Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants

Abstract

Candida glabrata, a yeast with intrinsically low susceptibility to azoles, frequently develops increased azole resistance during prolonged treatment. Transposon mutagenesis revealed that disruption of CgPDR1 resulted in an 8- to 16-fold increase in fluconazole susceptibility of C. glabrata. CgPDR1 is a homolog of Saccharomyces cerevisiae PDR1, which encodes a transcriptional regulator of multidrug transporters. Northern blot analyses indicated that CgPDR1 regulated both constitutive and drug-induced expression of CgCDR1, a multidrug transporter gene. In agreement with the Northern analysis, the Cgpdr1 mutant had increased rhodamine accumulation, in contrast to the decreased accumulation in the CgPDR1-overexpressing strain. Northern analyses also indicated the importance of CgPDR1 in fluconazole resistance arising during therapy. Two clinically resistant isolates had higher expression of CgPDR1 and CgCDR1 compared to their paired susceptible isolates. Integrative transformation of CgPDR1 from the two resistant isolates converted the Cgpdr1 mutant into azole-resistant strains with upregulated CgPDR1 expression. Two different amino acid substitutions, W297S in one isolate and F575L in the other, accounted for the upregulated CgPDR1 expression and the resistance. Finally, CgPDR1 was shown to be required for the azole resistance due to mitochondrial deficiency. Thus, CgPDR1 encodes a transcriptional regulator of a pleiotropic drug resistance network and contributes to the azole resistance of clinical isolates and petite mutants.

FIG. 1.

Southern hybridization analysis confirmed the disruption and targeted replacement of PDR1 in C. glabrata. (A) Restriction enzyme map. (B) Southern blot analysis. Total DNA was digested with BglII. The membrane was hybridized with the 2.9-kb Tn5<Cm URA3> (left panel) or 1.3-kb BglII CgPDR1 (right panel) probe. Lanes: 1, strain NCCLS84, C. glabrata laboratory wild-type strain; 2, strain CgB4, Cgpdr1 mutant; 3, strain Cg173C, CgB4 complemented with CgPDR1 of clinical fluconazole-resistant isolate Cg13928. An asterisk indicates the codon mutations in the clinical fluconazole-resistant isolates, W297S in Cg4672 and F575L in Cg13928.

FIG. 2.

Overexpression of CgPDR1 in the pdr1 mutant of S. cerevisiae reduced its sensitivities to fluconazole and rhodamine 6G. A disk diffusion assay was used to determine the drug sensitivities of S. cerevisiae strains. Strains: BY4741, wild type; 4381, pdr1 mutant; 4381/pH392, pdr1 mutant carrying the vector pH392; 4381/pHCgPDR1, pdr1 mutant carrying the CgPDR1 overexpression cassette ADH1_p-CgPDR1-ADH1_T. FLC, fluconazole; R6G, rhodamine 6G. Plates were photographed after 2 days of incubation at 30°C. WT, wild type.

FIG. 3.

Disruption or overexpression of CgPDR1 altered the expression of CgCDR1 and PDH1. Northern hybridization analyses were used to determine the expression of CgPDR1, CgCDR1, and PDH1. Ten micrograms of total RNA was used for Northern blot analysis. The membrane was hybridized with the following probe: 2.3-kb BglII CgPDR1 DNA (top panel), 1.1-kb NotI-BamHI CgCDR1 DNA from pCRScript-CDR1 (14) (middle panel), or 3.6-kb PDH1 DNA from pClaI (25) (bottom panel). Sizes of putative transcripts are indicated in kilobases on the right. The rRNA stained with ethidium bromide was used as the loading control. Lanes: 1, strain NCCLS84, wild type; 2, strain CgB4, Cgpdr1 mutant; 3, strain CgB4/pCgACU, Cgpdr1 mutant carrying the vector pCgACU; 4, strain CgB4/pCgPDR1, Cgpdr1 mutant overexpressing CgPDR1. WT, wild type.

FIG. 4.

Cycloheximide- and oligomycin-induced CgCDR1 expression depended on CgPDR1. Ten micrograms of total RNA was used for the Northern hybridization analysis to determine the level of drug-induced CgCDR1 expression. The membrane was hybridized with the 1.1-kb NotI-BamHI CgCDR1 DNA probe. The sizes of putative transcripts are indicated in kilobases. The rRNA stained with ethidium bromide was used as the loading control. Lanes: 1 to 4, NCCLS84 (wild type); 5 to 8, CgB4 (Cgpdr1 mutant). CHX, cycloheximide; OLI, oligomycin; R6G, rhodamine 6G. WT, wild type.

FIG. 5.

Altered drug sensitivity in C. glabrata caused by the disruption or overexpression of CgPDR1. Disk diffusion assay was used to determine the drug sensitivity of C. glabrata. Strains: NCCLS84, wild type; 84870, Cgcdr1 and pdh1 double disruptant; CgB4, Cgpdr1 mutant; CgB4u/pCgACU, Cgpdr1 carrying the vector pCgACU; CgB4u/pCgPDR1, Cgpdr1 mutant overexpressing CgPDR1. FLC, fluconazole; R6G, rhodamine 6G; CHX, cycloheximide; CHL, chloramphenicol; CRN, cerulenin. Plates were photographed after 2 days of incubation at 30°C. WT, wild type.

FIG. 6.

Clinical fluconazole-resistant isolates overexpressed CgPDR1 and CgCDR1. Two pairs of clinical isolates, Cg1660 versus Cg4672 and Cg12581 versus Cg13928, were analyzed by Northern blot hybridization along with NCCLS84. Probes used for each Northern blot are labeled on the left, and the estimated sizes of hybridized transcripts in kilobases are labeled on the right. The rRNA stained with ethidium bromide was used as the loading control. Probes: top, 2.3-kb BglII CgPDR1 DNA; bottom, 1.1-kb NotI-BamHI CgCDR1 DNA.

FIG. 7.

W297S or F595L single-amino-acid substitution of CgPdr1p led to upregulated expression of CgPDR1 and CgCDR1. The expression of CgPDR1 and CgCDR1 was analyzed by Northern blot analysis. The membrane was hybridized with the 2.9-kb HindIII CgPDR1 DNA or 1.1-kb NotI-BamHI CgCDR1 DNA probe. The rRNA stained with ethidium bromide was used as the loading control. All complementations were done with integrative transformation. (A) Lanes: 1, strain NCCLS84, wild type; 2, strain CgB4, Cgpdr1 mutant; 3 and 4, strain CgB4Ca₁₂₅₈₁ and strain CgB4Cb₁₂₅₈₁, CgB4 complemented by CgPDR1 from Cg12581; 5 and 6, strain CgB4Ca₁₃₉₂₈ and strain CgB4Cb₁₃₉₂₈, CgB4 complemented by CgPDR1 from Cg13928. (B) Lanes: 1, strain NCCLS84, wild type; 2, strain CgB4, Cgpdr1 mutant; 3 and 4, strain CgB4Ca₁₆₆₀ and strain CgB4Cb₁₆₆₀, CgB4 complemented by CgPDR1 from Cg1660; 5 and 6, strain CgB4Ca₄₆₇₂ and strain CgB4Cb₄₆₇₂, CgB4 complemented by CgPDR1 from Cg4672. WT, wild type.

FIG. 8.

CgPDR1 is required for upregulated expression of PDR genes caused by mitochondrial dysfunction. For Northern blot analysis, the membrane was hybridized with the 2.3-kb BglII CgPDR1 DNA (top) or 2.3-kb BamHI-NotI CgCDR1 (bottom) probe. The rRNA stained with ethidium bromide was used as the loading control. Lanes: 1, strain NCCLS84, wild type; 2 and 3, petite mutant strains derived from NCCLS84; 5, strain CgB4, Cgpdr1 mutant; 6 to 8, petite mutant strains derived from CgB4.