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Antimicrobial Agents and Chemotherapy, July 2009, p. 2982-2990, Vol. 53, No. 7
0066-4804/09/$08.00+0     doi:10.1128/AAC.00880-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Martine Florent,1 Thierry Noël,2 Gwenaël Ruprich-Robert,1 Bruno Da Silva,1 Valérie Fitton-Ouhabi,2 Christiane Chastin,1 Nicolas Papon,1 and Florence Chapeland-Leclerc1*

EA209 Eucaryotes Pathogènes, Transports Membranaires et Chimiorésistances, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 Avenue de l'Observatoire, 75006 Paris, France,1 Microbiologie Cellulaire et Moléculaire et Pathogénicité, UMR 5234 CNRS-Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France2

Received 3 July 2008/ Returned for modification 16 August 2008/ Accepted 14 April 2009

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{Delta} 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.

* Corresponding author. Mailing address: EA209 Eucaryotes Pathogènes, Transports Membranaires et Chimiorésistances, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 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}parisdescartes.fr

{triangledown} Published ahead of print on 4 May 2009.

Antimicrobial Agents and Chemotherapy, July 2009, p. 2982-2990, Vol. 53, No. 7
0066-4804/09/$08.00+0     doi:10.1128/AAC.00880-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • McManus, B. A., Moran, G. P., Higgins, J. A., Sullivan, D. J., Coleman, D. C. (2009). A Ser29Leu Substitution in the Cytosine Deaminase Fca1p Is Responsible for Clade-Specific Flucytosine Resistance in Candida dubliniensis. Antimicrob. Agents Chemother. 53: 4678-4685 [Abstract] [Full Text]  


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