Regulation of Efflux Pump Expression and Drug Resistance by the Transcription Factors Mrr1, Upc2, and Cap1 in Candida albicans

Abstract

Constitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeast Candida albicans. The zinc cluster transcription factor Mrr1 is a central regulator of MDR1 expression, but other transcription factors have also been implicated in MDR1 regulation. To better understand how MDR1-mediated drug resistance is achieved in this fungal pathogen, we studied the interdependence of Mrr1 and two other MDR1 regulators, Upc2 and Cap1, in the control of MDR1 expression. A mutated, constitutively active Mrr1 could upregulate MDR1 and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated the MDR1 promoter, and this activation depended on the presence of a functional MRR1 gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulate MDR1 in a partially Mrr1-independent fashion. The induction of MDR1 expression by toxic chemicals occurred independently of Upc2 but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling and in vivo DNA binding studies showed that a constitutively active Mrr1 binds to and upregulates most of its direct target genes in the presence or absence of Cap1. Therefore, Mrr1 and Cap1 cooperate in the environmental induction of MDR1 expression in wild-type C. albicans, but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance.The overexpression of efflux pumps that transport endogenous metabolites as well as xenobiotics out of the cell is a common mechanism of resistance to drugs and other toxic compounds in organisms from bacteria to humans. Fungi possess two types of efflux pumps, ABC transporters and major facilitators, which use ATP or the proton gradient across the cytoplasmic membrane, respectively, to drive active transport of their substrates (9). In the pathogenic yeast Candida albicans, multidrug resistance is mediated mainly by the ABC transporters Cdr1 and Cdr2 and the major facilitator Mdr1 (22). These efflux pumps are usually expressed at low or nondetectable levels and are upregulated in the presence of certain chemicals. Constitutive overexpression of Cdr1 and Cdr2 or Mdr1 is frequently observed in C. albicans strains that have become resistant to the antifungal drug fluconazole, which inhibits ergosterol biosynthesis, especially after long-term therapy of oropharyngeal candidiasis in AIDS patients (35). The analysis of deletion mutants lacking these transporters has confirmed that their overexpression contributes to the multidrug-resistant phenotype of such strains (34, 36).The transcription factors controlling the expression of multidrug efflux pumps in C. albicans have recently been identified. The zinc cluster transcription factor Tac1 mediates the upregulation of the CDR1 and CDR2 genes in response to inducing chemicals, and the constitutive overexpression of these efflux pumps in drug-resistant C. albic...

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans

“…In particular, the ZCF family appears well suited to give rise to adaptive mutation due to recent expansion of this family in the pathogenic Candida species. The role of ZCF15 and ZCF29 in oxidative stress appears newly acquired, based on the lack of conservation outside pathogenic fungi, and lack of similarity to conserved transcription factors known to regulate oxidative stress, such as CAP1 (Mogavero et al 2011;Schubert et al 2011;Jain et al 2013). Further study of additional related Zcf proteins may help to uncover how these novel regulatory patterns evolved.…”

Ascorbic Acid Inhibition of Candida albicans Hsp90-Mediated Morphogenesis Occurs via the Transcriptional Regulator Upc2

“…The data are means Ϯ standard deviations of triplicate experiments. One of the TFs that inhibited GdA-induced morphogenesis was Upc2, a Zn 2 Cys 6 transcription factor, a key regulator of drug efflux pump expression and ergosterol biosynthesis (15,35). Here, we show that Upc2 is required for ascorbic acid inhibition of cell elongation upon compromised Hsp90 function.…”

“…To determine the mechanism by which ascorbic acid and Upc2 affect elongated cell growth, we tested the roles of genes whose expression is regulated by Upc2, such as the drug efflux pump genes CDR1 and MDR1 (17,35). First, we hypothesized that ascorbic acid could increase the efflux of GdA, which was reported to be a substrate of the human ATPbinding cassette (ABC) transporter efflux pump, Mdr1 (36,37).…”

Mechanisms of Antifungal Drug Resistance

“…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).…”