2 and FCY1 alleles in 5FC-resistant fcy2{Delta} and fcy1{Delta} mutants, respectively, was sufficient to restore antifungal susceptibility.

Discussion and conclusion. Null mutants defective in the main enzymatic steps involved in the uptake and metabolism of 5FC were obtained in C. lusitaniae by using a "URA3-blaster" transformation system (1, 5) that we developed specifically for this Candida yeast species. This system allowed selection of fcy2{Delta}::URA3, fcy1{Delta}::URA3, and fur1{Delta}::URA3 mutants in a first round of transformation experiments. Mutants fcy2{Delta} and fcy1{Delta}, which had lost the URA3 marker, were then easily counterselected on 5FOA-containing medium and were used as recipient strains to successfully reintroduce functional FCY2 and FCY1 wild alleles. Nevertheless, a fur1{Delta} mutant could not be counterselected on 5FOA, probably because the combination of the ura3 and fur1{Delta} mutations resulted in synthetic lethality, as has already been described in S. cerevisiae (6).

Testing for the susceptibilities of the null mutants to 5FC, 5FU, FLC, and the association 5FC-FLC showed that they were all resistant to 5FC and as susceptible to FLC as reference strain 6936, from which they were derived. Mutant fur1{Delta} was cross-resistant to 5FU, demonstrating that a single block in UPRTase is sufficient for total prevention of the synthesis of toxic fluorinated compounds in the fungal cell. However, the fur1{Delta} mutant did not exhibit the 5FC-FLC cross-resistant phenotype, indicating that the 5FU which accumulated in the mutant cells did not play any role in cross-resistance to FLC. On the other hand, mutants harboring a fcy1{Delta} or a fcy2{Delta} allele were resistant to 5FC, susceptible to 5FU, and 5FC-FLC cross-resistant when both antifungals were used in combination. This study demonstrates that the 5FC-FLC cross-resistance phenotype in C. lusitaniae was promoted not only by disruption of the FCY2 gene encoding purine-cytosine permease, as described previously (3), but also by inactivation of the FCY1 gene encoding cytosine deaminase. Both mutations result in the accumulation of 5FC, indicating that the molecular events that lead to cross-resistance to FLC are mediated by the fluorinated cytosine.

It is now possible to assign the mutations responsible for 5FC resistance into two functional groups according to their cross-resistance pattern in C. lusitaniae. Those affecting the FUR1 gene can be responsible for a 5FC-5FU cross-resistance pattern and can confer a very high level of resistance (MICs, ≥512 µg/ml) to both drugs. Those affecting FCY1 or FCY2 confer a 5FC-FLC cross-resistance pattern and confer a lower level of resistance to 5FC (MICs, 64 to 128 µg/ml) either because in fcy1 mutants 5FC can behave as an imperfect substrate of cytidine deaminase (2) or can be subjected to spontaneous nonenzymatic deamination (2) or because in fcy2 mutants 5FC can enter the cell through low-affinity permeases, as recently reported in S. cerevisiae (9).

Nucleotide sequence accession numbers. The FCY1 and FUR1 sequences have been deposited in GenBank database under accession nos. DQ372926 and DQ372917, respectively.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire des Sciences Végétales, EA209, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris 5, 4 avenue de l'Observatoire, 75006 Paris, France. Phone: (33) 1 53 73 96 41. Fax: (33) 1 53 73 96 40. E-mail: florence.leclerc{at}univ-paris5.fr. Back

{triangledown} Published ahead of print on 23 October 2006. Back


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Antimicrobial Agents and Chemotherapy, January 2007, p. 369-371, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00824-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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