Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.     

Characterization of Cdr1p, a major multidrug efflux protein of Candida albicans: purified protein is amenable to intrinsic fluorescence analysis

Candida drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, confers multidrug resistance in immunocompromised and debilitated patients. A member of the ATP-binding cassette (ABC) superfamily of membrane transporters, Cdr1p contains two nucleotide binding/utilization sites (NBDs) and two transmembrane domains (TMDs). We had earlier characterized Cdr1p by its overexpression as a GFP-tagged fusion protein that elicits oligomycin-sensitive ATPase activity and is linked to drug extrusion. However, it is essential to have highly purified Cdr1p to understand the detailed molecular basis of structure and functions of this protein. In this study, we have developed a two-step purification protocol using stably overexpressed His-tagged Cdr1p in Saccharomyces cerevisiae. Purified Cdr1p exhibited divalent cation-dependent ATPase activity [approximately 1.2 micromol (mg of protein)(-)(1) min(-)(1)] with an apparent K(M) in the range of 1.8 to 2.1 mM and V(max) between 1.0 and 1.4 micromol (mg of protein)(-)(1) min(-)(1). Unlike its close homologue human P-gp/MDR1, purified Cdr1p only moderately displayed drug stimulated ATPase activity. By exploiting intrinsic fluorescence intensity of purified Cdr1p, which contains 24 tryptophan residues, we could monitor defined conformational changes upon substrate drug and ATP binding. It is observed that ATP binding to Cdr1p (K(d) = approximately 1.7 mM) is not a prerequisite for drug binding, and both the mechanisms of drug as well as ATP binding, which induce specific conformational changes, occur independent of each other. Our study for the first time provides a catalytically active purified ABC transporter from a fungal pathogen, which is amenable to fluorescence measurements and thus would be useful in understanding the molecular basis of antifungal transport.     

Rationally Designed Transmembrane Peptide Mimics of the Multidrug Transporter Protein Cdr1 Act as Antagonists to Selectively Block Drug Efflux and Chemosensitize Azole-resistant Clinical Isolates of Candida albicans

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.     

Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicans

To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([alpha-(32)P]8-azidoATP; IC(50)=0.76 microM) and drug-substrates ([(3)H]azidopine and [(125)I]iodoarylazidoprazosin; IC(50) approximately 12 microM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 microM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy.     

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.     

Membrane fluidity and lipid composition in clinical isolates of Candida albicans isolated from AIDS/HIV patients

In this study, we describe the membrane lipid composition of eight clinical isolates (azole resistant and sensitive strains) of Candida albicans isolated from AIDS/ HIV patients. Interestingly, fluorescence polarization measurements of the clinical isolates displayed enhanced membrane fluidity in fluconazole resistant strains as compared to the sensitive ones. The increase in fluidity was reflected in the change of membrane order, which was considerably decreased (decrease in fluorescence polarization "p" value denotes higher membrane fluidity) in the resistant strains. The ergosterol content in azole susceptible isolates was greater, almost twice as compared to the resistant isolates. However, no significant alteration was observed in phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of phosphatidylethanolamine exposed to the membrane's outer leaflet was higher in the resistant strains as compared to the sensitive strains, indicating increased floppase activity of the two major ABC drug efflux pumps, CDR1 and CDR2 possibly due to their overexpression in resistant strains. The results of the present study suggest that changes in the status of membrane lipid phase especially the ergosterol content and increased activity of drug efflux pumps by overexpression ofABC transporters, CDR1 and CDR2 might contribute to fluconazole resistance in C. albicans isolated from AIDS/HIV patients.     

Alanine scanning of all cysteines and construction of a functional cysteine-less Cdr1p, a multidrug ABC transporter of Candida albicans

Herein, we discuss the role of the native cysteines present in a major multidrug ABC transporter of Candida albicans, Cdr1p, and describe the construction of this transporter's functional cysteine-less (cysless) protein version for cross-linking studies. In the experiments in which all 23 cysteines were replaced individually, we observed that most of the cysteine replacements were tolerated by the protein, but the replacement of C1056, C1091, C1106, C1294 or C1336 resulted in an enhanced drug susceptibility together with an abrogated drug efflux. Notably, the ATPase activity was uncoupled, which largely remained unaffected in these variants. The substitution of the critical cysteines with serines restored the normal expression and functionality of Cdr1p because serine can effectively mimic the hydrogen bonding properties of cysteine. Finally, we constructed a functional cysless His-tagged Cdr1p in which all the cysteines of the native protein were replaced with alanines and the critical cysteines were replaced with serines. Notably, cysless GFP-tagged variant of Cdr1p was non-functional. The cysless His-tagged variant of Cdr1p is the first example of a cysless ABC transporter in yeast, and it will lead to a greater understanding of the architecture of this important protein and provide insight into the nature of drug binding and interdomain communication.     

Differential dynamics of membrane proteins in yeast

Lateral diffusion of lipids and proteins in yeast plasma membranes has been reported to be anomalously slow, and implicated as a possible reason for polarization in yeast. In order to gain insight into the observed slow diffusion in yeast membranes, we explored lateral diffusion of two proteins of different origin. We compared lateral dynamics of the Candida drug resistance protein-1 (Cdr1p), and the human serotonin_1A receptor (5-HT_1AR) by fluorescence recovery after photobleaching (FRAP). Our results show that while Cdr1p-GFP displays slow diffusion, the diffusion of 5-HT_1AR-EYFP is significantly faster. Interestingly, upon ergosterol depletion, the mobility of Cdr1p-GFP did not exhibit appreciable change, while 5-HT_1AR-EYFP mobility showed an increase. On the other hand, upon actin cytoskeleton destabilization, the mobile fraction of 5-HT_1AR-EYFP showed considerable increase, while the mobility of Cdr1p-GFP was not altered. Our results represent the first report on the dynamics of the important drug resistance protein Cdr1p and provide novel insight on diffusion of membrane proteins in yeast membranes.     

Candida glabrata ATP-binding cassette transporters Cdr1p and Pdh1p expressed in a Saccharomyces cerevisiae strain deficient in membrane transporters show phosphorylation-dependent pumping properties

The expression and drug efflux activity of the ATP binding cassette transporters Cdr1p and Pdh1p are thought to have contributed to the recent increase in the number of fungal infections caused by Candida glabrata. The function of these transporters and their pumping characteristics, however, remain ill defined. We have evaluated the function of Cdr1p and Pdh1p through their heterologous hyperexpression in a Saccharomyces cerevisiae strain deleted in seven major drug efflux transporters to minimize the background drug efflux activity. Although both Cdr1p- and Pdh1p-expressing strains CDR1-AD and PDH1-AD acquired multiple resistances to structurally unrelated compounds, CDR1-AD showed, in most cases, higher levels of resistance than PDH1-AD. CDR1-AD also showed greater rhodamine 6G efflux and resistance to pump inhibitors, although plasma membrane fractions had comparable NTPase activities. These results indicate that Cdr1p makes a larger contribution than Phd1p to the reduced susceptibility of C. glabrata to xenobiotics. Both pump proteins were phosphorylated in a glucose-dependent manner. Whereas the phosphorylation of Cdr1p affected its NTPase activity, the protein kinase A-mediated phosphorylation of Pdh1p, which was necessary for drug efflux, did not. This suggests that phosphorylation of Pdh1p may be required for efficient coupling of NTPase activity with drug efflux.     

Phosphorylation of candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability

Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae. Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration. Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress. The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation. Studies of pump function suggested conformational interaction between Ser(484) and Ser(307). An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain. Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant. Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.     

Functional characterization of Candida albicans ABC transporter Cdr1p

In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

The Candida albicans Cdr2p ATP-binding cassette (ABC) transporter confers resistance to caspofungin

Multidrug resistance may pose a serious problem to antifungal therapy. The Candida albicans Cdr2p is one of two ATP-binding cassette (ABC) transporters mediating antifungal resistance in vivo through increased drug efflux. Echinocandins such as caspofungin represent the newest class of antifungals that target cell wall synthesis. We show here by agar plate resistance assays that cross-resistant clinical isolates of C. albicans display high minimal inhibitory concentrations (MICs) to caspofungin when compared with a sensitive ATCC reference strain. Northern analysis and immunoblotting indicate that these isolates also show high levels of CDR1 and CDR2 expression. To determine a possible contribution of Cdr1p or Cdr2p to caspofungin resistance, we have functionally expressed Cdr1p and Cdr2p in appropriate recipient strains of the yeast Saccharomyces cerevisiae. Yeast cells expressing Cdr1p or Cdr2p exhibit cross-resistance to established antifungal drugs such as azoles and terbinafine. However, Cdr2p and, to a much lesser extent, Cdr1p confer caspofungin hyper-resistance when expressed in yeast. Likewise, Cdr2p confers caspofungin resistance when constitutively overexpressed in a drug-sensitive C. albicans strain. We therefore propose that Cdr2p may contribute to clinical candin resistance. Finally, our data suggest that cross-resistance phenotypes of clinical isolates are the consequence of distinct mechanisms that may operate simultaneously.     

Chimeras of Candida albicans Cdr1p and Cdr2p reveal features of pleiotropic drug resistance transporter structure and function

Members of the pleiotropic drug resistance (PDR) family of ATP binding cassette (ABC) transporters consist of two homologous halves, each containing a nucleotide binding domain (NBD) and a transmembrane domain (TMD). The PDR transporters efflux a variety of hydrophobic xenobiotics and despite the frequent association of their overexpression with the multidrug resistance of fungal pathogens, the transport mechanism of these transporters is poorly understood. Twenty-eight chimeric constructs between Candida albicans Cdr1p (CaCdr1p) and Cdr2p (CaCdr2p), two closely related but functionally distinguishable PDR transporters, were expressed in Saccharomyces cerevisiae. All chimeras expressed equally well, localized properly at the plasma membrane, retained their transport ability, but their substrate and inhibitor specificities differed significantly between individual constructs. A detailed characterization of these proteins revealed structural features that contribute to their substrate specificities and their transport mechanism. It appears that most transmembrane spans of CaCdr1p and CaCdr2p provide or affect multiple, probably overlapping, substrate and inhibitor binding site(s) similar to mammalian ABC transporters. The NBDs, in particular NBD1 and/or the ～150 amino acids N-terminal to NBD1, can also modulate the substrate specificities of CaCdr1p and CaCdr2p.     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p’s ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

A new function of isonitrile as an inhibitor of the Pdr5p multidrug ABC transporter in Saccharomyces cerevisiae

Pdr5p in Saccharomyces cerevisiae is a functional homologue of mammalian P-glycoprotein implicated in multidrug resistance (MDR). In order to obtain useful inhibitors to overcome MDR in clinical tumors, screening of Pdr5p inhibitors has been carried out. We isolated a fungal strain producing Pdr5p inhibitors using our original assay system, and it was classified as Trichoderma sp. P24-3. The purified inhibitor was identified as isonitrile, 3-(3'-isocyano-cyclopent-2'-enylidene)-propionic acid, a compound whose carboxyl residue is essential for the inhibitory activity. A non-toxic concentration of the isonitrile (41.5 microg/ml, 255 microM) inhibited Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. In addition, addition of the isonitrile led to accumulation of rhodamine 6G, a substrate of Pdr5p, in the Pdr5p-overexpressing cells. The inhibitory profiles of the isonitrile against S1360 mutants (S1360A and S1360F) of Pdr5p were different from those of FK506 and enniatin. The isonitrile did not influence PDR5 gene expression and the amount of Pdr5 protein, nor did it inhibit the function of Snq2p, a homologue of Pdr5p. Interestingly, the isonitrile inhibited the function of Cdr1p and Cdr2p, Pdr5p homologues in pathogenic yeast Candida albicans. Thus, it was found that the isonitrile shows a different inhibitory spectrum from that of FK506 and enniatin as a potent inhibitor for Pdr5p, Cdr1p, and Cdr2p.     

Functional characterization of N-terminal nucleotide binding domain (NBD-1) of a major ABC drug transporter Cdr1p of Candida albicans: uncommon but conserved Trp326 of Walker B is important for ATP binding

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.     

Cdr2p contributes to fluconazole resistance in Candida dubliniensis clinical isolates

The development of resistance to azole antifungals used in the treatment of fungal infections can be a serious medical problem. Here, we investigate the molecular mechanisms associated with reduced susceptibility to fluconazole in clinical isolates of Candida dubliniensis , showing evidence of the trailing growth phenomenon. The changes in membrane sterol composition were studied in the presence of subinhibitory fluconazole concentrations. Despite lanosterol and eburicol accumulating as the most prevalent sterols after fluconazole treatment, these ergosterol precursors still support growth of Candida isolates. The overexpression of ABC transporters was demonstrated by immunoblotting employing specific antibodies against Cdr1p and Cdr2p. The presence of a full-length 170?kDa protein Cdr1p was detected in two isolates, while a truncated form of Cdr1p with the molecular mass of 85?kDa was observed in isolate 966/3(2). Notably, Cdr2p was detected in this isolate, and the expression of this transporter was modulated by subinhibitory concentrations of fluconazole. These results suggest that C.?dubliniensis can display the trailing growth phenomenon, and such isolates express similar molecular mechanisms like that of fluconazole-resistant isolates and can therefore be associated with recurrent infections.     

Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux

The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.     

W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.