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

Papon, Nicolas; Noël, Thierry; Florent, Martine; Gibot‐Leclerc, Stéphanie; Jean, Dorothée; Chastin, Christiane; Villard, Jean; Chapeland-Leclerc, Florence

doi:10.1128/aac.00824-06

Cited by 70 publications

(56 citation statements)

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“…We then disrupted the SLN1, NIK1, and CHK1 genes by a transformation system based upon the "URA3 blaster" strategy that we developed for C. lusitaniae in a previous study (58). In this way, we then selected sln1, nik1, and chk1 simple mutants as well as sln1 chk1 and nik1 chk1 double mutants.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2007

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Fungal histidine kinase receptors (HKRs) sense and transduce many extracellular signals. We investigated the role of HKRs in morphogenetic transition, osmotolerance, oxidative stress response, and mating ability in the opportunistic yeast Candida lusitaniae. We isolated three genes, SLN1, NIK1, and CHK1, potentially encoding HKRs of classes VI, III, and X, respectively. These genes were disrupted by a transformation system based upon the "URA3 blaster" strategy. Functional analysis of disruptants was undertaken, except for the sln1 nik1 double mutant and the sln1 nik1 chk1 triple mutant, which are not viable in C. lusitaniae. The sln1 mutant revealed a high sensitivity to oxidative stress, whereas both the nik1 and chk1 mutants exhibited a more moderate sensitivity to peroxide. We also showed that the NIK1 gene was implicated in phenylpyrrole and dicarboximide compound susceptibility while HKRs seem not to be involved in resistance toward antifungals of clinical relevance. Concerning mating ability, all disruptants were still able to reproduce sexually in vitro in unilateral or bilateral crosses. The most important result of this study was that the sln1 mutant displayed a global defect of pseudohyphal differentiation, especially in high-osmolarity and oxidative-stress conditions. Thus, the SLN1 gene could be crucial for the C. lusitaniae yeast-to-pseudohypha morphogenetic transition. This implication is strengthened by a high level of SLN1 mRNAs revealed by semiquantitative reverse transcription-PCR when the yeast develops pseudohyphae. Our findings highlight a differential contribution of the three HKRs in osmotic and oxidant adaptation during the morphological transition in C. lusitaniae.Like bacteria, fungi and plants sense and transduce many extracellular signals through histidine kinase receptors (HKRs). In eukaryotic cells, these phosphorelay proteins often carry both a histidine kinase component and a response regulator domain. In response to an external signal, the histidine kinase component autophosphorylates a conserved histidine residue. The phosphate on this histidine is then transferred to a conserved aspartic acid in the response regulator domain. Such histidine-to-aspartate phosphotransfers initiate intracellular pathways mediated in particular by mitogen-activated protein (MAP) kinases (6, 40). In fungi, it is now accepted that HKRmediated transduction pathways are implicated in regulating diverse processes, including osmoregulation, morphogenesis, and virulence expression (62). Based on results from the fungal genome sequencing project (http//www.broad.mit.edu), a recent study provided a classification of several HKR genes identified in the Ascomycota and revealed that fungal HKRs fall into 11 classes (15). Most of these classes are encountered in each examined filamentous fungus, such as Neurospora crassa and Botryotinia fuckeliana, whereas Saccharomyces cerevisiae encodes only one HKR and the yeast species Schizosaccharomyces pombe and Candida albicans only three.The best-documented HKR-mediat...

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confidence: 99%

“…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.…”

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

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“…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-…”

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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

Florent

Noël

Ruprich-Robert

et al. 2009

Antimicrob Agents Chemother

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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 fcy1⌬ 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.Treatment of fungal infections is challenged by a limited number of available antifungal agents and by the emergence of antifungal resistance. Therefore, during the last few years, research has focused on elucidating the molecular mechanisms of antifungal resistance (4, 28). The haploid opportunistic yeast Candida lusitaniae (teleomorph, Clavispora lusitaniae) is a good model for studying antifungal resistance. Although much less common than other Candida species, this yeast is characterized by its propensity to develop resistance to antifungal agents during treatment, mainly to amphotericin B, but also to azole drugs and to flucytosine (5FC) (11,16,17,22,26).5FC is a fluorinated pyrimidine that is used in combination with other antifungals to treat human cryptococcosis and sometimes candidiasis (3, 33). 5FC is actively transported into the cell through the action of purine-cytosine permease (PCP), encoded by the FCY2 gene. Once inside the cell, 5FC is rapidly converted to 5-fluorouracil (5FU) by cytosine deaminase (encoded by the FCY1 gene) and to 5-fluorouridine monophosphate (5FUMP) by uracil phosphoribosyltransferase (UPRTase; encoded by the FUR1 gene). 5FUMP is then converted to 5-fluorouridine triphosphate, which, when incorporated into fungal RNA instead of uridylic acid, disrupts protein synthesis. 5FUMP is also converted to 5-fluorodeoxyuridine monophosphate, which is a potent i...

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The Need for New Antifungal and Antimalarial Compounds

Spížek

Demain

2014

Natural Products Analysis

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

Cited by 70 publications

References 11 publications

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

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

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 Need for New Antifungal and Antimalarial Compounds

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