Fluconazole resistance associated with drug efflux and increased transcription of a drug transporter gene, PDH1, in Candida glabrata

Abstract

Sequential Candida glabrata isolates were obtained from the mouth of a patient infected with human immunodeficiency virus type 1 who was receiving high doses of fluconazole for oropharyngeal thrush. Fluconazole-susceptible colonies were replaced by resistant colonies that exhibited both increased fluconazole efflux and increased transcripts of a gene which codes for a protein with 72.5% identity to Pdr5p, an ABC multidrug transporter in Saccharomyces cerevisiae. The deduced protein had a molecular mass of 175 kDa and was composed of two homologous halves, each with six putative transmembrane domains and highly conserved sequences of ATP-binding domains. When the earliest and most azole-susceptible isolate of C. glabrata from this patient was exposed to fluconazole, increased transcripts of the PDR5 homolog appeared, linking azole exposure to regulation of this gene.

FIG. 1

Cloned fragments of the C. glabrata PDH1 gene. Cloned genomic fragments CGlib18 and Cla1-4 are shown (the hatched areas indicate the ORFs). Five PCR subclones (H1213, H2223, H3228, H4546, and H3058) are shown, and each is aligned to the corresponding genomic sequence. Triangles at the end of PCR subclones show degenerate primers.

FIG. 2

(A) RAPD gel analysis of C. glabrata DNA. Isolates were obtained at the start of the study (isolate 35a [lanes 2, 5, and 8]), at 2 weeks (isolate 36a [lanes 3, 6, and 9]), and at 4 weeks (isolate 37a [lanes 4, 7, and 10]). Primer S (lanes 2 to 4), primer 6 (lanes 5 to 7), and primer 7 (lanes 8 to 10) were used. A molecular ladder is shown in lane 1. (B) CHEF gel using isolates from weeks 0, 2, 4, 6, 8, and 10 (isolates 35a, 36a, 37a, 38a, 40a, and 43a, respectively) are shown in lanes 2 to 7, respectively. The Saccharomyces control is in lane 1.

FIG. 3

Intracellular fluconazole concentrations. Isolates Y33.90 and Y33.91 were azole-susceptible and -resistant C. glabrata isolates from the previous report (22). Isolates 35a, 36a, 37a, 38a, 40a, and 43a were obtained at 0, 2, 4, 6, 8, and 10 weeks, respectively. Fluconazole accumulation without CCCP (white bars) and with CCCP (black bars) is shown. The standard errors are indicated by the error bars. OD₆₀₀, optical density at 600 nm.

FIG. 4

Comparison of the amino acid sequences of PDH1 and PDR5. For each pair of sequences, the top sequence is PDH1 and the bottom one is PDR5. Lines between the sequences indicate perfect matches of amino acids. Periods and colons between the sequences show similarity based on the Dayhoff table (30a) as described elsewhere (7a). There was 72.5% identity between the two sequences, with two gaps. Putative Walker A and B sites (double underlines) and putative ATP-binding sites (single underline) are indicated.

FIG. 5

Southern analysis and restriction map for PDH1 gene. EcoRV digestion of C. glabrata genomic DNA demonstrated a single 1.8-kb fragment in the Southern analysis as expected by the restriction map. PstI digestion showed a single fragment of about 10 kb. The sequenced area of the genome (white bar) and the PDH1 ORF (arrow) are shown.

FIG. 6

Northern blots of PDH1 transcripts in our series of C. glabrata strains. Actin transcripts were used as controls for gel loading. The amount of PDH1 transcript relative to actin (ACT1) was higher in the azole-resistant strains (strains 36a, 37a, 38a, and 40a) than in the susceptible strain (strain 35a).