Within the limited antifungal armamentarium, the azole antifungals are the most frequent class used to treat Candida infections. Azole antifungals such as fluconazole are often preferred treatment for many Candida infections as they are inexpensive, exhibit limited toxicity, and are available for oral administration. There is, however, extensive documentation of intrinsic and developed resistance to azole antifungals among several Candida species. As the frequency of azole resistant Candida isolates in the clinical setting increases, it is essential to elucidate the mechanisms of such resistance in order to both preserve and improve upon the azole class of antifungals for the treatment of Candida infections. This review examines azole resistance in infections caused by C. albicans as well as the emerging non-albicans Candida species C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata and in particular, describes the current understanding of molecular basis of azole resistance in these fungal species.

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.

Antifungal therapy is a central component of patient management for acute and chronic mycoses. Yet, treatment choices are restricted because of the sparse number of antifungal drug classes. Clinical management of fungal diseases is further compromised by the emergence of antifungal drug resistance, which eliminates available drug classes as treatment options. Once considered a rare occurrence, antifungal drug resistance is on the rise in many high-risk medical centers. Most concerning is the evolution of multidrug-resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. The mechanisms responsible are mostly shared by both resistant strains displaying inherently reduced susceptibility and those acquiring resistance during therapy. The molecular mechanisms include altered drug affinity and target abundance, reduced intracellular drug levels caused by efflux pumps, and formation of biofilms. New insights into genetic factors regulating these mechanisms, as well as cellular factors important for stress adaptation, provide a foundation to better understand the emergence of antifungal drug resistance.

Antifungal agents exert their activity through a variety of mechanisms, some of which are poorly understood. We examined changes in the gene expression profile of Candida albicans following exposure to representatives of the four currently available classes of antifungal agents used in the treatment of systemic fungal infections. Ketoconazole exposure increased expression of genes involved in lipid, fatty acid, and sterol metabolism, including NCP1, MCR1, CYB5, ERG2, ERG3, ERG10, ERG25, ERG251, and that encoding the azole target, ERG11. Ketoconazole also increased expression of several genes associated with azole resistance, including CDR1, CDR2, IFD4, DDR48, and RTA3. Amphotericin B produced changes in the expression of genes involved in small-molecule transport (ENA21), and in cell stress (YHB1, CTA1, AOX1, and SOD2). Also observed was decreased expression of genes involved in ergosterol biosynthesis, including ERG3 and ERG11. Caspofungin produced changes in expression of genes encoding cell wall maintenance proteins, including the ␤-1,3-glucan synthase subunit GSL22, as well as PHR1, ECM21, ECM33, and FEN12. Flucytosine increased the expression of proteins involved in purine and pyrimidine biosynthesis, including YNK1, FUR1, and that encoding its target, CDC21. Real-time reverse transcription-PCR was used to confirm microarray results. Genes responding similarly to two or more drugs were also identified. These data shed new light on the effects of these classes of antifungal agents on C. albicans.Candida albicans is the most common human fungal pathogen and is the fourth leading cause of bloodstream infections in the United States (6,14,24). Currently only four antifungal drug classes are available for the management of systemic infections due to Candida species. Recently we examined changes in the genome-wide expression profile of Saccharomyces cerevisiae in response to representatives of the polyene, pyrimidine, azole, and echinocandin antifungal agents in an effort to identify class-specific and mechanism-independent changes in gene expression (1). In the present study, we extend this analysis to the pathogenic fungus C. albicans. By using the same representative drugs and similar growth conditions as in our previous study, we are able to show similarities and differences in the responses to these antifungal agents between S. cerevisiae and C. albicans. Gene expression profiling experiments revealed drug-specific responses consistent with their mechanisms of action, responses indicative of other pathways that may be affected by these agents, and responses that reflect known and potential mechanisms of resistance to these antifungal drugs. MATERIALS AND METHODSAntifungal agents. Ketoconazole (KTZ) and flucytosine (5-FC) were obtained from Sigma (St. Louis, MO). Amphotericin B (AMB) was obtained from ICN Biomedicals (Aurora, OH). The commercially available preparation of caspofungin (CPF) acetate for injection (Cancidas) was used. Stock solutions of various concentrations were made in dimethyl sulfoxide (DMS...

SummaryCandida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. We previously reported that azole-resistant mutants (MIC Ն 64 mg ml ) to both F15 and 66032 and eliminated both constitutive and fluconazole-induced CDR1-PDH1 expression. Reintroduction of wild-type or F15 PDR1 fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild-type susceptibility (MIC = 16 mg ml -1 ) to F15. In an azole-resistant clinical isolate, PDR1 disruption reduced azole MICs eight-to 64-fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata microarrays were generated and used to analyse genome-wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1-Pdr3 targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c and YMR102c along with CDR1, PDH1 and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. However, expression of 99 additional genes was specifically altered in C. glabrata F15; their roles include transport (e.g. QDR2, YBT1), lipid metabolism (ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3) and cell wall function (MKC7, MNT3). These azole resistance-associated changes could affect C. glabrata tissue-specific virulence; in support of this, we detected differences in F15 oxidant, alcohol and weak acid sensitivities. C. glabrata provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence.

SummaryOverexpression of the MDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily, is a major cause of resistance to the widely used antifungal agent fluconazole and other toxic substances in the fungal pathogen Candida albicans. We found that all tested clinical and in vitro generated C. albicans strains that had become fluconazoleresistant by constitutive MDR1 upregulation contained mutations in the MRR1 gene, which encodes a transcription factor that controls MDR1 expression. Introduction of the mutated alleles into a drugsusceptible C. albicans strain resulted in activation of the MDR1 promoter and multi-drug resistance, confirming that the amino acid substitutions in Mrr1p were gain-of-function mutations that rendered the transcription factor constitutively active. The majority of the MDR1 overexpressing strains had become homozygous for the mutated MRR1 alleles, demonstrating that the increased resistance level conferred by two gain-of-function alleles provides sufficient advantage to select for the loss of heterozygosity in the presence of fluconazole both in vitro and within the human host during therapy. Loss of heterozygosity usually occurred by mitotic recombination between the two chromosome 3 homologues on which MRR1 is located, but evidence for complete loss of one chromosome and duplication of the chromosome containing the mutated MRR1 allele was also obtained in two in vitro generated fluconazoleresistant strains. These results demonstrate that gain-of-function mutations in MRR1 are the major, if not the sole, mechanism of MDR1 overexpression in fluconazole-resistant strains and that this transcription factor plays a central role in the development of drug resistance in C. albicans.

Antifungal compounds exert their activity through a variety of mechanisms, some of which are poorly understood. Novel approaches to characterize the mechanism of action of antifungal agents will be of great use in the antifungal drug development process. The aim of the present study was to investigate the changes in the gene expression profile of Saccharomyces cerevisiae following exposure to representatives of the four currently available classes of antifungal agents used in the management of systemic fungal infections. Microarray analysis indicated differential expression of 0.8, 4.1, 3.0, and 2.6% of the genes represented on the Affymetrix S98 yeast gene array in response to ketoconazole, amphotericin B, caspofungin, and 5-fluorocytosine (5-FC), respectively. Quantitative real time reverse transcriptase-PCR was used to confirm the microarray analyses. Genes responsive to ketoconazole, caspofungin, and 5-FC were indicative of the drug-specific effects. Ketoconazole exposure primarily affected genes involved in ergosterol biosynthesis and sterol uptake; caspofungin exposure affected genes involved in cell wall integrity; and 5-FC affected genes involved in DNA and protein synthesis, DNA damage repair, and cell cycle control. In contrast, amphotericin B elicited changes in gene expression reflecting cell stress, membrane reconstruction, transport, phosphate uptake, and cell wall integrity. Genes with the greatest specificity for a particular drug were grouped together as drug-specific genes, whereas genes with a lack of drug specificity were also identified. Taken together, these data shed new light on the mechanisms of action of these classes of antifungal agents and demonstrate the potential utility of gene expression profiling in antifungal drug development.

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...