Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata

Sélène Ferrari,Dominique Sanglard,Maurizio Sanguinetti,Brunella Posteraro,Riccardo Torelli,Robert Cramer

doi:10.1371/journal.pone.0017589

Sélène Ferrari, Dominique Sanglard + Show 4 more

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https://doi.org/10.1371/journal.pone.0017589

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Abstract

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1 upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance.

Highlights

Candida glabrata is a haploid member of Ascomycetes normally not found in the environment but which has rather adapted to conditions found in mammals [1]
GOF mutations are found within several domains of the transcription factor corresponding to putative functional elements inferred from comparison to the S. cerevisiae PDR1 and PDR3 and including the transcriptional activation domain, a regulatory domain and a socalled middle homology region (MHR) which is found in several zinc finger proteins [13,16]
In order to achieve this goal, labeled cRNA from mRNA isolated in triplicates from strains containing seven different CgPDR1 GOF was applied to oligonucleotides custom arrays

Summary

Introduction

Candida glabrata is a haploid member of Ascomycetes normally not found in the environment but which has rather adapted to conditions found in mammals [1]. We and others showed that azole resistance in C. glabrata was mediated almost exclusively by enhanced drug efflux and overexpression of multidrug transporters of the ATP Binding Cassette (ABC) transporters. Several genes encoding these transporters were identified including CgCDR1, CgCDR2 (PDH1) and CgSNQ2 [8,9,10,11,12]. CgPDR1 exhibits mutations, so called gain-of-function (GOF) mutations, which are responsible for intrinsic high expression of ABC transporters and constitute the molecular basis of azole resistance in C. glabrata [13,14,15]. GOF mutations are found within several domains of the transcription factor corresponding to putative functional elements inferred from comparison to the S. cerevisiae PDR1 and PDR3 and including the transcriptional activation domain, a regulatory domain and a socalled middle homology region (MHR) which is found in several zinc finger proteins [13,16]

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