Isogenic Strain Construction and Gene Targeting in Candida dubliniensis

Abstract

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA R -flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by 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. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extractpeptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.Several yeast species within the genus Candida are opportunistic pathogens of humans, Candida albicans being by far the most frequently isolated and also the most pathogenic species (20). Recently, a previously unrecognized Candida species, Candida dubliniensis, has been described (28). C. dubliniensis is closely related to C. albicans, and before the phylogenetic separation of the two species was recognized and reliable phenotypic and molecular markers to distinguish them became established, C. dubliniensis was often misidentified as C. albicans (27). Whereas C. albicans is a member of the normal microflora in most healthy persons, C. dubliniensis has been isolated primarily from the oral cavities of human immunodeficiency virus (HIV)-infected individuals and AIDS patients. However, recent epidemiological studies have also identified C. dubliniensis in cases of oral infection in non-HIV-infected individuals and in cases of septicemia, although at a lower incidence than C. albicans (3,13,21,23,29). These observat...

Efficient neocentromere formation is suppressed by gene conversion to maintain centromere function at native physical chromosomal loci in Candida albicans

“…We constructed a URA auxotrophic strain CdJ3 by transforming Cd36 using the MPA flipper cassette (Staib et al 2001;see Methods). Next we replaced the CENPA-binding region of Chr7 (coordinates: CdChr7 434001-440600) of C. dubliniensis with the 1.4-kb URA3 sequence in the CdJ3 strain (Supplemental Fig.…”

Role of Calcineurin in Stress Resistance, Morphogenesis, and Virulence of a Candida albicans Wild-Type Strain

“…When MPA R was used in C. albicans, sometimes a high proportion of MPA-resistant transformants apparently had substituted the MPA R marker for the genomic IMH3 gene but did not contain the mutagenesis cassette inserted into the target gene locus (16). In contrast, when the MPA R marker was used for targeted gene inactivation in the related species Candida dubliniensis, virtually all MPA-resistant transformants had specifically integrated the cassette into the target locus, because the sequence divergence between the two species prevented integration of the C. albicans-derived MPA R marker into the orthologous C. dubliniensis IMP dehydrogenase gene IMD1 (14). These observations suggested that a similar MPA R marker derived from C. dubliniensis should also improve targeted insertion into the C. albicans genome.…”

TheCandida dubliniensis CdCDR1Gene Is Not Essential for Fluconazole Resistance

“…This product was also ligated with the SacII/XhoI fragment from pSFI1 to create the plasmid pCDR⌬2. C. dubliniensis was sequentially transformed by using approximately 1 g of the linear SacI/SphI fragments from plasmids pCDR⌬1 and pCDR⌬2 by electroporation as described by Staib et al (27). MPA-resistant transformants were selected on minimal medium agar plates containing 10, 15, or 20 g of MPA per ml.…”

“…4), and this isolate was chosen in order to determine the influence of a CdCDR1 gene disruption on fluconazole susceptibility in this genetic background. For disruption of the CdCDR1 gene, a cassette (MPA R -flipper) containing the MPA resistance gene and the FLP site-specific recombinase fused to the inducible C. albicans SAP2 promoter (27), was inserted into the CdCDR1 ORF to create plasmids pCDR⌬1 and pCDR⌬2, thereby deleting the regions from nucleotide coordinates ϩ538 to ϩ4248 and ϩ1468 to ϩ3532, respectively (Fig. 5A).…”