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Summary: Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.
The fungal kingdom comprises an estimated 1.5 million species, about 200 of which have been associated with humans (40). Some of these fungi coexist with humans as commensals without causing harm, and others are overt pathogens. Certain commensal fungi, such as Candida species, however, are also opportunistic pathogens and cause infections when their human hosts become immunocompromised (43). These infections can be superficial and affect the skin or mucous membranes or can be hematogenously disseminated with serious consequences. Paradoxically, medical advances over the last 30 years have led to a significant increase in the incidence of life-threatening invasive fungal infections (IFIs) (275), a result of factors such as the AIDS epidemic, the rise in the number of people receiving organ transplantations, and the burgeoning range of new treatment options for cancer patients (171, 321, 327).
Systemic fungal infections are often hard to diagnose, which contributes to their high attributable mortality. In addition, there are far fewer classes of antifungal agents (Table (Table1)1) than antibacterial drugs, limiting therapeutic options. The azole antifungals are commonly used to treat fungal infections, as they are conveniently administered and have few side effects (325). Fungal azole drug resistance, however, can be a problem in some patient groups (380). The major mechanism responsible for high-level azole resistance in clinical Candida isolates is overexpression of plasma membrane efflux pumps (135, 300, 307, 378). There are two main families of efflux proteins, the ATP-binding cassette (ABC) pumps and the major facilitator superfamily (MFS) transporters (4, 23, 74, 89, 212, 267, 299, 334). MFS pumps appear to have a limited range of substrates, whereas ABC transporters have, in general, broader specificity (Table (Table2)2) and are of greater clinical significance, and they will be the focus of this review. The heterologous expression of efflux pumps in model systems, such as Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi (87, 90, 91, 109, 159, 181, 190, 306, 318, 362). This has indicated the range of substrates for individual pumps (74, 169, 334) and has begun to identify the amino acid residues involved in substrate and inhibitor recognition (87, 90, 91, 306, 318, 362). There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, for which efflux-mediated resistance is not an issue, may be used, but not in all cases. Future development of new therapeutic approaches targeting modulation by the efflux pump transcriptional regulators or the fungal stress response pathways may prevent resistance from developing. It may also be possible to overcome azole resistance by inhibiting efflux pumps directly, a scenario that parallels efforts to develop inhibitors of human P-glycoprotein (ABCB1) (P-gp) (188).
Fungi commonly cause superficial infections of the skin and mucous membranes. When they penetrate the tissues of an immunocompromised host, however, they can cause IFIs, which are associated with much greater morbidity and mortality. The fungal species most often associated with fatal IFIs belong to the genera Candida, Aspergillus, and Cryptococcus. Candida species are the fourth most common cause of nosocomial bloodstream infections (157) and the leading cause of IFIs. In the United States, Candida albicans causes most candidemias, followed by Candida glabrata, Candida tropicalis, Candida parapsilosis, and, in fifth place, Candida krusei (275). The excess treatment cost attributable to candidemia in the United States is between $1 and $2 billion per year (281, 382). IFIs are also a leading cause of infection-related mortality among patients with cancer and prolonged neutropenia and among allogeneic hematopoietic stem cell transplant (HSCT) (bone marrow transplant) recipients with graft-versus-host disease (321). The occurrence of IFIs in such patients is increasing as a result of changes in clinical procedures, for example, multiple transplantation, treatment of patients in higher age groups, and chemotherapy that affects the integrity of the gastrointestinal tract. The widespread use of fluconazole (FLC) prophylaxis in HSCT patients means that invasive aspergillosis (IA) and other filamentous fungal infections, rather than infections by Candida species, cause the majority of deaths from IFI (215, 321). In this patient population, the risk for invasive Aspergillus infection is declining but would range from 10% to 20% if no prophylaxis was employed. Fungal infections are also a serious complication for burn patients, for whom Aspergillus and Candida species are the most common cause of infections, with an attributable mortality of 33% (236).
Geography affects the fungal species detected in the clinic. Cryptococcus gattii, for example, is found predominantly in tropical and subtropical regions (27). A notable exception was a recent cryptococcosis outbreak caused by C. gattii on Vancouver Island in the Canadian Pacific Northwest (165). C. glabrata has emerged as an important opportunistic pathogen in the United States, whereas other non-albicans Candida species, such as C. parapsilosis and C. tropicalis, commonly cause infections in other countries, notably C. parapsilosis in Latin America (275).
The patient population and the clinical setting also influence the types of fungi detected (275). For example, pediatric patients are most likely to be infected with C. albicans or C. parapsilosis (262) and patients with leukemia with C. albicans or C. tropicalis, while HSCT patients are likely to be infected with naturally azole-resistant species such as C. krusei, C. glabrata, or Candida lusitaniae (275) which occurs mainly in AIDS patients or otherwise severely immunocompromised individuals. Moreover, previous exposure to, or the prophylactic use of, fungistatic azoles such as the well-tolerated drugs FLC and voriconazole (VCZ) is associated with an increasing frequency of novel drug-resistant clinical isolates, including non-albicans Candida species, non-fumigatus Aspergillus species, zygomycetes, and hyaline molds (172, 275). Clinically significant azole resistance is mainly due either to acquired resistance in commensal opportunistic fungal pathogens or to the selection of strains of species that show innate resistance.
C. albicans can cause serious infections of the oral mucosa, as well as disseminated infection in debilitated patients. Severe oropharyngeal candidiasis (OPC) afflicts many AIDS patients (15) and is a significant infection in cancer patients being treated with chemotherapy and/or radiotherapy (66, 327). OPC is frequently the first clinical symptom recognized in human immunodeficiency virus-positive patients prior to the onset of overt AIDS (327). In cancer patients, the increased incidence of OPC results both from the debilitating effects of the cancer itself and from the immunosuppressive treatment for the cancer. Administration of broad-spectrum antibiotics for the management of bacterial infections in these patients may further predispose them to OPC (338). Radiotherapy for oral cancer results in permanent damage to the salivary glands and frequently to lifelong xerostomia (115), another predisposing factor for OPC.
In the United States, C. glabrata is the most prevalent yeast pathogen after C. albicans, and it causes both superficial (oral, esophageal, vaginal, or urinary) infections and IFIs in humans. Systemic Candida infections caused by non-albicans Candida species have increased in the past 2 decades; C. glabrata and C. parapsilosis currently rank as the second and third most frequently isolated species from reported cases of candidiasis (53, 96, 123, 274). Bloodstream infection caused by C. glabrata accounts for approximately 16% of Candida fungemia worldwide (277) and occurs predominantly in patients with solid tumors and lymphoma (371) or in HSCT recipients (383, 384). C. krusei is the fifth most common cause of candidiasis (278) and accounts for about 2% of all clinical Candida isolates. C. krusei infections are more prevalent in the elderly (276).
Aspergillus species are widely distributed in the environment and are often found in association with rotting vegetation. They are also opportunistic pathogens of humans that can cause primary invasive lung infections and disseminate to other organs. Their spores can be present in high concentrations in the atmosphere, and Aspergillus species grow rapidly at elevated temperatures (>40°C). These attributes, together with the weak defenses of the immunocompromised host, are considered the main reasons for their pathogenicity, rather than specific virulence traits (348). IA is the most common form of invasive mold infection, accounting for 60% to 80% of all reported cases (172). A systematic review of the literature has identified the most common underlying disease or predisposing factor among patients with IA as leukemia (43%), followed by HSCT (26%), lung disease (20%), organ transplantation (13%), and human immunodeficiency virus/AIDS (4%) (191). The leading cause of IA is Aspergillus fumigatus (85%), followed by Aspergillus flavus (5 to 10%) and Aspergillus terreus (2 to 10%) (47, 120, 170, 281, 332). Aspergillus niger (2 to 3%), Aspergillus nidulans, and Aspergillus ustus are only rarely isolated. IA affects a narrower range of patients than invasive candidiasis, and one of the major risk factors is severe neutropenia (281, 325). Although the crude mortality rates of both IA and invasive candidiasis remained constant over the period 1997 to 2003 (275), the rates are high, with IA mortality exceeding 50% in most reports, and there are higher values in HSCT patients than in solid-organ transplant recipients (170, 191, 325, 332). The efficacies of current antifungal therapies for IA have improved with the introduction of newer antifungals, such as VCZ (343), or combinations of antifungals (47, 234, 331) and immunomodulatory strategies (332, 343), but they are still suboptimal. VCZ is superior to amphotericin B (AMB) for the treatment of IA and has become the primary treatment choice (129).
Cryptococcosis is an IFI caused predominantly by Cryptococcus neoformans and C. gattii. Cryptococcal meningitis is the most common fungal disease of the central nervous system (80, 192, 285). Excellent reviews of cryptococcal biology, life cycle, and virulence attributes and the different manifestations of cryptococcosis are available elsewhere (145, 192). C. gattii is rarely isolated from immunocompromised hosts but instead causes most (about 80%) cases of cryptococcal infection in the general population in tropical and subtropical regions, such as Australia, Papua New Guinea, and parts of Africa (46).
C. neoformans can be classified into three serotypes based on capsular agglutination: A (C. neoformans var. grubii), D (C. neoformans var. neoformans), and AD hybrids (145, 192, 193). C. neoformans var. grubii (serotype A) is the most pathogenic form, causing the vast majority of crytpococcal infections worldwide (95%) (46). In Europe, about 5 to 30% of cryptococcal infections are caused by AD hybrids, and this is likely an underestimate due to limitations in the serotyping technique (54, 193). C. neoformans is a heterothallic yeast with two mating types, MATa and MATα (178, 179, 192). While most (>95%) C. neoformans isolates of serotypes A and D are haploid and of the α mating type, some have been determined to be diploid (54, 193). In contrast, most serotype AD strains are diploid. Alpha mating-type isolates are also more virulent (178, 179). C. gattii (serotype B and C) strains from diverse sources are predominantly sterile in the laboratory, and no evidence of recombination between isolates has been observed (124). Notable exceptions are the C. gattii isolates from a recent Vancouver outbreak, which are clonal, mainly of the α mating type, and able to mate robustly (101, 102, 165).
Although the number of cryptococcal infections in AIDS patients has remained low or even decreased in most developed countries since the introduction of highly active antiretroviral therapy (HAART) in the mid 1990s (7, 36, 139, 208, 209, 222, 279), the disease remains responsible for up to 30% of the attributable mortality in AIDS patients in regions such as Southeast Asia, Africa, and Spain (28, 126, 138, 177, 271, 317). Cryptococcosis is also a significant opportunistic infection in solid-organ transplant recipients, with a prevalence rate ranging from 0.26% to 5% and overall mortality of 42% (330). In the most severe cases of cryptococcal infections of the central nervous system, combination therapy for 2 weeks with AMB and flucytosine (5-FC) is the gold standard, followed by treatment with FLC for a minimum of 10 weeks, which should be continued for life (305). Cryptococcal infections are considered incurable because the fungal cells can remain dormant for many years and relapse occurs when the host becomes immunocompromised (192).
Some of the main factors contributing to the high mortality rates associated with IFIs are problems with slow diagnosis and choosing the appropriate treatment. The early symptoms of IFI are nonspecific, and the most widely used detection methods make timely diagnosis difficult. In addition, species-specific variations in antifungal susceptibilities make selection of an effective therapy problematic. Conventional fungal detection methods include direct microscopy of clinical specimens and culture-based, and non-culture-based, techniques (297). Visual examination of fungi in tissue samples allows presumptive identification based on cellular morphology and staining properties. However such identification requires a skilled mycologist, and identification can often be equivocal. Culture-based techniques can employ chromogenic primary isolation media, such as Chromagar, for the presumptive identification of the most prominent pathogenic Candida species, including C. albicans, Candida dubliniensis, C. glabrata, C. krusei, C. tropicalis, and, in some instances, C. parapsilosis (249, 344). Other culture-based growth, morphology, and biochemical tests are available in kit format for the identification of fungi isolated from clinical samples. These kits include API ID 32C, API 20C AUX, and RapID Yeast Plus (297). Although the kits are relatively easy to use, the results often show poor discrimination between possible species, and the process involves two culturing steps that can take 36 to 48 h. In addition, Aspergillus cannot be cultured from a significant proportion of sputum (66 to 92%) or bronchoalveolar lavage (38 to 55%) samples from patients with IA (265).
Non-culture-based identification techniques include immunological detection of antigens and molecular detection of DNA or RNA. The galactomannan from the cell wall of Aspergillus species can be detected with a double-sandwich enzyme-linked immunosorbent assay (332). In HSCT patients, this method has been shown to diagnose IA with a sensitivity of 67 to 100% and a specificity of 86 to 99% (332). The cryptococcal capsular polysaccharide can be detected in patients with cryptococcal meningitis by latex agglutination with a sensitivity of 90% and a specificity of 95% (377). Immunological methods that detect Candida β(1,3)-glucan, mannan, or the Cand-Tec test antigen are of limited value because the levels of circulating antigens are low and the transient nature of the antigenemia requires sensitive assays and frequent sampling of at-risk patients (283).
The prospect of highly specific, highly sensitive, and rapid fungal detection and identification is offered by a range of molecular methods that are currently being tested in the laboratory. These methods include quantitative real-time PCR (166), fluorescence in situ hybridization (256; reviewed in reference 356), and multilocus sequence typing (MLST). Quantitative real-time PCR can be used to measure the amount of fungal DNA present, and the use of primers, or molecular beacons, specific for mutations that confer antifungal resistance can rapidly detect resistant fungal isolates (106). MLST, which uses PCR and DNA sequence analysis to detect nucleotide polymorphisms within several housekeeping genes, provides a rigorous molecular method for species and strain identification. The typing of strains can identify relationships between isolates that can help trace sources of infection and their transmission and microevolution and indicate if a drug-resistant infection is likely to occur (147, 252, 253). Thus, molecular detection methods, combined with short-term culture of clinical samples, has the potential not only to accurately and rapidly identify fungal pathogens, but also to indicate whether the pathogen is likely to respond to conventional antifungal treatment.
In order to evaluate the impact of efflux-mediated antifungal drug resistance, it is necessary to be aware of the range of antifungal agents currently available and the mechanisms of antifungal drug resistance. These topics are reviewed elsewhere (4, 61, 122, 171, 196, 251, 307) and can be summarized as follows. There are five main antifungal drug classes (Table (Table1).1). The fluorinated pyrimidine analog 5-FC causes aberrant RNA synthesis and interferes with DNA replication (4, 307). The polyenes, such as nystatin and AMB, are heterocyclic amphipathic molecules that insert into lipid bilayers, bind to ergosterol, and aggregate in annuli to form pores. These pores disrupt plasma membrane integrity and permit the efflux of cations, such as K+, which results in cell death. Polyenes are also thought to cause oxidative damage (171, 196, 307). The allylamines terbinafine and naftifine inhibit squalene epoxidase (encoded by ERG1), which catalyzes the first step in the biosynthesis of ergosterol from squalene (Table (Table1).1). Although they have the greatest potency against dermatophytes, they are fungistatic for the majority of Candida species (171, 251, 307). The azole antifungals, such as FLC, also interfere with sterol biosynthesis (Table (Table1).1). They inhibit the cytochrome P450 14α-lanosterol demethylase, encoded by the ERG11 gene (also known as CYP51), which is the rate-limiting step of the ergosterol biosynthetic pathway. Inhibition of Erg11p depletes the membranes of ergosterol and results in the increase of toxic sterol pathway intermediates, which inhibit growth (4, 307). Azoles are thus usually fungistatic for C. albicans. The first azole drugs developed were the imidazoles such as miconazole (MCZ) and ketoconazole (KTC) (326). These drugs have problems with solubility. The triazole FLC has increased water solubility and improved pharmacokinetic properties but is ineffective against A. fumigatus. Itraconazole (ITC) and VCZ are more effective and also fungicidal against Aspergillus, and the newer triazoles, such as posaconazole (POS), ravuconazole, and albaconazole, appear to be effective against Aspergillus species, Cryptococcus species, and other fungi, such as Malassezia species (263, 325). The most recently developed class of antifungals is the cyclic lipopeptides, the echinocandins. They were originally obtained from soil fungi in the 1970s, and semisynthetic derivatives have been developed, such as caspofungin, micafungin, and anidulafungin. These drugs interfere with wall biosynthesis by inhibiting β(1,3)-glucan synthase (Table (Table1).1). The echinocandins are fungicidal for C. albicans but are not active against Cryptococcus species and are fungistatic for Aspergillus and other filamentous fungi (154, 251, 307). Despite weak fungistatic activity against A. fumigatus, caspofungin has been approved as salvage therapy for patients with IA (202).
The extents of antifungal drug resistance vary for the different drug classes (Table (Table1).1). There is fairly limited resistance to the polyenes, allylamines, and echinocandins, whereas resistance to 5-FC, imidazoles, and triazoles is more common. The rare occurrence of resistance to polyenes can be caused by a reduction in the amount of plasma membrane ergosterol, to which polyenes bind. There is primary resistance in some isolates of C. lusitaniae, Candida lipolytica, and Candida guilliermondii (196). A. terreus and A. flavus are frequently associated with AMB resistance in both in vitro and in vivo studies (47, 118, 340, 341, 374). Although the molecular mechanisms are not well understood, it is clear that A. terreus has a much lower ergosterol content than most other fungal species (47, 374), and alterations in cell wall glucans have been shown to lead to AMB resistance in A. flavus (196). Mutations in C. albicans ERG3, which encodes a C-5 sterol desaturase, an enzyme in the ergosterol biosynthetic pathway, lower the concentration of ergosterol in the membrane and cause AMB resistance (161). These mutations also confer cross-resistance to azoles (161, 307). There is little evidence of fungal resistance to allylamines. This may reflect the fact that these drugs target an early step in ergosterol biosynthesis, precluding compensatory mutations elsewhere in the pathway conferring resistance. Echinocandin resistance also appears to be rare (154). This may be due to their limited use to date, because resistance events are inherently uncommon, or because mutations in multiple genes are required to obtain clinically significant resistance. Echinocandin-resistant Candida isolates have point mutations in the β(1,3)-glucan synthase subunit that is orthologous to S. cerevisiae Fks1p (17, 272).
There is significant intrinsic and acquired resistance of Aspergillus and Candida species to 5-FC, limiting its utility. Resistance of clinical C. albicans isolates to 5-FC most often correlates with mutations in the enzyme uracil phosphoribosyltransferase (Fur1p) that prevent the conversion of 5-fluorouracil to 5-fluorouridine monophosphate (4). C. albicans strains can be grouped into clades according to their genetic relatedness based on DNA fingerprinting and MLST analysis (250, 337). Clade I clinical isolates (equivalent to the general-purpose genotype described by Schmid et al. ) are the most prevalent group of strains isolated from patients in all geographic regions studied (250, 288, 319). Interestingly, 5-FC resistance caused by mutations in C. albicans Fur1p (CaFur1p) are restricted to clade I isolates (81, 250, 288). Mutations in cytosine deaminase (CaFca1p) may also contribute to resistance (154). The incidence of 5-FC resistance in fungi has led to its use primarily in combination with other antifungals, such as AMB, which has become the gold standard for the treatment of cryptococcosis (305).
There are multiple mechanisms that can give rise to azole resistance in fungi (Table (Table1),1), and different combinations of these mechanisms operate in different fungi. The drug target, Erg11p, can be overexpressed or can develop point mutations that reduce FLC binding (4, 61, 307, 380). Common mutations in CaErg11p that confer moderate azole resistance are Y132H, S405F, G464S, and R467K (214, 269, 310). Azole-induced C. albicans growth inhibition is caused by reduction in the ergosterol content of membranes and also by the accumulation of toxic ergosterol precursors, such as 14α-methylergosta-8,24(28)-dien-3β,6α-diol. If Erg3p is inactivated by mutation, in the presence of FLC, these cells accumulate the nontoxic sterol 14α-methylfecosterol. High-level azole resistance correlates with overexpression in the plasma membrane of proteins that pump the drug out of the cell, thus reducing intracellular azole concentrations to levels at which Erg11p is not inhibited (269, 380). Transcriptional analysis has demonstrated that some azole-resistant C. albicans strains express multiple pumps in vitro (300, 378). These pumps may have an additive effect on drug efflux, but the level of pump expression in vivo and the levels of expression required to achieve clinically significant resistance have not been determined.
While it could be expected that the expression of efflux pumps might confer a fitness cost in terms of protein expression or energy utilization, there is some evidence that this may not be the case. C. albicans cells grown in the presence of FLC developed resistance by a number of mechanisms, including drug efflux, and any significant cost of resistance in terms of fitness was eliminated with further evolution in the presence of FLC (59). In C. glabrata, strains with hyperactive C. glabrata PDR1 (CgPDR1) alleles that upregulate CgCDR1, CgPDH1, and CgSNQ2 expression were more virulent in mice than strains with wild-type alleles and gained fitness in the animal model (93).
Some fungi form biofilms on host tissues or prostheses. C. albicans, for example, establishes biofilms on catheters and voice prostheses, and it is well known that such biofilms are resistant to azole antifungals (235, 293). Some studies have shown upregulation of C. albicans efflux pump genes and gene products during biofilm formation (216, 233), and others have not (107). In addition, C. albicans strains with the efflux pump genes CDR1, CDR2, and MDR1 deleted still formed azole-resistant biofilms (273, 293). Therefore, the azole resistance of biofilms is complex and multifactorial and is unlikely to be solely dependent on efflux pump expression.
There are two main classes of efflux pumps, ABC proteins and MFS pumps. These membrane proteins actively translocate compounds across cell membranes using different energy sources. The ABC proteins are primary transporters that use the hydrolysis of ATP. The MFS pumps are secondary transporters that utilize the proton-motive force across the plasma membrane. Both types of transporter contain distinctive protein domains: nucleotide binding domains (NBDs) in ABC pumps and transmembrane domains (TMDs) in both ABC and MFS pumps that confer substrate specificity (Fig. (Fig.11).
MFS transporters, like ABC transporters, comprise large superfamilies of proteins with high sequence similarity found in plants, animals, bacteria, and fungi. There are two subfamilies of MFS transporters involved in drug efflux that are defined by the number of transmembrane spans (TMS) within the TMD: DHA1 (drug:H+ antiporter 1; 12 TMS) and DHA2 (14 TMS) (110, 267). The first MFS transporter gene to be characterized from a pathogenic fungus was CaMDR1 (also named BENr). This gene was cloned by its ability to confer benomyl and methotrexate resistance on S. cerevisiae (99). Expression of CaMDR1 has been detected in both in vitro-derived FLC-resistant mutants (6) and azole-resistant clinical isolates (269, 378). CaMdr1p is a DHA1 MFS transporter, and heterologous overexpression in S. cerevisiae conferred resistance to FLC and KTC, but not to MCZ or ITC (181, 264). Experimental overexpression of CaMdr1p in C. albicans conferred resistance to cerulenin and brefeldin A, but high levels of expression were required to confer FLC resistance (133). Structural and functional analyses of CaMdr1p have indicated that amino acid residues located in TMS5 are critical for drug/H+ transport (264). Another DHA1 MFS gene from C. albicans is FLU1 (42). Disruption of FLU1 in C. albicans had little effect on FLC susceptibility but made cells sensitive to mycophenolic acid, suggesting that it might be a pump substrate. There is no evidence of FLU1 expression being associated with azole resistance in clinical isolates. Thus, despite the involvement of CaMdr1p in the azole resistance of certain clinical C. albicans isolates and a strong association between expression of the C. dubliniensis MFS transporter Mdr1p and FLC resistance (345), there is, in general, a much stronger association between azole resistance and the expression of ABC pumps (see below).
ABC transporters are found in all cells of all organisms, often in the plasma membrane, but also in the membranes of organelles. Their function is to transport substances across the membrane. Some ABC proteins transport a specific ligand, while others, notably mammalian P-gp, which is responsible for the resistance of cancer cells to chemotherapeutic agents, have evolved broad specificity for hydrophobic compounds, including drugs, which is usually referred to as multidrug resistance (MDR) (132). The basic structure of ABC transporters consists of two cytoplasmic NBDs and two TMDs (108, 130, 260) (Fig. (Fig.1).1). The NBDs are involved in ATP binding and hydrolysis, and the TMDs span the membrane, usually six times, via putative α-helices. The arrangement of the NBDs and TMDs within the pump polypeptide varies according to the type of ABC protein (Fig. (Fig.1).1). ABC proteins in S. cerevisiae have been classified into three main subfamilies, the pleiotropic drug resistance (PDR), MDR, and multidrug resistance-associated protein (MRP) (cf. human CFTR) subfamilies (23, 71, 334). The domain arrangement in most MDR and MRP ABC proteins is, from the NH2 terminus, (TMD-NBD)2, and for most PDR pumps the arrangement is reversed, (NBD-TMD)2 (71). An example of a PDR ABC protein from a pathogenic fungus is C. albicans Cdr1p (NBD-TMS6)2 (260), and C. neoformans Mdr1p (TMS6-NBD)2 is an MDR protein (Fig. (Fig.1).1). In these four-domain ABC transporters, there is often a high level of homology between the amino-terminal and carboxy-terminal halves of the protein, suggesting gene duplication and fusion. Indeed, in many organisms, there are “half-size” transporters consisting of one TMD and one NBD (260), although biochemical and crystallographic evidence indicates that they probably function as dimers (132). In several S. cerevisiae ABC transporters, for example, the MRP Ycf1p, there is an N-terminal extension containing an extra TMD that precedes the other four domains, giving the following arrangement: TMS5(TMS6-NBD)2. In contrast to the significant differences in the primary sequences of TMDs, each NBD contains conserved amino acid sequences for ATP binding, such as the Walker A and Walker B motifs and the ABC signature sequence (287).
The subfamily of fungal ABC proteins most often associated with antifungal drug resistance is the PDR group of transporters, with the archetype being S. cerevisiae Pdr5p (20, 31, 134). These fungal PDR proteins appear to share common features on both sides of the two TMDs that separate the cytosolic from the extracytosolic space (Fig. (Fig.1).1). The cytosolic side consists of a large N-terminal domain including NBD1, followed by two small intracellular loops (IL-1 and IL-2), a second large domain including NBD2, and another two small loops (IL-3 and IL-4). On the extracytosolic side they all appear to have four small (EL1, EL2, EL4, and EL5) and two large (EL3, between TMS5/6, and EL6, between TMS11/12) extracellular loops (Fig. (Fig.1).1). While the amino acid sequences for most of the cytosolic portions appear to be highly conserved, the diversity of individual members of the PDR family resides mainly in the TMDs and the ELs. This probably reflects the fact that the cytosolic part is the motor that drives the transport of a variety of substrates across the lipid bilayer through the core of the protein (formed by the 12 TMS) into the extracytosolic space or the outer layer of the lipid bilayer. Sequence comparison of PDR proteins revealed another unique feature called the PDR-CDR signature motif. This motif spans the EL3, TMS6, and the cytosolic linker region preceding NBD2. So far, the biological significance of the motif has remained obscure, but its presence only in this subfamily suggests either a topological constraint or a role in drug efflux. Another unique feature of PDR transporters is that while most fungal, as well as human, MDR or MRP transporters contain two symmetrical NBDs (conserved Walker A, Walker B, and ABC signature motifs in both NBDs), most PDR ABC transporters display asymmetrical NBDs (20, 90, 109, 373). That is, their N-terminal NBD1 contains a highly conserved ABC signature motif flanked by degenerate Walker A and B motifs while their C-terminal NBD2 consists of two highly conserved Walker A and B motifs and a degenerate ABC signature motif (20, 90, 109). Many biochemical studies have investigated the contributions of the highly conserved NBDs, the ILs, and the TMDs to function, but little or no attention has been paid to the significance of the ELs. We have performed extensive molecular mapping of S. cerevisiae Pdr5p (ScPdr5p) and CaCdr1p and found that their ELs appear to be very important for the interaction of these efflux pumps with small-molecule inhibitors, such as d-peptide derivatives, FK506, and the milbemycins (M. Niimi, unpublished results). To map suppressor mutations that attenuated the inhibition of ScPdr5p and/or CaCdr1p, we employed our specially modified host S. cerevisiae ADΔ (181) (derived from AD1-8 [see below]), which is exquisitely sensitive to a large array of xenobiotics. Overexpression of these efflux pumps in ADΔ led to highly FLC-resistant strains. We used these strains to identify broad-spectrum, as well as very specific, efflux pump inhibitors (181) (Table (Table2).2). Screening these strains for suppressor mutants that were no longer susceptible to the inhibitors allowed us to identify an array of point mutations in both ScPdr5p and CaCdr1p that were almost exclusively located in the TMDs and the ELs of these pumps (B. C. Monk, unpublished results).
There is much interest in how these proteins bind and transport substrates and in what their “normal” substrates are. Surprisingly, there is no evidence to indicate that any of the Pdrp proteins are essential. Even when as many as six S. cerevisiae PDR genes have been deleted, the cells grow normally, apart from a hypersensitivity to certain ions and xenobiotics, including the azole drugs (72). There are indications that particular ABC proteins, including CaCdr1p, CaCdr2p, and CaCdr3p (336), are involved in phospholipid transport across the lipid bilayer that may help maintain an asymmetry in the compositions of the two leaflets (131). Thus, the overexpression of these ABC proteins may indirectly cause antifungal resistance through the effects of membrane composition on membrane function and/or membrane protein activity.
An understanding of the role of fungal ABC transporters in drug resistance is hampered by the lack of high-resolution crystal structures for these proteins. Recently, however, a structure has been generated for the half-size ABC transporter Sav1866 of Staphylococcus aureus (67), which forms a homodimer in the membrane. It is thought that the two TMDs in the homodimer provide inward-facing sites that bind drugs from the lipid bilayer, or possibly the cytoplasm. The NBDs are then able to form interfacial contacts mediated by the binding of two ATP molecules to conserved features on both NBDs (including the Walker A and B motifs on one NBD and the signature motif on the other NBD). This induces the TMDs to undergo conformational change and results in a cavity that is open extracellularly and closed intracellularly. The bound drug is thus able to access the extracellular space and is effluxed from the cell. The structure of the transporter after the efflux of the drug will be similar to that observed when Sav1866 binds a nonhydrolyzable ATP analog. In the normal reaction cycle, hydrolysis of ATP then allows the transporter to reset to its drug-binding conformation (68). The schematic representation of the TMS in the ABC protein in Fig. Fig.11 is based on the Sav1866 crystal structure but does not take into account the finding that isolated ScPdr5p may occur as dimers (95). Although biochemical analysis of the full-size ABC transporter P-gp suggests a slightly different arrangement of the TMS (198), the interactions between the two TMDs is also proposed to occur between TMS2 and TMS11 and between TMS5 and TMS8. Despite the advent of structural information on ABC transporters, much remains to be understood about individual pumps and the functions of their respective families. Many transporters extract their substrates from the inner leaflet of the bilayer (132), but it is not known whether this holds true for fungal pumps. One way to systematically study the structure and function of efflux pumps involved in drug resistance, using information that is already available, is to determine phylogenetic relationships among the PDR family of efflux pumps.
The best-studied families of fungal efflux pumps are those from S. cerevisiae (23, 71, 89), in part because it was the first eukaryote to have its genome sequenced (114). The group of S. cerevisiae ABC transporters most closely associated with drug resistance is the PDR subfamily. There are 28 ABC transporter genes in S. cerevisiae, and 9 of these encode PDR transporters. There are also large clusters of PDR genes in closely related fungal pathogens (Fig. (Fig.2).2). We have investigated the relationships between these PDR transporters using classical phylogenetic analysis involving data mining of sequences using the BLAST method with S. cerevisiae PDR genes as queries.
We identified a total of 123 PDR transporters in 14 fungal species for which nearly complete genome sequences were available (see Table S1 in the supplemental material). Nine of the representative species are prominent human pathogens (Table (Table3).3). The analyzed proteins are all full-size ABC transporters (1,241 to 1,564 amino acid residues) that are predicted to have the typical PDR topology: (NBD-TMD)2 (Fig. (Fig.1).1). The topology of the A. terreus transporter 063.1 is an exception because it has two additional TMDs (each containing six TMS) at its C terminus. All the Pdrp proteins analyzed (with the possible exception of A. nidulans 952.3, which requires further examination due to sequence uncertainties) (see Table S1 in the supplemental material) contain variants of the Walker A1 motif, LGXPG(S/A)G(C/ K)STL (71). They are also predicted to include the two extended ECLs, ECL3 and ECL6, of at least 50 amino acid residues that connect TMS5/6 and TMS11/12, as previously detected in the founding member, ScPdr5p, which was identified simultaneously by several research groups, attesting to its broad substrate specificity (20, 31, 134, 173).
A phylogenetic tree was constructed from the 45 Pdr proteins identified in S. cerevisiae and five representative human pathogens: C. albicans, C. lusitaniae, Coccidioides immitis, A. fumigatus, and C. neoformans (Fig. (Fig.3).3). A total of eight Pdrp phylogenetic clusters, some of which had not been described before, were identified and named A to H. All 123 Pdrp proteins could be classified according to the clusters illustrated in Fig. Fig.3.3. The number of proteins contributed to different clusters by each fungal species varied considerably, with clusters A, B, and H containing the largest numbers of members (Table (Table3).3). Cluster A contains the well-known S. cerevisiae members Pdr5p, Pdr10p, and Pdr15p and their closely related Saccharomycetes orthologs, including CaCdr1p and CaCdr2p. Cluster B shares a common ancestry with cluster A and contains only members from the Eurotiomycetes (e.g., A. fumigatus and C. immitis) and Basidiomycota (e.g., C. neoformans). They can therefore be considered to have emerged from “precursors” of the Saccharomyces and Candida Pdr5-like transporters. Cluster C contains only Eurotiomycetes members, none of which has been characterized. Cluster D includes the well-known S. cerevisiae member Snq2p, which shares many substrates and transcriptional regulators with ScPdr5p (168). The weak organic acid transporter ScPdr12p (282) shares an ancestor with ScSnq2p. Other Saccharomycetes orthologs belong to cluster D, but there are no Eurotiomycetes or Basidiomycota members of this cluster.
Cluster E members have some particular properties. The cluster includes the sterol importers ScAus1p and ScPdr11p (190) and their ortholog CgAus1p in the closely related species C. glabrata (239). Both of these organisms are able to grow anaerobically when supplemented with unsaturated fatty acids and sterols. Interestingly, the GSGK/C residues of the Walker A1 motif are deleted in these three proteins. As the Walker A1 motif is involved in ATP binding, the deletion may have significant structural and mechanistic consequences. The Saccharomycetes are not represented in cluster H, which comprises members from Eurotiomycetes and Basidiomycota species. The large size of the cluster reflects the considerable expansion of Pdrp paralogs in the Eurotiomycetes. The basidiomycete C. neoformans contributes two Pdrp members that form cluster G, distant from the other Basidiomycota members of cluster H.
Of the eight discrete clusters identified by phylogenetic analysis of fungal pathogens, only cluster F contains representatives of all the Saccharomycetes, Eurotiomycetes, and Basidiomycota species tested. Although the function of its S. cerevisiae member Yol075p (SACE_L075C) (see Table S1 in the supplemental material) is unknown, all of its paralogs show the classical GSGK Walker A1 motif found in most ABC transporters instead of the K-to-C substitution (e.g., GSGC instead of GCGK) that characterizes the Pdrp members of the A, B, C, D, G, and H clusters (20, 71). We therefore consider cluster F to be the fungal Pdrp family ancestor. On the other hand, deletion of the GSGK core in Walker A1 and an established role in sterol import suggests that cluster E members are unlikely to be drug efflux pumps. Thus, only clusters A, B, C, D, G, and H can be considered in a strict sense Pdrp molecules. The overall phylogenetic pattern is consistent with the occurrence of multiple independent expansions of PDR genes that have assisted the successful environmental adaptation of individual fungal pathogens. It remains now to assess systematically the responses of cluster families to xenobiotics, including antifungals, and to determine how individual clusters contribute to the transport of endogenous substrates.
The ABC and MFS transporter families existed in ancestral fungal lineages that preceded a genome duplication event that occurred in the S. cerevisiae branch about 100 million years ago (Fig. (Fig.2)2) (110). In addition, elements determining transcriptional control of the PDR ABC subfamily found in S. cerevisiae appear to be conserved in several well-studied fungal pathogens.
PDR in S. cerevisiae is the best-understood fungal MDR mechanism. Many point mutations causing resistance to chemically diverse xenobiotics (including azoles) with differing targets have been mapped in isogenes encoding the Gal4-like zinc finger transcription factors ScPdr1p and ScPdr3p (45, 248). These gain-of-function mutations activate over 20 target genes, the major ones being drug efflux transporters of the ABC (ScPDR5, ScSNQ2, and ScYOR1) or MFS (ScTPO1 and ScFLR1) superfamilies (77, 299). Resistance to a wide range of drugs involves trans-activation of gene expression through the binding of the dimeric ScPdr1p and/or ScPdr3p transcription factor to promoters containing palindromic octanucleotide consensus binding sites (PDR elements [PDREs]) (160, 206). Mechanisms regulating the trans-activation include plasma membrane sphingolipid homeostasis, autoregulation of ScPdr3p and its specific activation on loss of mitochondrial respiration, chaperone-specific differential regulation of ScPdr1p and ScPdr3p (122), and ScPdr1p's ability to induce compensatory expression of efflux pumps (20, 387). Yeast cells incubated with antifungals or other xenobiotics transiently activate ScPdr1p/ScPdr3p (199), and this has been shown to be associated with drug efflux pump expression (349). Drugs like ITC and progesterone bind to a 250-amino-acid hydrophobic xenobiotic binding domain of ScPdr1p/ScPdr3p, enabling a specific association with the KIX domain (comprising three α helices) of the ScGal11p subunit of the mediator complex that recruits RNA polymerase II for expression of the ScPdr1/ScPdr3p-controlled genes (113, 349). Another feature of ScPdr1p regulation of efflux pump expression is compensatory induction. If individual efflux pump genes, such as ScPDR5, ScSNQ2, or ScYOR1, are deleted, there is a compensatory upregulation of the other drug efflux pumps (168). This induction requires ScPdr1p and is inhibited by ScPdr3p (168).
Other zinc finger transcription factors, such as ScYrr1p, ScStb5p, ScRdr1p, and ScYrm1p, also contribute, in a combinatorial fashion, to the expression of the various transporter genes (3, 186). Activation of a basic leucine zipper transcription factor, ScYap1p, controls a parallel MFS-mediated drug efflux pathway and protects yeast cells against oxidative damage (122).
In C. albicans, expression of the ABC transporters CaCdr1p and CaCdr2p is controlled by the ScPdr1/ScPdr3p-like zinc finger transcription factor CaTac1p (57). Expression is increased by gain-of-function mutations in CaTac1p, with high-level drug resistance occurring when this mutation is brought to homozygosity by loss of heterozygosity (55, 324). High doses of the female steroid hormone progesterone transiently upregulate, via steroid-specific PDREs, the same core of closely related ABC transporters induced by antifungal intervention or gain-of-function mutations in the transcription factors (21, 185). In some instances, FLC-resistant clinical isolates overexpress the MFS transporter CaMdr1p (232). Analogous to the resistance that requires homozygosity of a mutant CaTac1p, mutations in the zinc cluster transcription factor CaMrr1p, followed by loss of heterozygosity, cause CaMDR1-mediated azole resistance (85). CaMRR1 deletion diminishes drug resistance more strongly than deletion of the efflux pump, indicating that additional protective cellular mechanisms are involved (122). There are differences between the PDR pathways of C. albicans and S. cerevisiae. CaTac1p and ScPdr1p/ScPdr3p show less than 20% sequence identity, they use significantly different PDRE motifs (57), and CaTac1p appears more focused in effect than ScPdr1p/ScPdr3p (21, 195).
C. glabrata, which is closely related to S. cerevisiae, uses only one ortholog of the ScPdr1p/Pdr3p transcription factor pairing to control expression of its major ABC pumps, CgCdr1p and CgPdh1p. As in S. cerevisiae, expression of these pumps is induced rapidly by treatment with diverse drugs, and they are highly expressed in mutants defective in respiratory function. Antifungal binding to the CgPdr1p xenobiotic binding domain induces PDR via a CgGal11p homolog in the C. glabrata transcription mediator complex (349). Mutants overexpressing CgPdr1p coordinately regulate 11 genes that are homologous to ScPdr1/ScPdr3p targets (368). However, the differential expression of other genes, which are functionally linked with transport, cell wall biosynthesis, lipid metabolism, subcellular trafficking, and cell stress, by CgPdr1p probably reflects the adaptation of the two species to different environmental niches.
Mechanistic data on azole and related PDR phenomena identified in C. krusei (182) and C. neoformans (E. Lamping, unpublished data) suggest that the various drug resistance mechanisms found in C. albicans may also operate in these pathogenic fungi. While there will be differences and commonalities in the mechanisms of resistance to azole antifungals among these less well-studied pathogens, Pdr1p/Pdr3p-like transcriptional control systems probably contribute to their PDR and are potential targets for overcoming efflux-mediated azole resistance.
Analysis of the C. albicans genome has identified at least 27 ABC proteins classified into six distinct subfamilies (37, 108). Of these, there are seven open reading frames (CDR1, CDR2, CDR3, CDR4, CDR11 [CDR5], SNQ2, and YOR1) that are annotated in the Candida genome database (CGD) (http://www.candidagenome.org/) (14) as having confirmed or inferred xenobiotic-transporting ATPase activity that could be associated with MDR. In transcriptional-array studies of C. albicans gene expression, several of these genes were shown to be upregulated in the presence of FLC, ITC, or fluphenazine (70, 155, 300, 390). Upregulation of CaCDR1 and CaCDR2 was shown for four FLC-resistant isolates, relative to their susceptible parental strains, in a study combining transcriptional-array analysis with a TAC1 regulon location analysis (195). Indeed, only for CaCDR1 and CaCDR2 has functional transport of known antifungal drugs been demonstrated (37). Disruption of CaCDR1 makes C. albicans hypersusceptible to azoles (313), and controlled overexpression of CaCdr1p in a C. albicans CDR1-null mutant conferred resistance to FLC and other xenobiotics (245). When CaCDR2 was deleted in a C. albicans strain with CaCDR1 deleted, the resulting double cdr1Δ:cdr2Δ mutant was more susceptible to azoles than the single cdr1Δ mutant (312). Both CaCDR1 and CaCDR2, when heterologously expressed in S. cerevisiae, confer resistance to azoles and other xenobiotics, including the fluorescent compound and PDR substrate rhodamine 6G (181), suggesting that they are ABC pump substrates (Fig. (Fig.44 and Table Table2).2). CaCDR3 and CaCDR4 have been shown to encode phospholipid flippases. Despite a high degree of sequence conservation with CaCdr1p and CaCdr2p, CaCdr3p does not appear to be involved in resistance to antifungals, including FLC (19). Similarly, the results of gene disruption and cloning experiments showed that CaCDR4 was not involved in C. albicans FLC resistance (100). The annotation of putative xenobiotic-transporting activity for the other C. albicans ABC transporters is inferred by homology to transporter genes in other fungi; in S. cerevisiae, the orthologs of SNQ2 and YOR1 are involved in efflux-mediated resistance to 4-nitro-1-oxido-quinoline (63) and aureobasidin A, respectively (254). CaCDR11 is uncharacterized but was annotated in the CGD as an ABC transporter by sequence homology.
The other class of membrane transporters identified in C. albicans as putative drug efflux pumps is the MFS-type transporter proteins. Six genes are annotated as MFS-like in the CGD (MDR1, FLU1, TPO3, orf19.2350, NAG3, and MDR97), and of these, only CaMdr1p and CaFlu1p have substrates that are antifungals. CaFLU1 was identified by complementation of FLC hypersusceptibility in an S. cerevisiae strain lacking the ABC transporter gene PDR5 but was shown not to be required for the development of azole resistance in clinical isolates (42). Disruption of MDR1 in C. albicans resulted in reduced FLC susceptibility, and CaMDR1 was shown to be upregulated in some C. albicans strains with reduced FLC susceptibility (386).
An important question related to strategies to overcome efflux-mediated antifungal resistance is the relative contribution of each efflux pump protein to clinically significant antifungal resistance in C. albicans. It is now clear that the transporters CaCdr1p, CaCdr2p, and CaMdr1p are the main efflux pumps mediating resistance of C. albicans to azole drugs. However, CaMdr1p is relatively specific for FLC (167, 307), whereas many azole drugs can act as substrates for CaCdr1p and CaCdr2p (237) (Table (Table2).2). Interestingly, a number of FLC-resistant isolates of C. albicans overexpress just CaCDR1 and CaCDR2, but not CaMDR1, whereas other strains overexpress only CaMDR1, reflecting the existence of at least two different transcriptional pathways that are responsible for the upregulation of these genes in azole-resistant strains (395).
Although it is evident that multiple mechanisms contribute to clinical C. albicans FLC resistance (49, 269, 379), high-level resistance in clinical isolates most often correlates with overexpression of mRNA for CaCDR1 and CaCDR2, rather than for CaMDR1 (49, 201, 269, 300, 314, 378), and evidence is accumulating that CaCDR1 expression may be more critical than CaCDR2 expression. Several studies (70, 155, 300, 390) have used whole-genome transcriptional-array analysis of expression, but they show only comparative, not absolute, levels of mRNAs. In a study using haploinsufficiency phenotype assays (389), FLC-induced haploinsufficiency was observed only for the CaCDR1 transporter, not for CaCDR2. CaCDR1 was also the only transporter gene significantly upregulated in a study of C. albicans resistance development in FLC-treated, C. albicans-infected mice using genome-wide transcriptional analysis confirmed by Northern analysis (9, 10). Protein expression levels are more relevant to in vivo function than mRNA expression. A recent analysis of Cdr protein expression in a collection of C. albicans clinical isolates with reduced FLC susceptibilities demonstrated that CaCdr1p was expressed in greater amounts than CaCdr2p and that most FLC efflux function in these strains was mediated by CaCdr1p rather than CaCdr2p (135). An earlier study showing that the CaCDR2 gene possesses much higher heterozygosity than CaCDR1 (137) may reflect this differential function. It is possible that the less conserved CaCDR2 gene may have a role in adaptation to varying environmental conditions in the human host.
Thus, there is strong evidence for the dominant role of CaCdr1p in clinically significant C. albicans FLC resistance. It therefore represents a good target for combination therapies to substantially reverse, or even prevent, FLC resistance.
A significant proportion (30%) of clinical C. glabrata isolates show moderate innate resistance to azole antifungals (384). The organism can also acquire increased azole resistance during the course of therapy, or prophylaxis, with azole drugs, usually with FLC (24, 364). The acquisition of azole resistance is rapid, and the resistance phenotype is stable after the removal of FLC (34). Increasing use of FLC for the treatment of C. glabrata infections apparently results in the selection of an azole-resistant population, and resistant clinical isolates possess cross-resistance to other azoles (ITC, KTC, or VCZ) (315). This makes it more difficult to treat patients with C. glabrata infections, and FLC prophylaxis for immunocompromised patients may be a risk factor for the development of C. glabrata infections (1).
As with C. albicans, azole-resistance in C. glabrata clinical isolates is associated with increased expression of PDR ABC drug efflux pumps, in this case, CgCdr1p and CgPdh1p (also called CgCdr2p) (24, 309). There is in vitro evidence that exposure of C. glabrata cells to FLC induces expression of the drug target, CgErg11p (34, 128, 301). Mass spectrometric fingerprint analysis confirmed that the induction of CgCdr1p and CgErg11p in laboratory strains occurred several hours after the cells were exposed to FLC (244). However, unlike C. albicans, there is little evidence for changes in CgERG11 expression levels (369) or mutations in CgERG11 in azole-resistant clinical isolates (315).
The C. glabrata genome was sequenced as part of the Genolevures program (84) (http://www.genolevures.org/). The genome of this haploid yeast comprises 13 chromosomes encoding a total of 5,283 genes and shows a high degree of synteny with the genome of the closely related yeast S. cerevisiae (Fig. (Fig.2).2). Comparison of the C. glabrata and S. cerevisiae genomes identified putative MDR transport proteins belonging to both the ABC and MFS families. There are predicted to be 18 ABC transporters (6 in the PDR subfamily) (Table (Table3),3), 6 half transporters and 12 full transporters, and 15 MFS transporters, 10 of subfamily DHA1 and 5 of subfamily DHA2, in C. glabrata (110). Of these transporter genes, only a few have been studied with regard to drug resistance or transport activities. The best-characterized transporter is CgCDR1 (309), an ortholog of ScPDR5. CgCDR1 overexpression confers resistance to azole antifungals, and deletion of the gene renders cells susceptible to azoles. A wide range of structurally and functionally diverse compounds are substrates of this efflux pump (372) (Table (Table2).2). C. glabrata clinical isolates exhibiting azole resistance predominantly overexpress CgCDR1 (308) and CgPDH1 (146, 224). Both pumps require protein phosphorylation to pump xenobiotics out of the cells (372). The phosphorylation apparently affects drug efflux activity, and specifically CgCdr1p ATPase activity, as amino acid substitution in certain phosphorylation sites resulted in substantial reduction in the ATPase activity and cells became more susceptible to azoles (373). The ABC transporter CgSNQ2 is highly similar to ScSNQ2 and mediates resistance to azoles and an SNQ2-specific substrate, 4-nitroquinoline N-oxide (354).
Expression of these ABC transporters is controlled by the transcriptional regulator CgPDH1, an ortholog of ScPDR1. Azole-resistant clinical isolates of C. glabrata showed higher expression of CgCDR1 and CgPDR1 than the susceptible parent strains, indicating the importance of the MDR network to azole resistance in clinical isolates (93, 357, 368). The CgPdr1p from the resistant isolates also had amino acid substitutions that conferred upregulation of CgPDR1 and azole resistance (93, 357). Overexpression of the drug efflux transporters CgCDR1, CgPDH1, and CgSNQ2, as well as the regulator CgPDR1, was also demonstrated in a strain with deletion of the CgPGS1 gene, encoding phosphatidylglycerolphosphate synthase, an enzyme involved in the synthesis of phospholipids essential for functional mitochondria, possibly in response to an altered mitochondrial phospholipid composition (22). In C. glabrata, loss of mitochondrial function or respiratory deficiency (resulting in petite mutants) is also linked to the upregulation of CgCDR1 and CgPDH1 (38, 308).
CgAUS1, an ortholog of the S. cerevisiae ABC transporters AUS1 and PDR11, was recently described as a putative sterol importer that may help protect C. glabrata from azole toxicity (239). It has been proposed that cells use CgAus1p to incorporate exogenous sterol present in serum into cell membranes to compensate for the ergosterol depletion caused by azoles (238).
While most Candida species, with the exception of C. glabrata, as discussed above, are susceptible to azole antifungals, C. krusei is generally considered innately resistant to FLC, with about 80% of strains being susceptible dose-dependent to ITC. C. krusei is, however, susceptible to the newer triazoles, such as VCZ and POS (278, 280). While the genomes of important fungal pathogens, such as C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. neoformans, and A. fumigatus, have been sequenced and partially or fully annotated, the C. krusei genome remains largely undescribed. This is partly because of uncertainties surrounding the karyotypes of clinical isolates and because C. krusei is not amenable to genetic manipulation. Transformation protocols have yet to be reported, and no auxotrophic or dominant selection markers are available for the genetic modification of C. krusei. The innate azole resistance phenotype of C. krusei appears to be mainly due to the reduced susceptibility of the drug target Erg11p to azole antifungals (103, 258, 365). It has also been shown that efflux pumps can contribute to C. krusei FLC resistance (52, 158, 213, 296, 366). Using degenerate primers against a highly conserved region of NBDs, Katiyar and Edlind (158) isolated NBDs belonging to two ABC transporters, C. krusei ABC1 (CkABC1) and CkABC2 (158). These are the only C. krusei ABC transporters reported to date. While CkABC2 was minimally expressed under all growth conditions tested, CkABC1 was strongly induced with different azoles (158). We have isolated and characterized CkABC1 in detail. Heterologous overexpression of CkAbc1p in the S. cerevisiae host ADΔ (181) showed that Abc1p is indeed a multidrug efflux transporter able to transport a large array of xenobiotics (Table (Table2),2), including FLC, ITC, KTC, MCZ, and VCZ (182).
The genomes for A. fumigatus (241), the model filamentous fungus A. nidulans, and Aspergillus oryzae have recently been sequenced. The A. fumigatus genome is predicted to encode 49 ABC and 278 MFS transporters, more than four times the number of such transporters found in yeasts like S. cerevisiae or Schizosaccharomyces pombe (94). However, despite the large number of putative transporters, there is a dearth of evidence linking any particular ABC or MFS transporter with clinically relevant antifungal drug resistance. This is quite surprising and may indicate very limited specificity for all transporters (348) or simply that efflux is not necessary to confer antifungal resistance. However, the sequence data also revealed that all three species contain multiple copies of genes encoding several enzymes in the ergosterol biosynthesis pathway (94). It appears that many filamentous fungi possess two (218) or even three (e.g., A. oryzae) CYP51 (equivalent to ERG11) genes belonging to two distinct CYP51 gene clusters, the CYP51A or CYP51B cluster (94). They also possess two (A. nidulans and A. oryzae) or three (A. fumigatus) distinct ERG3 genes belonging to three separate gene clusters (ERG3A, ERG3B, and ERG3C) (94). Much progress has been made in deciphering the azole resistance mechanisms of A. fumigatus (reviewed in detail in references 47, 61, 231, and 290). A. fumigatus is innately resistant to FLC and KTC (140, 221, 231). Gene knockout experiments have shown that Cyp51p is essential for A. fumigatus and that A. fumigatus Cyp51Ap (AfuCyp51Ap), not AfuCyp51Bp, is responsible for the innate resistance to FLC and KTC (140, 221). Although the prevalence of ITC- or VCZ-resistant clinical A. fumigatus isolates is low, several studies have demonstrated that resistance is usually due to point mutations in AfuCyp51Ap that lead to different patterns of azole resistance for long-side-chain-containing azoles, such as ITC and POS, as opposed to VCZ and FLC (50, 64, 210, 220, 240, 370). Often mutations in amino acids G54 (G54V, G54W, G54R, G54K, or G54E) or M220 (M220I, M220V, M220T, or M220K) are found in AfuCyp51Ap, both in clinical isolates that are resistant to ITC or POS (50, 79, 210, 219) and in A. fumigatus cells mutagenized in vitro (64, 210, 240). Mutation of CaErg11p A61, the equivalent of G54 in AfuCyp51Ap, and overexpression in S. cerevisiae, however, have no effect on azole susceptibility (388).
In contrast, there are few data associating ABC or MFS multidrug efflux pumps with antifungal drug resistance in Aspergillus. Most in vitro studies attempting to identify candidate efflux pumps that could contribute to azole resistance in Aspergillus have been performed in the model filamentous fungus A. nidulans. Candidate ABC transporters isolated and characterized thus far include AtrA (73) and AtrB (11, 73), AtrC (12), AtrC2 (13), and AtrD (12). AtrA and AtrB are PDR ABC pumps; AtrC, AtrC2, and AtrD belong to the MDR class of transporters, sharing significant homology with human P-gp. AtrB (11, 73) and AtrD (12) are the pumps most likely to contribute to MDR in A. nidulans. AtrB (11) and AtrD (12) knockout strains were hypersusceptible to a number of fungicides and/or xenobiotics. In addition, overexpression of AtrB in either S. cerevisiae (73) or A. nidulans (11) caused increased resistance to a similar range of compounds. AtrA, AtrC, and/or AtrC2 may be multidrug efflux pumps, but perhaps with narrower substrate specificity. They might also be less efficient transporters, so that any gene knockout is masked by AtrB, AtrD, or other endogenous efflux pumps.
Few azole-resistant A. fumigatus clinical isolates have been found to overexpress efflux pumps. A. fumigatus AF72 showed a reduced accumulation of ITC, possibly due to increased drug efflux (76). The PDR-type ABC transporter atrF (an ortholog of the A. nidulans transporters AtrA and AtrB) was cloned from this strain, and atrF mRNA levels were found to increase fivefold in AF72 cells in response to sub-MIC levels of ITC (335). Other studies have used in vitro evolution of ITC resistance by A. fumigatus to identify possible azole resistance mechanisms (64, 207, 240). In two cases, multiple resistance mechanisms were discovered involving drug target alterations, as well as other, uncharacterized mechanisms that possibly involved efflux pumps (64, 240). A third study concluded that reduced ITC uptake was responsible for the ITC-resistant phenotype of two variants (207).
AfuMDR1 and AfuMDR2 were isolated using ABC pump NBD-specific degenerate primers (352). AfuMDR1 is an MDR-type transporter with a (TMD-NBD)2 protein topology closely related to both A. nidulans AtrD (78% identical with conserved intron positions) and A. flavus MDR1 (AflMDR1) (352). Overexpression of AfuMdr1p in S. cerevisiae conferred increased resistance only to cilofungin, an echinocandin B analog (352). AfuMdr2p is a half-size transporter with a TMD-NBD topology orthologous to ScMdl1p and ScMdl2p (352). Neither transporter is associated with an antifungal resistance phenotype. A further transporter, A. fumigatus abcA, belongs to the PDR family of transporters and shows the highest homology to ATR2 of Mycosphaerella graminicola (59% identity) and ABC1 of Pyricularia grisea (54% identity) (184). However, deletion of abcA in A. fumigatus did not result in increased susceptibility to any of the antifungals tested (184).
In a study that generated 26 ITC-resistant A. fumigatus mutants by UV mutagenesis, 8 had point mutations in the previously described azole resistance-associated amino acid G54 of AfuCYP51A (240). About half of the mutants, however, appeared to be ITC resistant due to the overexpression of the efflux pumps AfuMDR3, an MFS-type transporter of the DHA2 family, and AfuMDR4, a typical MDR-type ABC transporter with the (TMD-NBD)2 topology. However, there was no evidence linking either AfuMDR3 or AfuMDR4 directly with the ITC-resistant phenotype of any of the mutants analyzed. Although these in vitro studies can be used to infer a role for multidrug efflux in the development of Aspergillus azole resistance, all in vivo studies suggest that this resistance mechanism is unlikely to be of major clinical importance.
C. neoformans and C. gattii are important human fungal pathogens. AMB-resistant clinical isolates (162, 367), as well as variants obtained in vitro (152), have been reported, but AMB-resistant cryptococcal infections are rare (7, 28, 36, 139, 271). Analysis of a series of clinical isolates that showed cross-resistance between AMB and FLC (367) revealed that they all had defects in either C. neoformans Erg2p (CneErg2p) or CneErg3p and had reduced levels of ergosterol that could explain the AMB resistance. In another study, C. neoformans strains cross-resistant to AMB and FLC appeared to display a multidrug efflux pump-mediated phenotype (152). However, efflux pump-mediated FLC resistance has yet to be reported in clinical isolates. Cryptococci are considered resistant to echinocandins, such as caspofungin or micafungin. The mechanisms are not fully understood, given that the drug target Fks1p, (1-3)-β-d-glucan synthase, is both essential and sensitive to echinocandins in vitro (205). A possible role for the calcineurin stress response pathway mediating echinocandin resistance in Cryptococcus has been suggested (205).
FLC or ITC resistance can be acquired by Cryptococcus isolates during prolonged maintenance therapy (30, 65, 266, 268, 367). The mechanisms responsible for azole resistance are either mutations of the drug target, CneErg11p (180, 298, 367), or increased drug efflux (284, 316, 367). Although azole resistance development during maintenance therapy is rare in AIDS patients receiving HAART, these resistance mechanisms are particularly important for those patients with no access to HAART.
While clinical isolates with medium levels of FLC resistance mostly contain mutations in CneERG11 (180, 298, 367), the highest levels of resistance are likely caused by increased drug efflux. The genomes of C. neoformans var. grubii (strain H99 MATα) and var. neoformans (strains JEC21 MATα, JEC20 MATa, and B3501 MATα), as well as two C. gattii genomes (strains WM276 MATα and R265 MATα; serotype B) have recently been sequenced (for a review of the strains and links to websites, see reference 192). Analysis of the H99 genome sequence predicted 54 ABC transporters and 159 MFS transporters, suggesting enhanced transport capabilities of this environmental yeast (197). CneAfr1p (284, 316) and CneMdr1p (350) are the only two efflux pumps that have been linked to antifungal drug resistance in C. neoformans. CneAfr1p is a member of the PDR family of ABC transporters, with the highest homology to A. nidulans AtrBp, AfuAtrFp, ScSnq2p, and CgPdh1p with a (NBD-TMD)2 protein topology (284). Overexpression of CneAfr1p leads to azole-resistant C. neoformans that cannot be treated with azole antifungals, as shown in mouse models that used inhalation or intravenous infection to produce systemic cryptococcosis (316). The overexpression of CneAfr1p led to increased virulence in the mouse models and significantly improved the survival of C. neoformans during in vitro macrophage infection (316). About 5% of clinical isolates (5 of 107 isolates tested) exhibited a so-called FLC heteroresistance phenotype (391). Heteroresistant C. neoformans isolates grow on solid media at four- to eightfold-higher FLC concentrations than their liquid MICs would suggest. CneAFR1 appears to be associated with this heteroresistant phenotype (E. Sionov, H. S. Lee, Y. C. Chang, J. E. Bennett, and J. Kwon-Chung, presented at the 7th International Conference on Cryptococcus and Cryptococcosis, Nagasaki, Japan, 2008). Heteroresistant strains are aneuploid (discussed further below) and have increased copy numbers of some chromosomes under selective pressure. CneAFR1 is located on one of these chromosomes, and deletion of CneAFR1 in a heteroresistant strain led to a reversion of the resistance phenotype (E. Sionov, H. S. Lee, Y. C. Chang, J. E. Bennett, and J. Kwon-Chung, presented at the 7th International Conference on Cryptococcus and Cryptococcosis, Nagasaki, Japan, 2008).
Another ABC transporter, CneMdr1p, thought to be associated with azole resistance, belongs to the MDR-type ABC transporter family with a (TMD-NBD)2 protein topology (350). CneMDR1 shows high homology to AflMDR1, AfuMDR1, and human ABCB1 and to a lesser extent to ScSTE6 (350). However, CneMDR1 expression has yet to be linked with azole resistance in C. neoformans clinical isolates or in resistant mutants isolated in vitro.
Overexpression of both CneAFR1 and CneMDR1 homologs from C. neoformans strain CDC551 (serotype A) in S. cerevisiae ADΔ conferred MDR to a large array of xenobiotics, including all azole antifungals tested (181; E. Lamping, unpublished data) (Table (Table2).2). Despite differences in domain order placing these ABC transporters in different classes, their resistance profiles in S. cerevisiae were very similar.
Considering the mortality associated with cryptococcal meningitis and the incidence of azole-resistant breakthrough infections in AIDS patients without access to HAART, further investigation of the role of efflux-mediated azole resistance in C. neoformans is warranted.
Fungi inhabit a range of environments, including various niches on humans. Their environment can change as the fungus colonizes and spreads within a host or due to medication given to the human host. These environmental changes cause physiological stress in fungal cells, and fungi have evolved responses to ameliorate the harmful effects of the stress (61, 176). While the response of microorganisms to stresses such as temperature, pH, and changes in osmolarity have been well studied, it is important to acknowledge that the administration of antifungal drugs also represents a stress to which fungi respond (44, 61). The nature of the fungal responses to antifungal drugs depends on the fungus, the dose, its duration, and the mechanism of drug action. For fungicidal drugs, such as echinocandins (for Candida species) and polyenes at concentrations significantly above the MIC, the responses fail to prevent cell death. With fungistatic drugs, such as the azoles, above the MIC, growth is inhibited but the cells are not killed, a phenomenon referred to in this context as drug tolerance (311).
Most of the established antifungal resistance mechanisms are due to genetic mutation, usually point mutations in drug targets or enzymes in metabolic pathways or in transcription factors, leading to gene overexpression. Such mutations are stable, take time to be acquired, and can be thought of as long-term stress responses. Likewise, azole resistance can be caused by genetic rearrangements (270) or aneuploidy (323) affecting the expression of drug targets, pumps, or transcription factors. However, antifungal drugs also stimulate classic, immediate stress responses. These can be thought of as reversible phenotypic responses that do not involve mutation or chromosomal rearrangement. The short-term phenotypic stress responses that lead to drug tolerance are important in fungi because they may give cells time to develop long-term genetically stable resistance mechanisms that could also confer a fitness gain.
Antifungal drugs that target the wall or membrane sterol biosynthesis can induce osmotic or membrane stress. These stresses elicit responses through conserved signaling pathways, notably the mitogen-activated protein kinase (MAPK) signal transduction network (174, 227, 303) and the cyclic AMP-protein kinase A pathway (44, 61, 291). Membrane stress induced by azole exposure is transduced through the protein kinase C (Mkc1p) component of the MAPK pathway (J. Pla, personal communication). Oxidative stress in C. albicans induces responses, mainly via the transcription factor CaCap1p, which is also involved in MDR (5, 302). Activation of CaCap1p by a C-terminal truncation of the protein results in upregulation of the MFS transporter gene CaMDR1 (5). The cyclic AMP-protein kinase A signal transduction pathway is involved in the response of C. albicans to antifungal stress; adenylate cyclase (CaCDC35) mutants no longer respond to azoles with upregulation of CaCDR1 (148).
The serine/threonine protein phosphatase calcineurin is highly conserved in eukaryotes and is activated in response to several stresses. It has important physiological roles in C. albicans and is essential for survival during membrane stress (62). C. albicans calcineurin is a heterodimer composed of a catalytic subunit A (encoded by CaCMP1 [also called CaCNA1]) and a regulatory subunit B (encoded by CaCNB1). The phosphatase activity of calcineurin is activated when it binds calmodulin in the presence of calcium ions, and it affects gene expression via transcriptional regulators, such as CaCrz1p. Calcineurin mediates tolerance for a variety of stresses, including salt and high pH, in addition to membrane stress (342). Calcineurin is essential for C. albicans survival in serum (16), and it is the calcium component of serum that is toxic to the yeast in the absence of functional calcineurin (32). Calcineurin also plays an important role in the tolerance of C. albicans for certain antifungal drugs, in particular, azoles. If C. albicans calcineurin activity is inhibited, the fungistatic azoles become fungicidal; tolerance is abolished. Thus, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) have been found to act synergistically with FLC (311). They bind with either cyclophilin A (Cyp1p) or Rbp1p, respectively, and are thought to inhibit calcineurin by binding at the subunit A and B interface (62, 311, 342). Synergism of FK506 with FLC was also seen with C. tropicalis and C. parapsilosis, but not with S. cerevisiae or C. krusei (62). Calcineurin is activated, following an increase in intracellular Ca2+, by the binding of Ca2+-calmodulin. As in S. cerevisiae, one of the major substrates of C. albicans calcineurin is the transcription factor Crz1p (156). Disruption of CaCRZ1, however, did not completely remove azole tolerance, suggesting that the tolerance is mediated by other substrates of calcineurin (156, 257). Protein kinase CaCka2p is involved in FLC susceptibility, possibly by inhibiting CaCDR1 and CaCDR2 expression (39). In a CaCKA2 knockout strain, CaCDR1, CaCDR2, and CaRTA3 were overexpressed. Although calcineurin contributes to Cka2p-mediated FLC sensitivity, CaCrz1p is not the CaCka2p substrate. Thus, the precise mechanism of calcineurin-dependent azole tolerance remains to be discovered. Calcineurin responds to changes in calcium ion concentration, and it is possible that exposure to azoles causes an intracellular calcium spike that induces the calcineurin tolerance pathway (311). The calcineurin pathway is also involved in the growth and pathogenicity of A. fumigatus and C. neoformans, and it has been proposed as a novel target for antifungal agents (342).
Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes a number of cellular proteins, many of which are involved in signaling pathways, including calcineurin (61). In vitro studies have demonstrated a role for Hsp90 in promoting the rapid acquisition of FLC resistance by S. cerevisiae and C. albicans (58, 60). Hsp90, together with calcineurin, could be targets for abolishing tolerance and thus preventing the development of azole resistance.
Population genetic analyses of C. albicans, C. neoformans, and A. fumigatus revealed that each fungus is mostly clonal with limited evidence of sexual recombination (192, 261, 351). Although these fungi contain the genetic machinery necessary for sexual reproduction (104, 192, 246), sexual recombination has been observed only in the laboratory (203, 255) or between modified strains in animal infection models (143). Thus, outside the laboratory, the fungi may rarely undertake sexual reproduction and will have to rely on other genetic mechanisms to adapt to changes in their environment. Whole-genome sequencing and physical mapping, chromosomal-genome hybridization, and haplotype analysis have shown that genetic recombination, chromosome deletions, and translocations, as well as the gain or loss of chromosomes, occur frequently in fungi. This can result in aneuploidy—an abnormal number of chromosomes—which can have profound effects on phenotypes through gene dosage or by uncovering recessive mutations. Widespread aneuploidy in S. cerevisiae has been revealed by using microarray profiling, which showed spurious gene expression for knockout strains (142). In this study, it was shown that 8% of 300 tested gene knockout strains contained chromosomal aneuploidies.
The genome of C. albicans is very “plastic,” and it has been found that specific, nonrandom chromosomal rearrangements occur during selective pressure, such as exposure to antimicrobials or growth on different carbon sources (e.g., l-sorbose) or even the genetic manipulation of cells (transformation and selection on 5-fluoroorotic acid medium) (2, 149, 187, 270, 304, 322). Aneuploidy is also common in C. albicans clinical isolates, and this can result in azole resistance due to overexpression of Erg11p and the ABC pumps Cdr1p and Cdr2p. It has been shown that 36% of 42 FLC-resistant C. albicans isolates possessed an increased copy number of chromosome 5 (either a trisomy or a segmental aneuploidy caused by the formation of an isochromosome containing two left arms of chromosome 5 separated by a centromere) (323). Further studies revealed that the left arm of chromosome 5 contained both the azole drug target CaERG11 and the transcription factor CaTAC1, which regulates CaCDR1 and CaCDR2 expression. All strains contained two or more copies of the CaTac1p gain-of-function mutation, mentioned above, that led to constitutively high levels of CaCdr1p and CaCdr2p expression. This, together with the increase in the CaERG11 copy number, was responsible for the FLC resistance phenotype observed in these clinical isolates (56, 324). We have recently reported aneuploidy in some C. krusei isolates that possess three CkERG11-containing chromosomes, but in this case, it was not sufficient to increase FLC resistance significantly (182).
Most C. neoformans clinical isolates are haploid serotype A strains, but it has been shown that some strains can become aneuploid, leading to a transient heteroresistant phenotype (226, 391). These strains possess an extra copy of chromosome A, and their increased resistance may be associated with the increased copy number of the multidrug transporter CneAFR1. Disruption of CneAFR1 removed the heteroresistant phenotype (E. Sionov, H. S. Lee, Y. C. Chang, J. E. Bennett, and J. Kwon-Chung, presented at the 7th International Conference on Cryptococcus and Cryptococcosis, Nagasaki, Japan, 2008).
Although aneuploidy can result in increased antifungal resistance, it usually confers a fitness cost that is more severe in haploid organisms. For example, haploid S. cerevisiae cells containing an extra copy of any of its chromosomes demonstrate a typical stress response that leads to reduced growth yields (355). Therefore, resistant aneuploid isolates are unstable and tend to lose these unfavorable chromosomal rearrangements in the absence of selective pressure. Indeed, the segmental aneuploidy responsible for the Tac1p-mediated FLC resistance of C. albicans is reversible. When FLC treatment ceases, isolates lose isochromosome 5 and become FLC sensitive (324). Chromosomal rearrangements are more prevalent in C. albicans than in S. cerevisiae, possibly because the fitness cost of aneuploidy in diploid organisms is significantly less than the cost to a haploid species and possibly because sexual recombination is not efficient in C. albicans. Therefore, aneuploidy, leading to overexpression of efflux pumps, may be a significant cause of drug resistance in diploid pathogenic fungi.
As efflux pumps play important roles in the biology of many eukaryotic organisms, not least the drug resistance of fungi, much effort has been expended in the study of pump structure and function. Although fungi have obtained multiple genes encoding efflux pumps during evolution, in common with most membrane proteins, the individual efflux pumps are normally expressed at low levels. In order to get sufficient transporter protein to study, they are often heterologously expressed in other organisms or in cell-free systems. The heterologous expression systems include Escherichia coli, insect cell lines, mammalian cell lines, and yeasts, such as Pichia pastoris or S. cerevisiae.
Significant differences in the synthesis of polypeptides in prokaryotes and eukaryotes place limitations on the use of E. coli for the expression of eukaryotic transporters. There has been some success in the expression of P-gp peptide motifs as antigens for antibody production (347), and the His-tagged NBD (NBD1) of murine P-gp has also been expressed and purified from E. coli (69). Although the expressed NBD was able to bind nucleotides, the authors reported low solubility and molecule half-life problems. Even when the full-length P-gp was expressed in E. coli deficient in OmpT protease, the problem of low yield and low activity (possibly due to the absence of glycosylation) persisted (26). Insect cell lines, such as Spodoptera frugiperda (18, 111) and Trichoplusia ni (cabbage looper moth) (163, 292), have also been used to express human P-gp with a higher yield of ~6 mg of purified protein from 4 × 109 baculovirus-infected insect cells.
Among cell-free expression technologies, the wheat germ system appears to be most suitable for eukaryotic proteins. This method, however, requires special techniques (such as separate mRNA synthesis), and despite substantial recent procedure improvements made by Promega (394), there have been few applications to membrane protein expression (247).
The yeast P. pastoris is considered by many a good choice for eukaryotic-protein expression and is widely used to produce secreted proteins (112, 200). It has advantages for fungal-protein expression due to its close relationship to the donor organisms and a similar membrane composition. It can use methanol as a sole carbon and energy source and grows under controlled fermentation conditions to very high cell densities of ~300 g (wet weight) of cells per liter of culture (51). The methylotropic pathway controlled by the AOX1 (alcohol oxidase) gene is highly induced by methanol (358), and the AOX1 promoter is often used to control the expression of heterologous genes (200). Disadvantages of using AOX1 induction include difficulties in monitoring methanol concentrations during induction and the need to switch between carbon sources at a precise growth stage. Cloning genes of interest in P. pastoris can also be problematic, with individual clones showing different levels of heterologous gene expression. The P. pastoris expression system has been widely used for producing microbial (8, 393) as well as animal, including human, transporters (41, 51, 82, 121, 189, 211). Although many of these proteins show appropriate activity and kinetic characteristics, there has been little success in obtaining the levels of purity and homogeneity (monodispersity) required for structural analysis by X-ray crystallography. This may be due to the fact that the Pichia cells are harvested in the late exponential (or stationary) phase, which increases the diversity of posttranslational modifications.
There are numerous advantages that make S. cerevisiae (baker's yeast) an attractive host for the expression of eukaryotic membrane proteins (29). Like P. pastoris, it is nonpathogenic and easy and inexpensive to culture. S. cerevisiae is able to grow at a range of temperatures, at various pHs, and under hypoxic (anaerobic) conditions. Baker's yeast can often be used both for the production of recombinant proteins and for in vivo assays and screening, as the expression of the heterologous transporter can complement host mutations and often gives a measurable phenotype. The S. cerevisiae genome is comprehensively annotated, and microarray analysis has provided extensive information on the expression of many of its ~6,000 genes. Haploid and diploid forms of many well-characterized strains are available, and the ease of mating and sporulation facilitates a variety of classic genetic approaches that can be used to confirm the predictions and veracity of molecular biological experiments. In addition to a panel of haploid mutants in which each nonessential gene has been knocked out (385), there is a panel of haploid strains in which the activities of many essential genes can be titrated using the Tet promoter (141, 225), and there is a panel of heterozygous diploid mutants in which individual essential or nonessential genes have been deleted (http://www.openbiosystems.com/GeneExpression/Yeast). Such mutants enable insightful study of phenotypes and the detection of gene and chemogenomic interactions. A number of other useful mutations/modifications are available in S. cerevisiae. They include the secretion/transport mutation sec6-4, which leads to the accumulation of post-Golgi network secretory vesicles (183, 375) or mutations that ensure protein quality based on the unfolded-protein response pathway (119). Strong evidence of the utility of S. cerevisiae as a host for heterologous membrane protein expression was obtained with the expression of functional rabbit Ca2+ ATPase SERCA1a (150). The incorporation of a C-terminal biotinylation domain allowed the purification and crystallization of the correctly folded enzyme (150).
Several researchers have studied ABC pumps heterologously expressed in S. cerevisiae (73, 175, 286, 294, 309, 312, 314, 352, 353). The analysis of any heterologously expressed protein can be complicated by the presence of endogenous host proteins with similar activities. The study of ABC protein function in S. cerevisiae is therefore made more difficult by the presence of numerous ABC genes (71), as described above. In 1998, Decottignies and coworkers developed an S. cerevisiae mutant (AD12345678 [AD1-8]) in which seven ABC pump genes, predominantly of the PDR family, were deleted in order to reduce background drug transport activity due to endogenous transporters (72). In addition, the strain has PDR3 deleted and has a gain-of-function mutation in PDR1. The pdr1-3 mutation in AD1-8, together with the disruption of pdr3, leads to the constitutive hyperexpression of PDR5 and the coordinated overexpression of other members of the PDR gene network that facilitate the biosynthesis and trafficking of membrane proteins (45, 77). This strain has been developed into an expression host in which heterologous genes are cloned downstream of the ScPDR5 promoter and then integrated into the S. cerevisiae genome at the PDR5 locus from a transformation cassette by homologous recombination (181, 237). When the C. albicans ABC transporter CaCdr1p was expressed in this system, it contributed approximately 30% of the plasma membrane protein. The CaCdr1p was functional and conferred resistance to azoles and other xenobiotics on AD1-8 (181). The S. cerevisiae expression system has been used to study the functions of several fungal ABC proteins, including CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, as well as the C. albicans MFS pump Mdr1p (181, 264, 329). As the genetic distance from S. cerevisiae increased, the level of heterologous pump expression, in general, decreased (181). This drawback could possibly be addressed through codon optimization of heterologous genes or by altering the membrane composition of the host yeast.
Heterologous expression can be used to examine the effects of point mutations in specific domains of the ABC transporters on pump function. Site-directed mutation has shown that F774 in TMS6 of CaCdr1p affects trafficking and localization (329), and a T1351F mutation in CaCdr1p affects substrate specificity (328). Other amino acid residues important for transporter function have been identified by cloning CaCdr1p alleles mutated by low-fidelity amplification of the CaCdr1p open reading frame (136). Two serine residues involved in phosphorylation of CgCdr1p and CgPdh1p were identified by heterologous expression in S. cerevisiae (372, 373), and recently, we have confirmed that at least one homologous phosphorylation site (S312) is important for the efflux function of CaCdr1p (A. R. Holmes, unpublished data). Alanine scanning mutagenesis of CaCDR1 and expression in S. cerevisiae have revealed the importance of TMS11 for pump function (306). Heterologous expression in S. cerevisiae was also used to map naturally occurring single nucleotide polymorphisms and to analyze allelic variation in CaCdr1p and CaCdr2p (125, 136). CaCdr1p sequences were shown to be highly conserved, but an intra-allelic differential function was demonstrated for CaCdr2p, resulting from two adjacent nonsynonymous single nucleotide polymorphisms in TMS12, present in 81% of 61 clinical isolates (135).
As well as allowing a functional analysis of heterologous efflux pumps, S. cerevisiae can provide a robust in vivo screening platform. The expression of particular efflux pumps in ADΔ can confer antifungal drug resistance. If these pumps are inhibited, the cells can be chemosensitized to the antifungal. Thus, an efflux pump inhibitor will not inhibit the normal growth of cells expressing the efflux pump but will sensitize the cells to sub-MIC levels of their antifungal substrates. Screening for chemosensitization in agar diffusion assays or liquid growth assays has been used to identify pump inhibitors, as discussed further below. The functional overexpression of heterologous transporter molecules in a background depleted of endogenous transporters can also provide the high signal-to-noise ratios required for in vitro ATPase assays and the measurement of energy-dependent drug efflux in whole cells. These secondary screens provide experimental confirmation of ABC transporter inhibition and increase the robustness of the drug discovery process (181).
An ideal antifungal agent, in addition to meeting pharmacological requirements, would not be susceptible to the development of resistance due to efflux mechanisms (48). There are four principal approaches to negating the impact of efflux, all of which depend on maintaining a high concentration of the antifungal agent at its site of action (Fig. (Fig.5).5). The simplest would be to use antifungals that are not substrates of efflux pumps; current examples are the polyenes and the echinocandins (Fig. (Fig.5,5, image 1). Another approach would be to protect the efficacy of antifungals that are subject to efflux by developing treatments that prevent efflux (Fig. (Fig.5,5, image 2a and b). A third approach would be to deplete cells of the energy required for drug efflux by inhibiting the plasma membrane H+ ATPase (Fig. (Fig.5,5, image 3). Alternatively, it might be possible to design drugs with an enhanced rate of uptake and thus shift the balance between uptake and efflux so that a high intracellular concentration of the drug is maintained despite any upregulation of efflux (Fig. (Fig.5,5, image 4).
The polyene and echinocandin antifungals in clinical use are probably not substrates of fungal efflux pumps at therapeutic concentrations. There is one report that the echinocandin caspofungin is pumped by the CaCdr2p ABC transporter (320), although the efflux activity detected was very weak, was subject to highly specific conditions, and does not appear to be an important feature in clinical isolates (243). As the polyenes span the lipid bilayer, they may not be able to enter the drug-binding pocket of the transporter. Significant hydrophobicity is currently considered a prerequisite for substrate-multidrug-transporter interaction (204), and size is also reported to be a factor determining whether a hydrophobic compound can act as a substrate of at least one fungal ABC transporter, ScPdr5p (117). Echinocandins are large lipopeptide molecules with a molecular weight of about 1,200, and although they have a lipid side chain (Fig. (Fig.4)4) that presumably intercalates with the phospholipid bilayer of the cell membrane (75), they may not be sufficiently hydrophobic or the right size to interact with the efflux pump. Thus, new drug classes designed by taking into account such size and hydrophobicity constraints could avoid being ABC transporter substrates (Fig. (Fig.5,5, image 1). However, because efflux pumps have evolved large and flexible drug-binding sites to protect cells against a wide variety of toxic compounds (132), such rational drug design will be challenging.
Another approach has been to develop pump inhibitors that chemosensitize cells to existing effective drugs. A major impetus for this work has come from cancer research, because the human drug efflux pumps, such as P-gp, are important mediators of resistance to many anticancer drugs. Since the discovery of P-gp in 1976 (153), a wide range of compounds that are pump pseudosubstrates or that can modify pump activity by other mechanisms have been identified. The P-gp-inhibitory compounds identified include natural and synthetic polymers (88, 376, 392); modulators of H bond acceptor capacity (97); a globotriaosylceramide analog (78); P-gp substrates, such as FK506 (359); calcium channel modulators, such as verapamil (361); calmodulin inhibitors (105); and quinine analogs (360). Clinical trials of promising synthetic P-gp inhibitors, such as Tarquidar, have been undertaken but have been discontinued because of side effects that may be related to the protective function of P-gp in sites such as the blood-brain barrier (289). Strategies for the development of P-gp inhibitors have been comprehensively reviewed by McDevitt and Callaghan (217) and include pharmacophore profiling, combinatorial chemistry, and structure-based design, in combination with high-throughput screening of lead compounds.
Such strategies can also be applied to the development of fungal ABC transporter inhibitors (Fig. (Fig.5,5, image 2). These inhibitors could affect the pump directly (Fig. (Fig.5,5, image 2a), either by binding as a pseudosubstrate (Pdr5p is thought to have at least three substrate-binding sites ), competitively or noncompetitively, and blocking access to the binding site, or by locking the molecule in a conformation that prevents the transport reaction cycle. A possible confounding factor for this approach is the observation that if one S. cerevisiae PDR transporter is inactivated, there is compensatory upregulation of other PDR pumps (168). In an alternative strategy, inhibitors could be designed that act indirectly on efflux, de-energizing the transporter by lowering the cytoplasmic ATP concentration (for ABC pumps) or depleting the electrochemical potential of the plasma membrane (for MFS transporters) (Fig. (Fig.5,5, image 3). The plasma membrane H+ ATPase Pma1p is the primary proton pump in fungi. It maintains intracellular pH and generates the electrochemical gradient of the plasma membrane that is required for drug transport by MFS pumps. Pma1p drives a range of secondary transporters, such as those involved in ionic balance and nutrient uptake required for ATP synthesis, so it indirectly affects drug efflux by ABC transporters (Fig. (Fig.5,5, image 3). This approach has been validated: it has been demonstrated that inhibitors of C. albicans Pma1p also inhibit azole resistance at concentrations below the MIC (230). However, lowering the membrane potential could also result in decreased uptake of the antifungal drug if active diffusion/transport is involved.
The heterologous expression of fungal efflux pumps in S. cerevisiae has been used to screen for compounds that inhibit the transporters and thus chemosensitize the host yeast cells to pump substrates. The application of this screen to panels of xenobiotics (181), combinatorial peptide libraries (242), and libraries of natural products (346) has identified tacrolimus (FK506), milbemycins, enniatins (181), beauvericin (M. Niimi, unpublished), unnarmicins (346), and some octapeptides (242) as specific ABC pump inhibitors (Table (Table33 and Fig. Fig.4).4). Ideally, these compounds will inhibit a range of clinically important efflux pumps, but not human orthologs. While some of the identified pump inhibitors may not be suitable for clinical application, they may be useful in pump protein crystallization and structural resolution. As the structures of fungal transporters become available, this will enable in silico modeling (25, 295) and structure-directed development of more effective pump inhibitors. It should be noted, however, that despite a great deal of promising investigation of pump inhibitors as chemosensitizers of tumor cells and bacteria, no successful products have entered clinical use.
Another way of inhibiting the activity of ABC transporter-mediated efflux, instead of directly targeting the ABC transporter protein, would be to modulate the transcription of the genes encoding ABC transporters (113, 349). In S. cerevisiae and C. glabrata, xenobiotic substrates (such as the antifungal azoles) of ABC transporters have recently been shown to directly bind to the Pdr1p family of transcription regulator proteins via a nuclear-receptor-like pathway, in a mechanism similar to the regulation of human MDR by the PXR (pregnane X receptor) nuclear receptor (349). The authors suggest that small-molecule antagonists could be developed that bind Pdr1p and its orthologs and thus prevent activation of the efflux pump genes (Fig. (Fig.5,5, image 2b). Alternatively, transcription of ABC transporter genes could be targeted by the use of RNA silencing (RNA interference [RNAi]). Human MDR genes have been successfully inhibited experimentally using RNAi (339). Limitations to anti-human MDR therapy with small RNAs include the longevity of small interfering RNA expression, the stability of the targeted message, and the efficient delivery of the RNAi. Delivery of RNAi as an adjunct to antifungal therapy with pump-susceptible drugs would have the same limitations. An additional problem is that although RNAi can be used in some fungi, such as A. fumigatus and C. neoformans (127, 194), it is ineffective in C. albicans. Target specificity might also be an issue. An advantage of directly inhibiting the pump protein is that inhibitors can be designed to act at the fungal cell surface and therefore are less likely to be subject to the development of efflux-mediated resistance.
One factor to be considered for all efflux-susceptible antifungals is that there can be a very rapid, reversible upregulation of efflux by fungi in response to such drugs. This is a stress response, as described above. Calcineurin mediates tolerance for several environmental stressors in fungal cells, and the use of calcineurin inhibitors has been suggested as a possible therapy in conjunction with azoles (342). Calcineurin inhibitors, such as FK506 (Fig. (Fig.4),4), render the normally fungistatic azole FLC fungicidal (363) and therefore would not only reduce the immediate upregulation of efflux and tolerance by negation of the calcineurin-mediated stress response, but would also act quickly enough to prevent the selection of mutants in the transporter regulatory pathway. Nonimmunosuppressive analogs of FK506, however, would have to be used for immuncompromised patients.
Finally, an alternative strategy for combating antifungal resistance due to efflux upregulation could be to increase uptake rather than decrease efflux of drugs (Fig. (Fig.5,5, image 4). This is a current strategy for combating human ABC transporter-mediated drug resistance; anticancer drugs can be more rapidly delivered to their intracellular targets by inclusion of multiple arginine residues, optimally eight (octaarginine [R8]) (83), thus overcoming the effects of MDR. R8 liposomes can be used to transport genes and drugs into cells by their rapid interaction with, and transport through, the plasma membrane and improved intracellular trafficking, avoiding lysosomal degradation (reviewed by Khalil et al. ). Fungal cell walls generally have an overall negative charge due to phosphate residues on mannoproteins. Although a positively charged triarginine motif prevents the uptake of peptides by yeast cells (229, 230), more than six arginines increase uptake (223, 229). The phosphomannan could, therefore, concentrate arginine-tagged drugs at the cell periphery, and provided the fungal transporter has a higher affinity for the drug than the high micromolar affinity shown by the cell wall site, the drug will be delivered to its target.
In summary, a number of ways to overcome antifungal resistance due to MDR can be envisaged. New antifungals that are fungicidal and not subject to efflux would be ideal. However, combination therapy with inhibitors of ABC transporters, some of which have already been used in clinical trials, based either on direct inhibition of activity or on inhibition of their expression, shows promise for future successful treatment of fungal infections (151).
IFIs are important diseases with high attributable morbidity and mortality that affect an ever-increasing population: the immunocompromised. Most life-threatening fungal infections could be treated more effectively if faster and more specific diagnostic technologies were available. For example, PCR amplification of rRNA intervening transcribed sequences followed by DNA pyrosequencing has the potential to halve the time needed for species-level fungal identification (33, 35). Translation of this technology into the clinic should allow the early identification of fungal species, including innately resistant species and those that are susceptible to the development of MDR. Pyrosequencing is already being applied in the laboratory to the detection of mutations responsible for fungal echinocandin resistance (381). This could be extended to detect transcriptional-regulator mutations responsible for efflux-mediated resistance, although multiple mutations of this type occur in CgPdr1p (93).
Molecular techniques could be adapted for clinical microbiology laboratories by automating aspects of the methodology, converting to a microfluidic format for PCR, and using robotic recovery of amplimers for DNA sequence analysis. DNA and RNA amplification systems that do not require PCR technology may assist DNA detection and automation. Finally, the hybridization of the amplified products to high-density oligonucleotide microarrays could be used to discriminate between matched sequences and their variants in a format suitable for rapid species identification by computer analysis. This could be achieved with a microfluidic technology that has been used to identify bacteria in complex microbial ecosystems (259).
Many fungal species, mostly Candida species, acquire azole resistance by the overexpression of efflux pumps, predominantly ABC transporters. This is not so for all fungi. In Aspergillus species, azole resistance appears to be dictated more by the drug target, Cyp51Ap (equivalent to Erg11p), than efflux pumps. Also, the contribution of efflux to the azole resistance of C. neoformans clinical isolates needs to be confirmed. The ability to functionally express individual fungal transporters in model organisms, such as S. cerevisiae, has enabled the analysis of pump function and screening for pump inhibitors. Although it may be difficult to identify broad-spectrum pump inhibitors with minimal toxicity, the structural resolution of fungal efflux pumps will make a major contribution to the understanding of these important eukaryotic membrane proteins and may help in rational drug design. As the structures for fungal transcription factors become available, it may even be possible to design multifunctional drugs that inhibit conventional targets, such as Erg11p, together with the transcription factors responsible for the overexpression of efflux pumps and the pumps themselves (228).
We gratefully acknowledge funding from the National Institutes of Health (R01DE016885-01-RDC); the Japan Health Sciences Foundation; the Health Science Research Grants for Research on Emerging and Re-Emerging Infectious Diseases (H16-Shinko-6 and H19-Shinko-8) from the Ministry of Health, Labor and Welfare of Japan; the Fundação para Ciénca e a Tecnologia of Portugal; the New Zealand Lottery Grants Board; and the Foundation for Research Science and Technology of New Zealand (IIOF grant UOOX0607-RDC).
Richard Cannon received a B.A. in Natural Sciences (Biochemistry) from Cambridge University, United Kingdom, in 1984 and a Ph.D. in Microbiology, also from Cambridge University, in 1987. He then travelled to the University of Otago, Dunedin, New Zealand, for a postdoctoral fellowship and is now Professor of Molecular Microbiology in the Department of Oral Sciences. Richard has retained an interest in Candida albicans since his Ph.D. studies of yeast-mycelial dimorphism. He conducted some of the early work on the genetic transformation of C. albicans and has investigated C. albicans virulence mechanisms. More recently he has focused on the oral adhesion of C. albicans, fungal drug resistance, particularly efflux-mediated resistance, and antifungal drug development.
Erwin Lamping was educated in Austria. He obtained an engineering degree (Dipl. Ing.) in Technical Chemistry in 1988 and a Ph.D. in Biochemistry and Genetics from the Graz University of Technology (TU Graz) in 1992. He was an Assistant Professor at the Department of Biochemistry, TU Graz, from 1991 to 1994. Between 1992 and 1996, he was an Erwin-Schrödinger postdoctoral fellow at the Department of Biochemistry, University of Otago, Dunedin, New Zealand. Since 2001, he has been working in the Department of Oral Sciences, University of Otago, and is currently a Senior Research Fellow. He is experienced in yeast molecular biology and the study of drug resistance mechanisms in fungal pathogens. In the past eight years, he has focused on studying the structure, function, and regulation of expression of the azole drug target Erg11p and ABC and MFS efflux pumps that are associated with resistance to azole antifungals.
Ann Holmes was educated in the United Kingdom and received a B.Sc. (Hons) in Microbiology from Queen Elizabeth College, University of London in 1970 and a Ph.D. in Virology (1974) from the Wright Fleming Institute, University of London. She held postdoctoral fellowships at Stanford University, CA, and the University of Otago, New Zealand, and was a visiting scientist at CAMR, Porton Down, United Kingdom. Ann is currently a Senior Research Fellow at the University of Otago. She has studied Candida albicans since 1986, and her current research in adherence mechanisms and in the efflux pump-mediated drug resistance of C. albicans reflects an interest in developing new approaches to the treatment of patients with C. albicans infections.
Kyoko Niimi was educated in Japan, gaining a Doctor of Veterinary Medicine degree from Nihon Veterinary and Zoo Technical College (Tokyo) in 1971. Her early career, up to 1980, was in mycology and drug discovery. Kyoko then had a break from science to raise her family before starting Ph.D. studies at the University of Otago. She gained her Ph.D. in 1996, and Kyoko is currently a Senior Research Fellow at the University of Otago. Her research interest is the study of antifungal resistance mechanisms in pathogenic fungi and development of heterologous membrane protein expression systems, using the nonpathogenic yeast Saccharomyces cerevisiae, that could be used for antifungal drug discovery.
Philippe Baret is a bioengineer and obtained his Ph.D. in quantitative genetics from the Université Catholique de Louvain, Belgium. As a postdoctoral research fellow, he studied quantitative trait locus (QTL) and linkage disequilibrium mapping in the William G. Hill laboratory at the University of Edinburgh, United Kingdom. In 1997, he was appointed as a Professor of Genetics and System Analysis at the Université Catholique de Louvain. He has set up a statistical genetics research team to study genetic diversity, bioinformatics, and systems of innovation. With André Goffeau, he is interested in the evolution of transporter proteins and uses a combination of approaches such as similarity and synteny analyses. His team is a member of the French consortium Genolevures that provides annotated sequence data and classifications for the genomes of 18 species of hemiascomycete yeasts, including nine complete genomes.
Mikhail Keniya graduated from the Biological Faculty of Rostov State University (RSU; Russia) in 1986. In 1991, he completed a Ph.D. in biochemistry and enzymology at the same university. Mikhail worked in the Biological Research Institute of RSU as a Research Scientist and as a Lecturer in the Department of Biochemistry and Microbiology. His main scientific interests were investigating mechanisms of microbial resistance to extreme environments and the associated biomedical problems. He began work on the structural biology of fungal membrane transporters in 2001 as a Postdoctoral Research Associate in the Public Health Research Institute, University of Medicine and Dentistry of New Jersey. At present, Mikhail is a Research Fellow in the Department of Oral Sciences, University of Otago, New Zealand, where he is studying CaCdr1p and the plasma membrane H+-ATPase as a novel antifungal target.
Koichi Tanabe was educated in Japan, receiving a B.Sc. (1996) and Master's degree (1998) in Biochemistry from Kyoto University and a Ph.D. in Biochemistry from Kyoto University in 2001. He has had a postdoctoral fellowship in Biochemistry from Kyoto University, and he is currently a Research Fellow in the National Institute of Infectious Diseases, Tokyo, Japan. Koichi has studied Candida albicans since 2003. His current research in fungal sterol metabolism and the efflux pump-mediated drug resistance of Candida spp. reflects an interest in developing new approaches to the treatment of patients with fungal infections.
Masakazu Niimi was educated in Japan and obtained a D.D.S. from Kyushu Dental College in 1974 and a Ph.D. in Microbiology (1984) from Kyushu University. He held a research assistant position at Kyushu Dental College and at Kagoshima University, Japan. This was followed by a postdoctoral fellowship at the University of Otago, New Zealand, for 10 years. He is currently the Chief of the Mycology Laboratory, Department of Bioactive Molecules, at the National Institute of Infectious Diseases in Tokyo. Masakazu has studied Candida albicans since 1975. His current research is in virulence factors, mode of action of antifungals, and the efflux pump-mediated drug resistance of pathogenic fungi. This reflects an interest in dissecting fungal ABC membrane protein functions and developing new classes of antifungal agents.
André Goffeau obtained his Ph.D. from the University of Louvain (UCL) in Belgium. He studied mitochondrial ATP synthesis as a postdoctoral fellow of Albert Lehninger in Johns Hopkins, Baltimore, MD. In 1970, he started a new laboratory as Professeur Extraordinaire from UCL in Louvain-la-Neuve to study the proton and drug ATPases from yeast plasma membranes. He has contributed to the setting up of the early Biotechnology Research Programmes of the Commission of the European Communities and of the basic Human Frontier Science Programme. He has initiated, contributed to, and coordinated the international yeast genome sequence project that was completed in 1996. From 1999 to 2002, he was awarded a Rothschild-Mayen fellowship and then a Blaise Pascal International Chair at the Institut Curie and Ecole Normale Superieure in Paris. He is now formally retired but collaborates with numerous international projects on the comparative genomics of fungal transport ATPases.
Brian Monk received a B.Sc. (Hons) from Victoria University of Wellington, Wellington, New Zealand, and a Ph.D. from Monash University, Melbourne, Australia, in 1977. He has held postdoctoral fellowships in the Department of Biochemistry and Biophysics, University of California, Davis, and the Department of Biology, Washington University, St. Louis, MO. He has been a Visiting Scientist at EMBL, Heidelberg, Germany; the PHRI, New York, NY; the Max Planck Institute for Biophysics, Frankfurt, Germany; the FYSA, Universite Catholique de Louvain, Louvain-la-Neuve, Belgium; and the Instituto Superior Técnico, Lisbon, Portugal. He is currently Senior Lecturer, Department of Oral Sciences, Faculty of Dentistry, University of Otago, Dunedin, New Zealand. Brian's long-term interest in the structure and function of P-type ATPases and drug efflux pumps is aimed at the discovery of novel anti-infectives that circumvent drug resistance in bacteria, fungi, parasites, and weeds.
†Supplemental material for this article may be found at http://cmr.asm.org/.