The Transcription Factor Mrr1p Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans

Abstract

Constitutive overexpression of the MDR1 (multidrug resistance) gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to fluconazole and other toxic compounds in clinical Candida albicans strains, but the mechanism of MDR1 upregulation has not been resolved. By genome-wide gene expression analysis we have identified a zinc cluster transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately upregulated with MDR1 in drug-resistant, clinical C. albicans isolates. Inactivation of MRR1 in two such drug-resistant isolates abolished both MDR1 expression and multidrug resistance. Sequence analysis of the MRR1 alleles of two matched drug-sensitive and drug-resistant C. albicans isolate pairs showed that the resistant isolates had become homozygous for MRR1 alleles that contained single nucleotide substitutions, resulting in a P683S exchange in one isolate and a G997V substitution in the other isolate. Introduction of these mutated alleles into a drug-susceptible C. albicans strain resulted in constitutive MDR1 overexpression and multidrug resistance. By comparing the transcriptional profiles of drug-resistant C. albicans isolates and mrr1Δ mutants derived from them and of C. albicans strains carrying wild-type and mutated MRR1 alleles, we defined the target genes that are controlled by Mrr1p. Many of the Mrr1p target genes encode oxidoreductases, whose upregulation in fluconazole-resistant isolates may help to prevent cell damage resulting from the generation of toxic molecules in the presence of fluconazole and thereby contribute to drug resistance. The identification of MRR1 as the central regulator of the MDR1 efflux pump and the elucidation of the mutations that have occurred in fluconazole-resistant, clinical C. albicans isolates and result in constitutive activity of this trancription factor provide detailed insights into the molecular basis of multidrug resistance in this important human fungal pathogen.

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

“…Tac1p was also shown to activate the transcription of CDR1 and CDR2 upon cell treatment with different compounds such as fluphenazine (FPZ) and steroids (estrogen, progesterone) (6), but the mechanisms by which these compounds trigger Tac1p activity are still unknown. Similarly, gain-of-function mutations in two other zinc cluster transcription factors, Mrr1p and Upc2p, have recently been shown to be responsible for the constitutive upregulation of Mdr1p and Erg11p, respectively, in clinical A r isolates (10,22). These data confirmed the involvement of different transcriptional pathways in the upregulation of the CDR1/CDR2, MDR1, and ERG11 genes in A r isolates.…”

Mechanisms of Antifungal Drug Resistance

“…MRR1 was discovered by genome-wide transcriptional analysis of C. albicans isolates that up-regulated MDR1. Deletion of MRR1 in clinical strains blocked MDR1 up-regulation (Morschhauser et al 2007). Fifteen different MRR1 GOF mutations have been reported (Sanglard 2011;Eddouzi et al 2013), causing constitutive up-regulation of MDR1 (Dunkel et al 2008).…”

“…Nineteen different TAC1 mutations have been confirmed as GOF, whereas others remain candidates Siikala et al 2010;Sanglard 2011). The TAC1 regulon was further elucidated by genome-wide transcriptional profiling and DNA occupancy analysis of Tac1 (Liu et al 2007), demonstrating that Tac1 binds to the DRE of CDR1 and CDR2, likely via a consensus-binding motif (CGGN 4 CGG). Recently, another CDR1 regulator was discovered by systematic overexpression of modified Zn 2 Cys 6 genes; MRR2 (orf19.6182) is required for the basal expression of CDR1; however, it does not regulate CDR2 (Schillig and Morschhauser 2013).…”

“…Fifteen different MRR1 GOF mutations have been reported (Sanglard 2011;Eddouzi et al 2013), causing constitutive up-regulation of MDR1 (Dunkel et al 2008). Genome-wide transcriptional studies using clinical strains revealed a core set of 14 genes involved in diverse functions that are up-regulated in isolates carrying MRR1 GOF mutations (Morschhauser et al 2007). Genome-wide in vivo occupancy studies of a hyperactive form of Mrr1 identified 701 binding sites and a putative binding motif, DCSGHD (Schubert et al 2011), which was also found in MDR1 regulatory regions (Hiller et al 2006;Rognon et al 2006).…”

Role of the Yap Family in the Transcriptional Response to Oxidative Stress in Yeasts

“…Recently, it was proposed that drug resistance and the response to oxidative stress are interconnected processes in C. albicans (Znaidi et al, 2009). This connection exists through the activity of several TFs like Cap1p, Tac1p, Mrr1p or Upc2p for which (i) gain-of-function mutations were observed as being related to clinical azole resistance and (ii) genome-wide studies identified several of their target genes as being involved in the general response to oxidative stress Morschhauser et al, 2007;Znaidi et al, 2009;Znaidi et al, 2008). Also, it is interesting to remind that, in S. cerevisiae, YAP1 was initially described as a gene involved in pleiotropic drug resistance (Wu et al, 1993).…”