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. 2004 Oct;48(10):3773-81.
doi: 10.1128/AAC.48.10.3773-3781.2004.

Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor

John-Paul Vermitsky  1 , Thomas D Edlind
Affiliations

Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor

John-Paul Vermitsky et al. Antimicrob Agents Chemother. 2004 Oct.

Abstract

Candida glabrata has emerged as a common cause of fungal infection. This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented. Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target. Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C. glabrata these include CDR1 and PDH1. By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains. In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed. Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants). Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold. Effects of antifungal treatment on gene expression in susceptible C. glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold. These findings suggest that these transporter genes are regulated by a common mechanism. In support of this, a mutation associated with laboratory resistance was identified in the C. glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae. The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation. Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

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Figures

FIG. 1.
FIG. 1.
Expression of ERG11, ABC transporters CDR1 and PDH1, and ACT1 loading control in azole-susceptible and -resistant C. glabrata clinical isolates. (A) RNA was isolated from log-phase cultures, blotted to membranes, and hybridized to the indicated gene probes as described in Materials and Methods. S, susceptible isolates; R, resistant isolates. Refer to Table 1 for complete strain numbering. (B) Histogram of ERG11, CDR1, and PDH1 gene expression in individual resistant isolates relative to average expression in a panel of susceptible isolates (R/S). Expression was quantified by densitometric scanning of RNA blots with normalization to ACT1 expression. Bars (left to right) represent the resistant isolates shown in panel A (top to bottom, left to right).
FIG. 2.
FIG. 2.
Expression of ERG11, ABC transporters CDR1 and PDH1, and ACT1 in laboratory-derived fluconazole-resistant mutants (R; F15 to F26), their parent 66032, and three additional azole-susceptible strains (S). (A) RNA was isolated from log-phase cultures, blotted to membranes, and hybridized to the indicated gene probes as described in Materials and Methods. (B) Histogram of ERG11, CDR1, and PDH1 gene expression in individual resistant isolates relative to their susceptible parent 66032 (R/S). Expression was quantified by scanning and normalized to ACT1. Bars (left to right) represent the resistant isolates shown in panel A (top to bottom, left to right).
FIG. 3.
FIG. 3.
Expression of ERG11, ABC transporters CDR1 and PDH1, and ACT1 in C. glabrata 66032 cultures treated for 0.5 or 2.5 h with itraconazole (ITR, 0.25 or 1 μg/ml), amphotericin B (AMB, 0.25 or 1 μg/ml), terbinafine (TER, 1 or 8 μg/ml), fluconazole (FLU, 64 μg/ml), or no drug (control). RNA was isolated from log-phase cultures, blotted to membranes, and hybridized to the indicated gene probes as described in Materials and Methods.
FIG. 4.
FIG. 4.
Alignment of amino acid sequences encoded by S. cerevisiae transcriptional activator genes PDR1 and PDR3 (ScPdr1 and ScPdr3) and their C. glabrata homolog (CgPdr1). Underlined CgPdr1 residues represent amino acids conserved in ScPdr1, ScPdr3, or both. Bars represent characterized domains involved in DNA binding (zinc cluster), the inhibitory domain defined by deletions which lead to constitutive activation, and the activation domain which recruits the transcriptional apparatus (25, 37). Previously reported gain-of-function mutations in ScPdr1 and ScPdr3 (11, 25, 32, 43) are indicated by amino acids above or below their respective wild-type sequence. The CgPdr1 mutation (P927 to L) identified here in laboratory-derived fluconazole-resistant mutant F15 is indicated. Alignment was generated by ClustalW (http://clustalw.genome.ad.jp). S. cerevisiae sequences were from GenBank files AAA34849 (A1036 to L as per reference 11) and CAA56198. C. glabrata Pdr1 is from the protein database for strain CBS138 (http://cbi.labri.fr/Genolevures/C_glabrata.php; CAGL-CDS0315.1) with the following changes specific to strain 66032: S76 to P, V91 to I, L98 to S, and T143 to P (GenBank accession number AY700584).
FIG. 5.
FIG. 5.
Expression of ACT1, CDR1, and PDR1 in azole-susceptible (S), clinical resistant (CR), and laboratory resistant (LR) C. glabrata strains. RNA was isolated from log-phase cultures, blotted to membranes, and hybridized to the indicated gene probes as described in Materials and Methods.
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