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 study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, AD⌬. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K m value and the V max value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.The resolution and exploitation of protein structure and function are among the greatest biological challenges in the postgenomic era. These challenges, and their potential dividends, are greatest for membrane proteins, which are notoriously difficult to functionally express and purify in the quantities and forms needed for drug discovery or for high-resolution X-ray crystallography (1, 16). About a quarter of the cellular proteome consists of membrane proteins (5), which often play vital physiological roles: from environmental sensing to energy transduction, from nutrient uptake to drug efflux, and from cellular proliferation to programmed cell death. Membrane proteins are involved in many prominent diseases, including cystic fibrosis (48), type 2 diabetes (49), heart disease (52), and the drug resistance of numerous cancers (57). Hence, they are the targets for many therapies and constitute up to 70% of the drug targets used in medicine today. Membrane proteins also play key roles in drug modification, detoxification, and resistance in a wide variety of prokaryotic and eukaryotic systems (7). A fundamental understanding of cell biology, cell physiology, and cell-drug interactions therefore requires a detailed analysis of membrane protein function. Fu...
Analysis of the transport functions of individual Candida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic PDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u ؊ ) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u؊ was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC 80 s] were 0.625 g/ml for fluconazole, <0.016 g/ml for ketoconazole, and <0.016 g/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC 80 s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 g/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicans Cdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.Candida albicans is an asexual diploid fungus that causes opportunistic infections commonly seen in immunocompromised and debilitated patients (9, 30). An estimated 33 to 55% of patients with human immunodeficiency virus infection and AIDS contract oropharyngeal candidosis (34), and the synthetic triazole fluconazole has been the mainstay of their treatment. The widespread use of prolonged fluconazole therapy has increased the incidence of treatment failure due to fluconazole-resistant C. albicans (3,14,21,34,42). A number of studies have identified the major azole resistance mechanisms (1,20,38,41,42,(44)(45)(46). These include overexpression of, or mutations in, the drug target, 14␣-sterol demethylase; mutations in other parts of the sterol biosynthesis pathway; and, most commonly, overexpression of drug efflux proteins.C. albicans possesses transporters such as Cdr1p and Cdr2p with homology to proteins of the ATP-binding cassette (ABC) family (10,16,18,19,31), as well as Ben r p, which has homology to the major facilitator superfamily (MFS) class of drugproton antiport efflux pumps (1,5,36,46). The BEN r gene encodes a transporter associated with resistance to benomyl and methotrexate when it is expressed in Saccharomyces cerevisiae. The C. albicans CDR1 gene is a homologue of S. cerevisiae PDR5, which encodes a multidrug efflux pump, and CDR1 is the gene most often associated with energy-dependent drug efflux in fluconazole-r...
Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.
Fluconazole (FLC) remains the antifungal drug of choice for non-life-threatening Candida infections, but drug-resistant strains have been isolated during long-term therapy with azoles. Drug efflux, mediated by plasma membrane transporters, is a major resistance mechanism, and clinically significant resistance in Candida albicans is accompanied by increased transcription of the genes CDR1 and CDR2, encoding plasma membrane ABC-type transporters Cdr1p and Cdr2p. The relative importance of each transporter protein for efflux-mediated resistance in C. albicans, however, is unknown; neither the relative amounts of each polypeptide in resistant isolates nor their contributions to efflux function have been determined. We have exploited the pump-specific properties of two antibody preparations, and specific pump inhibitors, to determine the relative expression and functions of Cdr1p and Cdr2p in 18 clinical C. albicans isolates. The antibodies and inhibitors were standardized using recombinant Saccharomyces cerevisiae strains that hyper-express either protein in a host strain with a reduced endogenous pump background. In all 18 C. albicans strains, including 13 strains with reduced FLC susceptibilities, Cdr1p was present in greater amounts (2-to 20-fold) than Cdr2p. Compounds that inhibited Cdr1p-mediated function, but had no effect on Cdr2p efflux activity, significantly decreased the resistance to FLC of seven representative C. albicans isolates, whereas three other compounds that inhibited both pumps did not cause increased chemosensitization of these strains to FLC. We conclude that Cdr1p expression makes a greater functional contribution than does Cdr2p to FLC resistance in C. albicans.Factors identified as affecting the susceptibility of Candida albicans to azole antifungal drugs such as fluconazole (FLC) include overexpression or mutation of the drug target 14␣ lanosterol demethylase, mutations in other enzymes of the ergosterol pathway and increased expression of drug efflux pumps (reviewed in references 4, 40, and 53). Mediators of azole efflux from C. albicans include the major facilitator superfamily pumps Mdr1p (28) and Flu1p (1) and the ATPbinding cassette (ABC) transporters Cdr1p and Cdr2p (4, 52). Although FLC resistance clearly can be multifactorial, highlevel, clinically relevant resistance (MIC Ն 64 g ml Ϫ1 ) is most often associated with increased expression of mRNAs from the ABC genes CDR1 and CDR2 (3, 34, 37, 38). Analysis of resistance in clinical isolates has, to date, focused almost exclusively on measuring gene transcription, initially by Northern analysis (22,41,53), and more recently by transcript profiling and quantitative reverse transcription-PCR (16,34,38,55) and the use of reporter genes (24). However, the ability to compare the amounts of expressed Cdr polypeptides and, more importantly, the efflux activities of Cdr1p and Cdr2p, is crucial if the contribution of each pump protein to drug efflux function in clinical resistance is to be determined. Unfortunately, proteomic approaches using...
Fungi comprise a minor component of the oral microbiota but give rise to oral disease in a significant proportion of the population. The most common form of oral fungal disease is oral candidiasis, which has a number of presentations. The mainstay for the treatment of oral candidiasis is the use of polyenes, such as nystatin and amphotericin B, and azoles including miconazole, fluconazole, and itraconazole. Resistance of fungi to polyenes is rare, but some Candida species, such as Candida glabrata and C. krusei, are innately less susceptible to azoles, and C. albicans can acquire azole resistance. The main mechanism of high-level fungal azole resistance, measured in vitro, is energy-dependent drug efflux. Most fungi in the oral cavity, however, are present in multispecies biofilms that typically demonstrate an antifungal resistance phenotype. This resistance is the result of multiple factors including the expression of efflux pumps in the fungal cell membrane, biofilm matrix permeability, and a stress response in the fungal cell. Removal of dental biofilms, or treatments to prevent biofilm development in combination with antifungal drugs, may enable better treatment and prevention of oral fungal disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.