Characterization of the Candida glabrata Transcription Factor CgMar1: Role in Azole Susceptibility.

Pedro Pais,Michiyo Okamoto,Romeu Viana,Mónica Galocha,Hiroji Chibana,Geraldine Butler,Miguel C Teixeira,Raquel Califórnia,Mihaela Ola

doi:10.3390/jof8010061

Abstract

The prevalence of antifungal resistance in Candida glabrata, especially against azole drugs, results in difficult-to-treat and potentially life-threatening infections. Understanding the molecular basis of azole resistance in C. glabrata is crucial to designing more suitable therapeutic strategies. In this study, the role of the transcription factor encoded by ORF CAGL0B03421g, here denominated as CgMar1 (Multiple Azole Resistance 1), in azole susceptibility was explored. Using RNA-sequencing, CgMar1 was found to regulate 337 genes under fluconazole stress, including several related to lipid biosynthesis pathways. In this context, CgMar1 and its target CgRSB1, encoding a predicted sphingoid long-chain base efflux transporter, were found to contribute to plasma membrane sphingolipid incorporation and membrane permeability, decreasing fluconazole accumulation. CgMar1 was found to associate with the promoter of CgRSB1, which contains two instances of the CCCCTCC consensus, found to be required for CgRSB1 activation during fluconazole stress. Altogether, a regulatory pathway modulating azole susceptibility in C. glabrata is proposed, resulting from what appears to be a neofunctionalization of a Hap1-like transcription factor.

Highlights

CgMar1 Affects Azole Susceptibility in Candida glabrata In C. albicans, the transcription factors (TFs) CaTac1 and CaMrr1 are the main azole resistance regulators, while in C. glabrata, only CgPdr1 is recognized as taking this role
We searched for a possible CaMrr1 counterpart in C. glabrata by first assessing protein sequence similarity: the protein encoded by the uncharacterized C. glabrata ORF CAGL0B03421g shows the highest similarity hit for C. albicans Mrr1, with limited identity (E-value = 10−20 ; 25.55% identity)
C. glabrata CAGL0B03421g/CgHap1 and C. albicans Mrr1 share some degree of sequence similarity on the DNA binding domain and a regulatory middle homology domain (Figure S1B)

Summary

Introduction

Using RNA-sequencing, CgMar was found to regulate 337 genes under fluconazole stress, including several related to lipid biosynthesis pathways. In this context, CgMar and its target CgRSB1, encoding a predicted sphingoid long-chain base efflux transporter, were found to contribute to plasma membrane sphingolipid incorporation and membrane permeability, decreasing fluconazole accumulation. A regulatory pathway modulating azole susceptibility in C. glabrata is proposed, resulting from what appears to be a neofunctionalization of a Hap1-like transcription factor. Azoles inhibit ergosterol biosynthesis by targeting the Erg enzyme, leading to loss of plasma membrane properties (e.g., fluidity, stability, structure, asymmetry, and function) by ergosterol depletion and incorporation of the toxic sterol dimethylcholesta-8,24(28)-dien-3β,6α-diol (DMCDD) in the membrane [8,9].

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Results

Conclusion