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.

Genomewide Expression Profile Analysis of the Candida glabrata Pdr1 Regulon

“…While the manuscript was under review, Tsai et al reported the investigation by microarray analysis of seven paired azoleresistant and -susceptible C. glabrata oropharyngeal isolates from hematopoietic stem cell transplant recipients (34). All seven resistant isolates carried nonsynonymous mutations in CgPDR1 occurring in one of three domains, five of which were verified to be activating mutations directly influencing azole resistance.…”

Fluconazole impacts the extracellular matrix of fluconazole-susceptible and -resistant Candida albicans and Candida glabrata biofilms

“…Candida albicans is the main species associated with candidiasis, while Candida glabrata is one of the most predominant non- albicans species that has been linked to the development of oral infections [1–6]. It is an opportunistic pathogen in immunocompromised individuals [7,8], and it has an innate resistance to azoles [9,10]. C. glabrata differs from other pathogenic yeasts because of its phylogenetic position and haploid state with unconfirmed sexual cycle and diploid phase, contrary to C. albicans [11].…”

“…Intrinsic or primary resistance occurs when a microorganism has low susceptibility to a medication, before its exposure to the agent. Some Candida species possess intrinsic resistance to antifungal drugs, especially to FLZ [7,10]. In contrast, secondary resistance is one that can be developed by yeasts after long periods of exposure to antifungal drugs [16].…”

Prion-Forming Ability of Ure2 of Yeasts Is Not Evolutionarily Conserved

“…S. cerevisiae microarrays were purchased from Agilent. For C. glabrata, Agilent custom microarrays were used as described (Tsai et al 2010) C. albicans microarrys (Brown et al 2006) were purchased from the Genome Tsai et al (2010). Statistical calculations were performed on the processed signal data by using the mAdb analysis system provided by the BIMAS group at the Center for Information Technology, NIH.…”