Isogenic strain construction and gene targeting in Candida dubliniensis

Citation
P. Staib et al., Isogenic strain construction and gene targeting in Candida dubliniensis, J BACT, 183(9), 2001, pp. 2859-2865
Citations number
32
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF BACTERIOLOGY
ISSN journal
0021-9193 → ACNP
Volume
183
Issue
9
Year of publication
2001
Pages
2859 - 2865
Database
ISI
SICI code
0021-9193(200105)183:9<2859:ISCAGT>2.0.ZU;2-G
Abstract
Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respec t to epidemiology, certain virulence characteristics, and the ability to de velop fluconazole resistance in vitro. A comparison of C. albicans and C. d ubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In cont rast to C. albicans, no auxotrophic C. dubliniensis strains are available f or genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate by targeted gene deletion. The tw o URA3 alleles were sequentially inactivated using the MPA(R)-flipping stra tegy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again b y site-specific? FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albican s-adapted GFP reporter gene. Uridine-prototrophic transformants were obtain ed with high frequency, and all transformants of two independent ura3-negat ive parent strains had correctly integrated the reporter gene fusion into t he CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficie nt and specific targeting of recombinant DNA into the C. dubliniensis genom e. Transformants carrying the reporter gene fusion did not exhibit detectab le fluorescence during growth in yeast extract-peptone-dextrose medium in v itro, suggesting that CdMDR1 is not significantly expressed under these con ditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-depe ndent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.