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Antimicrobial Agents and Chemotherapy, May 2004, p. 1788-1796, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1788-1796.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata

Sophie Brun,1 Thierry Bergès,2 Pascal Poupard,3 Carole Vauzelle-Moreau,2 Gilles Renier,4 Dominique Chabasse,1 and Jean-Philippe Bouchara1*

Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie,1 Laboratoire d'Immunologie, Centre Hospitalier Universitaire, 49033 Angers Cedex,4 Laboratoire de Génétique de la Levure, CNRS UMR 6161, Faculté des Sciences, 86022 Poitiers Cedex,2 UMR Pathologie Végétale 77, Faculté des Sciences, 49045 Angers Cedex, France3

Received 25 August 2003/ Returned for modification 22 September 2003/ Accepted 20 January 2004

We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA. Here, we analyzed the mechanisms of azole resistance in petite mutants of C. glabrata obtained by exposure to fluconazole or induced by ethidium bromide. The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA. Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C. glabrata CDR1 (CgCDR1) and CgCDR2 genes, encoding efflux pumps. Conversely, the expression of CgERG11, which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants. Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates. Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content. Thus, resistance or decreased susceptibility to azoles in petite mutants of C. glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2. In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes.

* Corresponding author. Mailing address: Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, 4 rue Larrey, 49033 Angers Cedex, France. Phone: 33 02 41 35 34 72. Fax: 33 02 41 35 36 16. E-mail: jean-philippe.bouchara{at}univ-angers.fr.

Antimicrobial Agents and Chemotherapy, May 2004, p. 1788-1796, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1788-1796.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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