Allelic variants of ABC drug transporter Cdr1p in clinical isolates of Candida albicans

Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.     

Membrane fluidity affects functions of Cdr1p, a multidrug ABC transporter of Candida albicans

Earlier, we have shown that the overexpression of an ABC transporter, CDR1, is involved in the emergence of multidrug resistance in Candida albicans. In this study, we checked its function in vivo by expressing it in different isogenic Saccharomyces cerevisiae erg mutants, which accumulated various intermediates of the ergosterol biosynthesis and thus altered the membrane fluidity. Functions like the accumulation of rhodamine 123, beta-estradiol, fluconazole and floppase activity associated with Cdr1p were measured to ascertain their responses to an altered membrane phase. The floppase activity appeared to be favoured by an enhanced membrane fluidity, while the effluxing of substrates and Cdr1p's ability to confer multidrug resistance were significantly reduced. We demonstrate that only some of the functions of Cdr1p were affected by an altered lipid environment.     

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.     

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.     

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a d-octapeptide derivative inhibitor

Overexpression of the Candida albicans ATP‐binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ～ 1.89 × 10⁶ member d ‐octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4‐methoxy‐2,3,6‐trimethylbenzenesulphonyl derivative of the d ‐octapeptide d ‐NH₂‐FFKWQRRR‐CONH₂, as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization‐resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug‐like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3.     

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.     

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.     

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.     

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.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

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.     

白念珠菌铁通透酶基因CaFTH1的功能研究

[目的]白念珠菌CaFTH1是一种铁通透酶编码基因.为了研究CaFTH1对胞内铁代谢和液泡功能的影响,构建fth1△/△单基因缺失菌株和fth1△/△fet33△/△双基因缺失菌株.[方法]利用生物信息学软件对CaFTH1进行序列比对和分析;通过实时荧光定量PCR技术研究铁离子丰度对CaFTH1表达的影响;利用PCR介导的同源重组方法构建基因缺失菌株;利用原子吸收光谱方法测定基因缺失菌株胞内铁含量的变化,并对基因缺失菌株在缺铁条件和菌丝诱导条件下的生长状况进行研究;通过代谢转换实验,研究CaFTH1对细胞液泡功能的影响.[结果]序列比对结果表明白念珠菌CaFth1蛋白属于铁通透酶Ftr1超家族,与酿酒酵母液泡膜蛋白ScFth1具有最高的同源性.铁匮乏条件会诱导CaFTH1的表达,而富铁条件则会抑制其表达.白念珠菌CaFTH1的缺失会导致胞内铁含量的降低,fth1△/△突变菌株基础上CaFET33的缺失则会进一步降低胞内铁含量.在缺铁条件下,fth1△/△fet33△/△双基因缺失菌株在一定程度上表现出代谢转换能力的缺陷.另外,在某些固体菌丝诱导培养条件下,fth1△/△fet33△/△缺失菌株菌落表面形成褶皱能力显著增强;而在液体菌丝诱导条件下,则表现为增强的菌丝聚集能力.[结论]CaFTH1是一种低铁应答基因,在维持白念珠菌胞内铁离子稳态及液泡功能方面具有重要作用.CaFTH1和CaFET33基因的双缺失会对白念珠菌的菌落形态和菌丝聚集产生影响.     

Functional characterization of the Shigella dysenteriae heme ABC transporter

The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.     

The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter.

We report the cloning and functional analysis of a third member of the CDR gene family in Candida albicans, named CDR3. This gene codes for an ABC (ATP-binding cassette) transporter of 1,501 amino acids highly homologous to Cdr1p and Cdr2p (56 and 55% amino acid sequence identity, respectively), two transporters involved in fluconazole resistance in C. albicans. The predicted structure of Cdr3p is typical of the PDR/CDR family, with two similar halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six predicted transmembrane segments. Northern analysis showed that CDR3 expression is regulated in a cell-type-specific manner, with low levels of CDR3 mRNA in CAI4 yeast and hyphal cells, high levels in WO-1 opaque cells, and undetectable levels in WO-1 white cells. Disruption of both alleles of CDR3 in CAI4 resulted in no obvious changes in cell morphology, growth rate, or susceptibility to fluconazole. Overexpression of Cdr3p in C. albicans did not result in increased cellular resistance to fluconazole, cycloheximide, and 4-nitroquinoline-N-oxide, which are known substrates for different transporters of the PDR/CDR family. These results indicate that despite a high degree of sequence conservation with C. albicans Cdr1p and Cdr2p, Cdr3p does not appear to be involved in drug resistance, at least to the compounds tested which include the clinically relevant antifungal agent fluconazole. Rather, the high level of Cdr3p expression in WO-1 opaque cells suggests an opaque-phase-associated biological function which remains to be identified.     

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.