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...
The overexpression of pleiotropic drug resistance (PDR) efflux pumps of the ATP-binding cassette (ABC) transporter superfamily frequently correlates with multidrug resistance. Phylogenetic analysis of 349 full-size (~160 kDa) PDR proteins (Pdrps) from 55 fungal species, including major fungal pathogens, identified 9 separate protein clusters (A, B, C, D, E, F, G, H1a/H1b and H2).
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...
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