Microarray and molecular analyses of the azole resistance mechanism in Candida glabrata oropharyngeal isolates

Abstract

DNA microarrays were used to analyze Candida glabrata oropharyngeal isolates from seven hematopoietic stem cell transplant recipients whose isolates developed azole resistance while the recipients received fluconazole prophylaxis. Transcriptional profiling of the paired isolates revealed 19 genes upregulated in the majority of resistant isolates compared to their paired susceptible isolates. All seven resistant isolates had greater than 2-fold upregulation of C. glabrata PDR1 (CgPDR1), a master transcriptional regulator of the pleiotropic drug resistance (PDR) network, and all seven resistant isolates showed upregulation of known CgPDR1 target genes. The altered transcriptome can be explained in part by the observation that all seven resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 open reading frame. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, and S391L) and one in the activation domain (G943S), while two mutations (N764I and R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive isolate and five resistant isolates into a laboratory azole-hypersusceptible strain (Cgpdr1 strain) via integrative transformation. The Cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic backgrounds, upregulation of CgPDR1 and CgPDR1 target genes varied between the five transformants, independent of the domain locations in which the mutations occurred. In summary, gain-of-function mutations in CgPDR1 contributed to the clinical azole resistance, but different mutations had various degrees of impact on the CgPDR1 target genes.

FIG. 1.

Microarray analysis of clinical sensitive/resistant paired isolates. (A) Heat map of hierarchical gene clustering. For the seven pairs of azole-susceptible and -resistant isolates, five arrays for each pair and the expression ratio of resistant isolate over sensitive isolate are shown in log₂ scale to show upregulation (in red) or downregulation (in green) of the genes in the resistant isolates. RD, regulatory domain; UD, undefined domain; AD, activation domain. Panel I, genes upregulated in the majority of seven groups; II, genes downregulated in 16R/15S. (B) Heat map of the genes with altered expression in 16R/15S compared to that of the complemented strains matched to the host wild-type strain 84. C1S, Cgpdr1 mutant complemented by CgPDR1 from Cg1S; C16R, Cgpdr1 mutant complemented by CgPDR1 from Cg16R. (C) Heat map and annotation of the 19 genes upregulated in a majority of the clinical resistant isolates (A, panel I). The C. glabrata locus tags of each ORF represented by the oligonucleotide as well as its S. cerevisiae homologs are listed on the right. ND, not determined. (D) Putative PDRE motif logo of CgPDR1. The 1-kb upstream sequences of 18 upregulated genes were used for the motif search with MEME version 4.3.0. The major motif was obtained with an E-value of 1.4 × 10⁻²⁸.

FIG. 2.

CgPDR1 mutations and fluconazole susceptibilities. (A) Distribution of CgPDR1 mutations. The domains shown were based on the homology between S. cerevisiae Pdr1p and CgPdr1p. Asterisks indicate the locations of mutations. FSTFD is fungus-specific transcription factor domain. Five mutations (boxed) were selected to represent each of the domain/region groups for complementation analysis. (B) Targeted gene replacement via double-crossover homologous recombination; the 2.9-kb HindIII DNA fragment of the CgPDR1 ORF from each of clinical isolates, Cg1S, Cg4R, Cg18R, Cg14R, Cg16R, and Cg6R, was transformed into the Cgpdr1 mutant (CgB4) for targeted gene replacement. Two transformants, a and b, were selected from each complementation for analysis. The solid bar indicates the CgPDR1 ORF. (C) Fluconazole susceptibilities. Solid boxes, clinical isolates; open boxes, laboratory complemented strains. Strains: 84, wild-type strain; Cgpdr1, Cgpdr1 mutant (CgB4); Cg1Sa and Cg1Sb, Cgpdr1 mutant complemented by CgPDR1 from clinical sensitive isolate Cg1S; Cg4Ra and Cg4Rb, Cgpdr1 mutant complemented by CgPDR1 from Cg4R; Cg18Ra and Cg18Rb, Cgpdr1 mutant complemented by CgPDR1 from Cg18R; Cg14Ra and Cg14Rb, Cgpdr1 mutant complemented by CgPDR1 from Cg14R; Cg16Ra and Cg16Rb, Cgpdr1 mutant complemented by CgPDR1 from Cg16R; Cg6Ra and Cg6Rb, Cgpdr1 mutant complemented by CgPDR1 from Cg6R. All susceptibility tests were repeated in triplicate. The standard error of the geometric mean is shown for the isolate in which susceptibilities differed.

FIG. 3.

qRT-PCR analysis of CgPDR1, CgCDR1, PDH1, CgSNQ2, and CgYOR1 expression. The expression of pleiotropic drug resistance genes was analyzed by qRT-PCR. CgACT1 was used as an internal control for normalization. The fold differences of gene expression were compared to the wild-type strain 84, for which the expression is considered 100% and is represented by 1 as the baseline for all.