The ATP-binding cassette transporter–encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata

ABSTRACTA crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases.Candida glabratais a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance.C. glabrataoften acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encodingCDR1locus. GOF alleles ofPDR1have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, anERGgene regulator. We show that Upc2A binds directly to thePDR1andCDR1promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.IMPORTANCEA likely contributor to the increased incidence of non-albicanscandidemias involvingCandida glabratais the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding geneCDR1. Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding geneERG11. These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of theERGpathway, Upc2A, is shown to directly bind to thePDR1andCDR1promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

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Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)31675-2 ◽

1994 ◽

Vol 269 (51) ◽

pp. 32592-32597 ◽

Cited By ~ 1

Author(s):

J A Wemmie ◽

M S Szczypka ◽

D J Thiele ◽

W S Moye-Rowley

Keyword(s):

Atp Binding ◽

Cadmium Tolerance ◽

Atp Binding Cassette Transporter ◽

Encoding Gene ◽

Atp Binding Cassette

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Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6875 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6875-6883 ◽

Cited By ~ 165

Author(s):

D J Katzmann ◽

T C Hallstrom ◽

M Voet ◽

W Wysock ◽

J Golin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Gene ◽

Sequence Similarity ◽

Yeast Cells ◽

Atp Binding ◽

Zinc Cluster ◽

Atp Binding Cassette Transporter ◽

Transporter Family ◽

Encoding Gene ◽

Atp Binding Cassette

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

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Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1174-1183.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1174-1183 ◽

Cited By ~ 169

Author(s):

Dominique Sanglard ◽

Francoise Ischer ◽

Jacques Bille

Keyword(s):

Abc Transporter ◽

High Frequency ◽

Candida Glabrata ◽

Antifungal Agents ◽

Clinical Isolates ◽

Azole Resistance ◽

Atp Binding ◽

Transporter Gene ◽

Atp Binding Cassette

ABSTRACT Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation ofCgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, calledCgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.

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