The first transcription factor discovered to be a determinant of multidrug resistance in S. cerevisiae was appropriately designated Pdr1 (Saunders and Rank, 1982). Early genetic work pointed to the existence of dominant alleles of the PDR1 gene as conferring resistance to a wide range of different toxic compounds (Rank and Bech-Hansen, 1973; Rank et al., 1975, 1976). This broad range resistance phenotype in S. cerevisiae was designated pleiotropic drug resistance (Pdr) and is functionally analogous to the multidrug resistance seen in mammalian cells and other fungi. This early work has been the subject of several reviews (Balzi and Goffeau, 1995; Bauer et al., 1999; Gulshan and Moye-Rowley, 2007) and will only be outlined here.

Isolation of the cloned PDR1 gene by Balzi et al. (1987) led to rapid advances in understanding of the molecular basis of the Pdr phenotype in S. cerevisiae. The DNA sequence of PDR1 led to two important findings. First, this gene encoded a Zn₂Cys₆ cluster-containing transcription factor similar to the previously described Gal4 (Johnston, 1987). Second, sequence of a dominant, multidrug resistant allele of PDR1 demonstrated that this hypermorphic or gain-of-function (GOF) behavior of Pdr1 was caused by a single amino acid substitution mutation in the central region of the protein. Many different amino acid substitution mutations were eventually discovered that caused Pdr1 to exhibit elevated, constitutive transcriptional activation of target gene expression (Carvajal et al., 1997).

The discovery that Pdr1 was a transcriptional regulator led to work from multiple laboratories to identify genes that were more directly responsible for the observed multidrug resistance phenotype. The first major class of Pdr1 target genes contained several different loci encoding ABC transporter proteins (Balzi et al., 1994; Bissinger and Kuchler, 1994; Hirata et al., 1994a; Katzmann et al., 1995; Mahe et al., 1996). These proteins were typically found in the plasma membrane and thought to act as broad specificity, drug efflux pumps as previously seen in mammalian tumor cells (Gottesman et al., 1995). The PDR5 locus was found to possess unique central importance among the ABC transporter-encoding genes as the expression level of this gene is the highest of the drug resistance-related ABC transporters and mutants lacking this gene exhibit profound drug hypersensitivities (Leppert et al., 1990; Decottignies et al., 1994). PDR5 transcription was strongly elevated in cells containing hyperactive PDR1 dominant alleles through the binding of the transcriptional regulatory protein to three sites in the promoter region (Katzmann et al., 1996). These sites are referred to as Pleiotropic Drug Response Elements or PDREs and have been found upstream of the majority of Pdr1-regulated genes.

A factor closely related to Pdr1 was discovered during genome sequencing experiments and designated PDR3 (Delaveau et al., 1994). GOF PDR3 alleles similar to those described for PDR1 were isolated and elevated PDR5 transcription and drug resistance found to occur in these strains (Dexter et al., 1994; Nourani et al., 1997; Simonics et al., 2000). Generally speaking, the phenotypic effects of activated alleles of PDR1 and PDR3 are very similar. However, a feature unique to the PDR3 gene was the presence of two PDREs in its promoter (Delahodde et al., 1995). This autoregulatory input was found to be essential for normal function of PDR3 and is a key component for the regulated expression of this gene (Delahodde et al., 1995; Zhang and Moye-Rowley, 2001).

Along with Pdr1 and Pdr3, several other zinc cluster-containing transcription factors have been associated with the Pdr phenotype. The first of these, Yrr1, was recovered in a screen for tolerance to a cell cycle inhibitor called reveromycin A (Cui et al., 1998). Later work indicated that Yrr1 was both autoregulated and transcriptionally induced by Pdr1/Pdr3 activity (Zhang et al., 2001). Several laboratories discovered that YRR1 mutants could be recovered that exhibited high constitutive transcriptional activation as previously discussed for similar alleles in PDR1 and PDR3 (Cui et al., 1998; Zhang et al., 2001; Keeven et al., 2002; Le Crom et al., 2002). Two homologs of Yrr1 have also been studied: Yrm1 (Lucau-Danila et al., 2003) and Pdr8 (Hikkel et al., 2003).

While the first genes identified that were controlled by the Pdr regulon were ABC transporters, more recent data make it clear that many different types of proteins are encoded by Pdr1/Pdr3 target genes (Derisi et al., 2000; Devaux et al., 2001, 2002; Traven et al., 2001). These other classes of proteins include 7 transmembrane domain-containing membrane proteins (Rsb1, Rta1) (Kihara and Igarashi, 2004; Panwar and Moye-Rowley, 2006; Kolaczkowska et al., 2012), enzymes involved in sphingolipid biosynthesis (Hallstrom et al., 2001; Kolaczkowski et al., 2004), phospholipid transfer (Van Den Hazel et al., 1999), and other proteins of poorly characterized function. Given the roles for many of these other proteins in lipid homeostasis it is reasonable to propose that the control of lipids has an important effect on drug resistance. Evidence in support of this notion is accumulating in S. cerevisiae and other fungi (Hallstrom et al., 2001; Kolaczkowski et al., 2004; Prasad et al., 2005). While it is important to recognize that the Pdr response involves more than simply transporter genes, these are the best understood in terms of their role in drug resistance. We will focus on the control of transporter genes in this review.

One of the earliest transcriptional regulators found to influence Pdr was the basic region-leucine zipper (bZip)-containing factor Yap1. Yap1 was isolated in a high-copy-plasmid suppressor screen as PDR4, a gene capable of elevating resistance to cycloheximide and the branched chain amino acid biosynthesis inhibitor sulfometuron methyl (Leppert et al., 1990). Later work demonstrated that YAP1 and PDR4 were allelic (Hussain and Lenard, 1991) and that Yap1 did not appear to act via PDR5 (Dexter et al., 1994). Work from several labs demonstrated that the bZip-containing protein Cad1/Yap2 also elevated drug resistance although this effect could only be seen when this protein was overproduced (Bossier et al., 1993; Wu et al., 1993; Hirata et al., 1994b). Similarly, high dosages of the Yap1 homologs Yap5 and Yap6 also conferred resistance to several different drugs (Fernandes et al., 1997; Furuchi et al., 2001).

While there are clear links between the bZip-containing factors and drug resistance, most of the drug resistance effects shown by this group of transcription factors occur when these proteins are overproduced. Disruption mutations rarely exhibit hypersensitivity suggesting either that extensive redundancy exists between these family members or that the normal role of these factors is not to modulate drug resistance. Yap1 is the best understood of these bZip-containing factors in terms of its regulation (reviewed in Toone et al., 2001; Moye-Rowley, 2003a; Rodrigues-Pousada et al., 2004). Loss of the YAP1 gene has a profound sensitivity to oxidative stress (Schnell et al., 1992; Kuge and Jones, 1994) but little to no consequence on drug resistance (Leppert et al., 1990; Wu et al., 1993; Alarco et al., 1997). An exception to this rule is provided by drugs such as the agrochemical mancozeb that proceed via an oxidative mechanism in which physiological levels of Yap1 are required for normal resistance (Teixeira et al., 2008). Drugs that act through a mechanisms that does not invoke an oxidative stress response typically are insensitive to the presence of Yap1 expressed at normal gene dosage. Yap1 has been thoroughly documented as an important regulator of major facilitator superfamily (MFS) gene expression in the case of FLR1 (Alarco et al., 1997) and ATR1 (Coleman et al., 1997). Activation of expression of these genes is likely to explain many of the effects of this transcription factor on drug resistance although little evidence exists that Yap1-dependent transactivation can be stimulated by the presence of drugs. Extensive documentation exists for activation of Yap1 function upon oxidative stress (Kuge et al., 1997; Coleman et al., 1999; Delaunay et al., 2000, 2002). We will focus our discussion on the Zn₂Cys₆ zinc cluster-containing proteins as the paradigms for drug-induced transcriptional activation in S. cerevisiae (and likely other fungi as well).

The identification of alleles of PDR1 as dominant, GOF mutations suggested the possibility of regulatory modulation of the Pdr1 protein. The determination that Pdr1 and Pdr3 were closely related Zn₂Cys₆ zinc cluster-containing transcription factors also argued that these proteins were most likely to be indirect determinants of multidrug resistance via their regulation of direct effectors of this phenotype (such as PDR5). Sequence analysis of a large number of these hyperactive mutant alleles of PDR1 and PDR3 demonstrated that this multidrug resistant phenotype was routinely caused by single amino acid substitutions in three main regions of these factors (Dexter et al., 1994; Carvajal et al., 1997; Nourani et al., 1997; Simonics et al., 2000). A model of Pdr1 structure is shown in Figure 1. Early analyses of these GOF alleles indicated that target genes were overexpressed in the presence of the mutant forms of the transcription regulators. For example, PDR5 mRNA levels are increased by roughly 10-fold in the presence of a PDR1 GOF allele compared to levels driven by the wild-type gene (Meyers et al., 1992). This correlates well with the observed increment in drug resistance.

While the characterization of the GOF mutants supported the view that the factors regulating Pdr were under some type of regulatory control, these alleles represent chronic functional alterations in these transcription factors. Here we will summarize more recent progress in elaboration of mechanisms controlling activity of either wild-type Pdr1 or Pdr3. To date, regulatory inputs to these two closely related proteins are unique to each factor and will be considered separately.

The first example of a trans-factor influencing Pdr1 activity came from the identification of the Hsp70 protein Ssz1 (originally Pdr13) as a positive regulator of Pdr1 function when overproduced (Hallstrom et al., 1998). Later work demonstrated that this Hsp70 was primarily associated with the ribosome (Hallstrom and Moye-Rowley, 2000a) and has an important role in folding of nascent polypeptide chains (Gautschi et al., 2001). Recently, these findings have been extended with the determination that the dnaJ protein Zuo1 (partner of the dnaK Ssz1 Michimoto et al., 2000) can also stimulate Pdr1 function, possibly by direct binding to this transcription factor (Eisenman and Craig, 2004). Zuo1 contains a short C-terminal region of 13 residues that is necessary and sufficient to induce Pdr1 transcriptional activity (Ducett et al., 2013).

Both Zuo1 and Ssz1 are present at much higher levels than Pdr1 and are thought to have their primary function at the ribosome (Gautschi et al., 2001). Work from the Craig lab has argued that the Zuo1/Ssz1 protein pair can independently activate Pdr1 and are found directly associated with this factor on target promoters (Prunuske et al., 2012). As these findings suggest, the action of Zuo1/Ssz1 in terms of control of Pdr1 activity represents an extra-ribosomal function of these proteins (Eisenman and Craig, 2004). Despite significant effort, the molecular rationale linking activity of these chaperone proteins to Pdr1 remains obscure. An interesting possible tie between Pdr1 and protein folding/degradation came from the finding that the gene encoding the key proteasomal transcriptional regulator Rpn4 is a transcriptional target of Pdr1 (Devaux et al., 2001; Owsianik et al., 2002). Activation of Pdr1 function by Ssz1 can suppress defects in endoplasmic reticulum-associated degradation (Bosis et al., 2010) further strengthening the association of Pdr1 activity with protein homeostasis.

An intriguing model for Pdr1 (and Pdr3) to act as xenobiotic receptor proteins came from studies led by Anders Näär (Thakur et al., 2008). This work provided evidence that the central regions of both these transcription factors could directly bind radioactive fluconazole and that this antifungal drug triggered an increase in Pdr1/3-dependent gene expression. These data support the attractive notion that Pdr1/3 were normally held in a low activity state but could be remodeled in the presence of an appropriate ligand to a more potent transcriptional stimulator.

The simplicity and precedence of this model make it a compelling view for the regulation of Pdr1/3 in physiology. It is still difficult to rationalize why overproduction of either Pdr1 or Pdr3, in the absence of any exogenous drug, can lead to increased gene expression (see for example: Katzmann et al., 1994; Carvajal et al., 1997). If activation of these factors required the presence of a small molecule, then overproduction would not be expected to lead to increase downstream gene expression. In cases where a small molecule is known to be required for activity of a zinc cluster-containing transcription factor, overproduction of the factor leads to downstream repression of gene expression. Examples of this behavior include the heme-dependent activator Hap1 (Zhang et al., 1998) and the leucine biosynthetic activator protein Leu3 (Sze et al., 1993). The genetic behavior of Pdr1/3 seem more easily fit to a situation in which a negative regulatory system is outcompeted when these proteins are overproduced. Further experiments are required to dissect the mechanism of Pdr1/3 control via by small molecules.

Although hyperactive alleles of PDR3 were identified at the inception of the study of the Pdr network in S. cerevisiae (Dexter et al., 1994), early analyses of the contribution of Pdr3 to drug resistance suggested that this factor was a less important contributor to transcriptional control than its homolog Pdr1 (Delaveau et al., 1994; Katzmann et al., 1994). Screening a library of random transposon-induced null mutations to identify negative regulators of PDR5 expression uncovered an array of gene involved in maintaining the mitochondrial genome (Hallstrom and Moye-Rowley, 2000b). Loss of mitochondrial DNA (ρ⁰) led to a strong induction of PDR5 gene expression that was strictly Pdr3-dependent. This was the first example of a situation in yeast in which the level of PDR5 expression and associated drug resistance rivaled those seen in the presence of a hyperactive allele of either PDR1 or PDR3.

Characterization of the requirements for this mitochondrial signal to trigger Pdr3 induction demonstrated that of the many types of mutations compromising mitochondrial function, only those that also elicited loss of mitochondrial DNA were able to elevate Pdr3 function (Zhang and Moye-Rowley, 2001). The autoregulatory loop of PDR3 transcription was also essential for the ρ⁰ response. However, driving Pdr3 expression from the PDR1 promoter was still able to produce a ρ⁰-dependent induction in drug resistance (Hallstrom and Moye-Rowley, 2000b) arguing that Pdr3 was regulated post-translationally. Together, these findings suggest that a mitochondrial genome-dependent signal leads to a release of Pdr3 from some negative regulatory system. This in turn allows Pdr3 to engage with its own promoter and positively autoregulate transcription. Pdr3 regulation is diagrammed in Figure 2.

Further genetic analyses identified several other factors that are required for the normal mitochondrial control of Pdr3. A protein of unknown function called Lge1 was identified as a participant in ρ⁰-induced gene expression (Zhang et al., 2005). Lge1 is involved in ubiquitination of histone H2B (Hwang et al., 2003) but this function seems to be distinct from its role in ρ⁰ induction of PDR5 expression (Zhang et al., 2005). Mutants lacking Lge1 fail to fully induce PDR5 transcription in ρ⁰ cells. Overproduction of an enzyme involved in biosynthesis of phosphatidylethanolamine (PE) was discovered to increase Pdr3-dependent transactivation even in ρ⁺ cells (Gulshan et al., 2008). This enzyme, called Psd1, is one of two enzymes typically used to produce PE and is located in the inner membrane space of mitochondria (reviewed in Voelker, 1997). Surprisingly, overproduction of a catalytically-inactive form of Psd1 was still capable of inducing PDR5 expression even though this mutant enzyme could no longer carry out PE biosynthesis (Gulshan et al., 2008). The presence of Psd1 was required for normal ρ⁰ signaling to occur consistent with this enzyme having a role in wild-type signal transduction.

Mass spectrometric analysis of purified Pdr3 led to the identification of the Hsp70 protein Ssa1 as a negative regulator of this transcription factor (Shahi et al., 2007). Overproduction of Ssa1 caused a decrease in Pdr3-dependent gene expression and hyperactive mutants exhibited less binding to Ssa1 in vivo than wild-type Pdr3. This reduction in Hsp70 association correlated with increased transcriptional activation capability is reminiscent of the regulation of steroid hormone receptors (Pratt and Toft, 1997); however, the presence of other heat shock proteins docked to Pdr3 has not been reported. Interestingly, both Pdr1 and Pdr3 have been reported to be induced by challenge with progesterone, furthering the analogy with a regulatory mechanism resembling steroid hormone receptors (Banerjee et al., 2008).

Further biochemical experiments seeking to identify interacting proteins capable of binding to the KIX domain of the transcriptional Mediator subunit Med15/Gal11 determined that both Pdr1 and Pdr3 interacted with this protein region (Thakur et al., 2008). The Mediator complex is composed of roughly 20 different proteins that act to provide a link between sequence-specific transcriptional regulatory proteins and the RNA polymerase II machinery (see Casamassimi and Napoli, 2007 for a review). Biochemical and genetic experiments have separated the Mediator complex into two different forms referred to as either S-Mediator (or core mediator) containing three and L-Mediator containing four subdomains (Poss et al., 2013). The three subdomains of S-Mediator are called Head, Middle, and Tail with Med15 contributing a component of the Tail complex of proteins (Beve et al., 2005). L-Mediator contains an additional subcomplex consisting of the cyclin-dependent kinase Cdk8 (or Med15/Srb8) and attendant cyclin: cyclinC (Srb11) (reviewed in Conaway and Conaway, 2011). Two other large proteins designated Med12 (Srb8) and Med13 (Srb9) complete the Cdk8 complex.

Interactions between Pdr3 (and Pdr1) and Med15 are crucial for normal levels of downstream gene expression under all conditions (Shahi et al., 2010). However, activation of Pdr3 function in ρ⁰ cells still occurs in med15Δ cells. Loss of the Med12 protein from the Cdk8 complex completely blocked ρ⁰ induction of PDR5 transcription but had a negligible effect on expression in ρ⁺ cells. These findings argue that Med12 is a key target for activated transcription supported by Pdr3, at least in ρ⁰ cells. Generally speaking, the Cdk8 complex is thought to serve repressive functions in terms of transcriptional control but more recent data argue that this complex also has positive influences on gene expression as illustrated by the case of Pdr3 activation in ρ⁰ cells.

The two best characterized genes involved in multidrug resistance in C. albicans are the ABC transporter-encoding CDR1 gene (Prasad et al., 1995) and the MFS protein-encoding MDR1 locus (Fling et al., 1991). Both of these resistance determinants are localized to the plasma membrane in C. albicans (Manoharlal et al., 2008; Basso et al., 2010; Kapoor et al., 2010). CDR1 was isolated on the basis of complementation of the drug hypersensitivity of a pdr5Δ mutant in S. cerevisiae. Loss of CDR1 led to a pronounced drug sensitive phenotype in C. albicans. A closely related ABC transporter-encoding gene, designated CDR2, also contributes to drug resistance when overproduced or when CDR1 is deleted (Sanglard et al., 1997).

MDR1 has been documented to be overexpressed in a number of different clinical isolates (Morschhauser, 2002); however, its loss does not appear to have significant effects on baseline drug resistance. The plasma membrane location of these transporter proteins supports the view that these proteins act as efflux pumps for a range of different drugs (Schuetzer-Muehlbauer et al., 2003; Lettner et al., 2010). Multidrug resistance in C. albicans is associated in large part with transcriptional induction of the genes encoding these membrane proteins.

The first insight into the molecular basis of transcriptional regulation of multidrug resistance in C. albicans came from the identification of the TAC1 locus. As with PDR1 and PDR3 in S. cerevisiae, genetic observations drove the discovery of the TAC1 gene. Analyses of azole tolerant C. albicans isolates led to the discovery of loss of heterozygosity/aneuploidy around the mating type loci (MTL) gene cluster (Rustad et al., 2002). Using this observation as a starting point, Sanglard and colleagues took a candidate gene approach by disrupting several different Zn₂Cys₆ zinc cluster-encoding genes in this chromosomal region (Coste et al., 2004). Loss of one of these genes, designated TAC1 (Transcriptional Activator of Cdr genes), resulted in a strain displaying enhanced azole susceptibility and depressed expression of the CDR1 ABC transporter-encoding gene.

Along with a requirement for TAC1 to confer normal wild-type expression of CDR1 and drug resistance, evidence was obtained that two different changes in the TAC1 gene influenced its function. First, a number of different substitution and even small deletion mutations strongly enhanced the function of Tac1 (Coste et al., 2007). Secondly, chromosomal rearrangements of chromosome 5 were detected that led to loss of heterozygosity and changes in the dosages of the linked ERG11 and TAC1 genes. These structural changes in chromosome 5 were found to lead to the amplification and likely overexpression of ERG11 and TAC1 (Coste et al., 2007; Selmecki et al., 2008). These findings are consistent with a model in which Tac1 can be activated either by changes in its primary sequence or simply by overproducing the wild-type factor. Consistent with the notion that increased expression of Tac1 leads to increased function comes from data that the TAC1 gene is positively autoregulated (Liu et al., 2007; Znaidi et al., 2007).

Although we do not yet know the nature of the signals that control Tac1, the currently available data suggest that its regulation may resemble that of Pdr3 in S. cerevisiae. Both of these factors can be activated by mutations along their respective primary sequence and the genes encoding these regulators are positively autoregulated.

A second zinc cluster-containing transcription factor that is an important contributor to drug resistance was identified in cells that were already known to overproduce MDR1 (Morschhauser et al., 2007). Transcriptional profiling experiments demonstrated that fluconazole resistant isolates shared a common overproduced transcript encoding a zinc cluster-containing transcription factor. This regulatory protein was designated Mrr1 (multidrug resistance regulator). The presence of Mrr1 is required for the observed overproduction of MDR1 in fluconazole resistant strains. While the MDR1 promoter contains binding sites for other positive regulators, loss of Mrr1 is sufficient to abrogate the overproduction that occurs in drug resistant isolates. This argues that Mrr1 is the key transcriptional regulator during development of drug resistance involving MDR1 overproduction (Dunkel et al., 2008). Importantly, azole resistant clinical isolates have been described that overproduce MDR1 and are dependent upon the presence of this MFS protein for this drug resistance (White, 1997; Wirsching et al., 2000).

Genetic activation of Mrr1 shows similar behavior to other zinc cluster-containing transcription factors in which single amino acid substitutions lead to dramatic induction of Mrr1 transcriptional activity (Dunkel et al., 2008). Detailed analyses of the functional subdomains within Mrr1 indicate that the regulation of this factor is likely to be complex with separable inhibitory and activation domains (Schubert et al., 2011b). Chromatin immunoprecipitation experiments also indicated that Mrr1 is likely to control its own expression (Schubert et al., 2011a) as mentioned above for Tac1. Both these C. albicans factors appear to share common overall features with ScPdr3 that is also autoregulated and can be genetically activated by a range of different mutations (reviewed in Moye-Rowley, 2003b).

Along with their important effects on multidrug resistance and critically azole tolerance, these transcription factors have been examined for their influence on virulence. Systematic introduction of GOF forms of TAC1 and MRR1 indicated a fitness cost in a gastrointestinal colonization model caused by either mutant gene individually that was exacerbated with their combination (Sasse et al., 2012). These authors suggested an interesting potential rationale for the common observation that clinical isolates of azole resistant C. albicans contain either TAC1 or MRR1 mutations but not both. Although the double TAC1 MRR1 strains were robustly azole tolerant, the fitness cost could be so great as to exclude this genetic combination in vivo. More recent data suggests that certain combinations of azole resistance mechanisms, including simultaneous presence of mutations in TAC1 and MRR1, may be tolerated (Morio et al., 2013).

A different infection model was used to investigate similar mutations in these same transcription factor genes (tail vein injection) (Lohberger et al., 2014). Using this bloodstream infection model, no significant fitness defect was found consistent with the interpretation that, at least under certain conditions, GOF forms of these transcription factors could be well-tolerated in vivo.

The intensive investigation of drug resistance in C. albicans has led to identification of a range of different transcriptional regulatory proteins that participate at some level in drug resistance including Ndt80 and Mcm1 among others. These factors in general have not been associated with clinically-relevant drug resistance and seem to play roles as expression modifiers of target genes involved in azole resistance (Mogavero et al., 2011; Sasse et al., 2011).

Two other transcription factors have been linked to certainly azole resistance if not multidrug resistance in C. albicans. The Upc2 transcriptional regulator is a positive modulator of sterol biosynthesis and mutations in this factor lead to pronounced azole resistance (Flowers et al., 2012). The effects of Upc2 on drug resistance occur via its role as a key regulator of sterol biosynthesis making this factor unlikely to serve as a true multidrug resistance determinant. CAP1 encodes the C. albicans ScYap1 homolog but does not appear to participate in drug resistance when expressed at normal levels. (Alarco et al., 1997; Alarco and Raymond, 1999). Cap1 clearly regulates MDR1 (Rognon et al., 2006) but this may have more to do with a role for Mdr1 in the oxidative stress response than drug resistance.

Consistent with the fact that C. glabrata is phylogenetically closer to S. cerevisiae than C. albicans, the multidrug resistance pathways in S. cerevisiae and C. glabrata share more extensive similarities than with the cognate C. albicans system. The molecular players involved in drug resistance, ρ⁰-induced PDR pathway activation, and the fact that the pivotal control of multidrug resistance in C. glabrata is at the transcriptional level are all shared with S. cerevisiae. However, there are interesting differences as well. Unlike S. cerevisiae, C. glabrata ABC transporters involved in multidrug resistance is regulated by the single transcription factor CgPdr1. CgPdr1 seems to be a blend of transcription factors ScPdr1 and ScPdr3 that modulate drug resistance in S. cerevisiae. Though CgPdr1 shares greater identity with ScPdr1 in terms of protein sequence, the C. glabrata transcription factor has binding elements in its own promoter contributing to its autoregulation, just as in the case of ScPdr3. Also like ScPdr3, CgPdr1 responds to Pdr pathway activation in ρ⁰ cells that lack mitochondrial DNA, as well as when the mitochondrial enzyme phosphatidylserine decarboxylase (ScPsd1) is overproduced (Paul et al., 2011). The role played by the Mediator component Gal11 in regulation of CgPdr1 is also interestingly different in C. glabrata when compared to its cognate role in S. cerevisiae. There are 2 GAL11 paralogs (CgGAL11A and CgGAL11B) in C. glabrata, of which only CgGAL11A seems to be important for CgPdr1 induced drug resistance (Thakur et al., 2008). The requirement for CgGal11A is however seen only in ρ⁺ and not in ρ⁰ cells, unlike in S. cerevisiae (Paul et al., 2011). Moreover, CgGal11A is required for induction of drug resistance upon azole challenge in ρ⁺ cells (Figure 3). This phenomenon of xenobiotic/drug-induced efflux pump activation, while not consistently seen in S. cerevisiae, is similar to that observed in C. albicans (Liu et al., 2005).

C. glabrata respiratory deficient mutants (ρ⁰) that have been generated in vitro by either ethidium bromide or azole treatment (where ρ⁰ cells occur at high frequency), exhibit a growth defect phenotype and reduced virulence (Brun et al., 2005). However, these same cells also possess robust drug resistance that is primarily based on activation of the transcription factor CgPdr1 and upregulation of its target gene CgCdr1 (Vermitsky and Edlind, 2004; Tsai et al., 2006). Interestingly, such mutants have, albeit rarely, been recovered from patients undergoing fluconazole treatment, suggesting that this mechanism of drug resistance may be clinically relevant (Bouchara et al., 2000). Recently, it was reported that an azole resistant C. glabrata ρ⁰ isolate from the clinic (containing a wild-type CgPDR1 gene) exhibited higher virulence and in vivo fitness in a murine infection model than its azole-susceptible and respiration-competent parental strain (Ferrari et al., 2011a).

Intrinsic low susceptibility and high frequency of resistance to widely used azole antifungals have led to greater use of other drugs, particularly amphotericin B and echinocandins in treatment of candidemias involving C. glabrata (Sanguinetti et al., 2005). Echinocandins such as caspofungin and micafungin are lipopeptide inhibitors of β-1,3-glucan synthase and interfere with fungal specific cell wall synthesis. This class of antifungals is active against most Candida species, including C. glabrata (Ostrosky-Zeichner et al., 2003; Pfaller et al., 2008) and has been used as first-line therapy against C. glabrata infections (Pappas et al., 2009). Echinocandin resistance in C. glabrata is rare by comparison to azole tolerance but increasing in frequency in response to increasing clinical use (Pfaller et al., 2012). Echinocandin resistance maps to genes encoding components of the β-1,3-glucan synthase (recently discussed in Alexander et al., 2013) and as such is considered outside the typical multidrug resistance determinants in this organism.

To overcome the intrinsic and high frequency acquisition of azole resistance in C. glabrata, alternative therapies have also been employed. As mentioned above, echinocandins are effective antifungal agents against C. glabrata but resistance to these drugs is on the rise. Combined therapies have also been explored but unexpected complications have arisen in some cases (Alves et al., 2012). An oral drug that represents an alternative to azoles is 5-flurocytosine (5-FC), although there are concerns regarding resistance and toxicity associated with high doses used to negate resistance. Surprisingly, 5-FC/azole combinations have been reported to have an antagonistic effect in C. glabrata (Te Dorsthorst et al., 2002; Alves et al., 2012). This antagonistic effect seems to be mediated through the Pdr pathway as it was found that this phenomenon was abrogated in Cgpdr1Δ and Cgcdr1Δ strains (Steier et al., 2013). 5-FC exposure resulted in 6-fold induction of CgCdr1 that was CgPdr1-dependent even though 5-FC is not a CgCdr1 substrate. Interestingly, 5-FC exposure induced high frequency formation of petite (ρ⁰) mutants that upregulate CgPdr1-dependent CgCdr1 activation. Though the molecular mechanism contributing to this antagonistic effect is not clearly understood, it seems that the mitochondrial retrograde signal that activate CgPdr1 is the likely basis for this phenomenon in C. glabrata.

As mentioned previously for C. albicans, C. glabrata also contains a number of MFS-encoding genes. While function of these membrane transporter proteins remains to be characterized, a C. glabrata homolog of ScFlr1 has been investigated for its potential role as a multidrug resistance determinant (Chen et al., 2007). The C. glabrata homolog was designated CgFLR1 and as in both C. albicans and S. cerevisiae, disruption mutations lacking this gene did have drug phenotypes but were unaffected in terms of fluconazole susceptibility. CgFLR1 is regulated by the C. glabrata homolog of ScYap1 (CgAP1) but this regulation does not appear to be involved in normal azole tolerance. More recent experiments have demonstrated that additional MFS proteins contributed to multidrug resistance including CgTPO3 and CgQDR2 (Costa et al., 2013, 2014).

The limited repertoire of antifungal drugs makes developing new modalities an important and pressing need. However, a goal of crucial importance to the enterprise of antifungal therapies in particular and drug-based therapies in general is to understand mechanisms of drug resistance. Multidrug resistance is an especially serious issue as a small number (often one) of genetic changes can lead to loss of susceptibility to multiple drugs simultaneously. In terms of antifungal multidrug resistance, our most developed picture applies to the yeast Saccharomyces cerevisiae, due in large part to the facile genetics of this organism. In recent years, important progress has been made in the two major pathogenic Candida species, C. albicans and C. glabrata. While there are significant similarities between the Candida species and S. cerevisiae, important differences have been described. The increasing development and application of genetic technologies in the Candida species will expedite direct study of the resistance mechanisms in these organisms. This is crucial to ensure continued utility of existing and future antifungal drugs.

While we have a fairly extensive catalogue of genes that are likely participants in the process of multidrug resistance, our understanding of how these genes are regulated is still very incomplete. In some cases, the identities of the transcription factors that control the multidrug resistance genes are well-established; yet a detailed picture of how the activity of these factors is regulated is not available. This will be an important future goal as interference with regulation of multidrug resistance gene expression has promise as a potential avenue to prevent development of this phenotype.

Improvements in medical care in regards to longer term survival in the face of immunosuppression has led to a greater chronic reliance on antimicrobial chemotherapy. Candida bloodstream infections are now the 4th most common form of nosocomial infection seen (Lewis, 2009). The occurrence of aspergillosis is also on the rise and outcomes associated with azole tolerant forms of this filamental fungal pathogen are dramatically worsened compared to azole susceptible fungi (Van Der Linden et al., 2011). Continued progress in the molecular understanding of antifungal drug resistance is an crucial step toward ensuring the sustainability of antifungal drug therapy and confidently expecting to treat these microbial pathogens in the future.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.