title: Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole.
abstract: We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved 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 was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Parasitologie-Mycologie, Bordeaux, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Gabriel
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Ayman
authors.initials: A
authors.lastname: Sabra
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Sofiane
authors.initials: S
authors.lastname: El-Kirat-Chatel
authors.affiliation: Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Parasitologie-Mycologie, Bordeaux, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Pujol
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Fitton-Ouhabi
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France Institut de Biochimie et Génétique Cellulaires, UMR 5095, Bordeaux, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Brèthes
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Dementhon
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Parasitologie-Mycologie, Bordeaux, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Accoceberry
authors.affiliation: University Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France thierry.noel@u-bordeaux2.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Noël
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology; D001692:Biological Transport; D002175:Candida / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D003433:Crosses, Genetic; D004336:Drug Antagonism; D025141:Drug Resistance, Fungal; D005437:Flucytosine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D008826:Microbial Sensitivity Tests; D033702:Nucleobase Transport Proteins / Q000235:genetics* / Q000378:metabolism; D033704:Nucleotide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D058886:Sterol 14-Demethylase / Q000235:genetics / Q000378:metabolism; D014498:Uracil / Q000494:pharmacology; D014529:Uridine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D033702:Nucleobase Transport Proteins; D033704:Nucleotide Transport Proteins; C001943:5-fluorouridine; D014498:Uracil; D005437:Flucytosine; D058886:Sterol 14-Demethylase; D005472:Fluorouracil; D014529:Uridine