Gain of Function Mutations in CgPDR1 of Candida glabrata Not Only Mediate Antifungal Resistance but Also Enhance Virulence

Sélène Ferrari,Dominique Sanglard,Maurizio Sanguinetti,Bettina Rohde,Oliver Bader,Giovanni Fadda,David Calabrese,Brunella Posteraro,Christopher Bauser,Françoise Ischer

doi:10.1371/journal.ppat.1000268

Abstract

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different “hot spots,” were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Highlights

Candida glabrata has recently emerged as the second most common cause of invasive candidiasis, and there are increasing numbers of reports showing its important role in mucosal or bloodstream infections [1,2]
Azole-resistant isolates upregulated at least one of the ATP Binding Cassette (ABC)-transporter genes including CgCDR1, CgCDR2 or CgSNQ2
CgPdr1p is a Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2 and CgSNQ2, which are responsible for azole resistance in C. glabrata

Summary

Introduction

Candida glabrata has recently emerged as the second most common cause of invasive candidiasis, and there are increasing numbers of reports showing its important role in mucosal or bloodstream infections [1,2]. In C. glabrata, the constitutive upregulated expression of ABC-transporter genes CgCDR1 and, to a lesser extent, CgCDR2 ( known as PDH1) plays a dominant role in azole resistance [4,8,9,10,11]. Each of these genes can be upregulated in C. glabrata clinical isolates and disruption of CgCDR1 or CgCDR1/CgCDR2 leads to hypersusceptibility to fluconazole, cycloheximide, and chloramphenicol [4,9,10,12,13]

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Results

Conclusion