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Keywords:

  • concentrative nucleoside transporter;
  • Candida albicans;
  • CaCNT;
  • purine nucleoside;
  • drug transport;
  • Xenopus laevis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References

Human and other mammalian concentrative (Na+-linked) nucleoside transport proteins belong to a membrane protein family (CNT, TC 2.A.41) that also includes Escherichia coli H+-dependent nucleoside transport protein NupC. Here, we report the cDNA cloning and functional characterization of a CNT family member from the pathogenic yeast Candida albicans. This 608 amino acid residue H+/nucleoside symporter, designated CaCNT, contains 13 predicted transmembrane domains (TMs), but lacks the exofacial, glycosylated carboxyl-terminus of its mammalian counterparts. When produced in Xenopus oocytes, CaCNT exhibited transport activity for adenosine, uridine, inosine and guanosine but not cytidine, thymidine or the nucleobase hypoxanthine. Apparent Km values were in the range 16–64 µM, with Vmax : Km ratios of 0.58–1.31. CaCNT also accepted purine and uridine analogue nucleoside drugs as permeants, including cordycepin (3′-deoxyadenosine), a nucleoside analogue with anti-fungal activity. Electrophysiological measurements under voltage clamp conditions gave a H+ to [14C]uridine coupling ratio of 1 : 1. CaCNT, obtained from logarithmically growing cells, is the first described cation-coupled nucleoside transporter in yeast, and the first member of the CNT family of proteins to be characterized from a unicellular eukaryotic organism. Copyright © 2003 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References

Nucleoside transporters (NTs) are specialized integral membrane proteins that mediate cellular uptake and release of nucleosides and nucleoside analogue drugs (Griffith and Jarvis, 1996; Mackey et al., 1998). In higher organisms (humans and rodents), nucleoside transport is mediated by members of the ENT (equilibrative, Na+-independent) and CNT (concentrative, Na+-dependent) protein families (TC 2.A.57 and 2.A.41, respectively) (Baldwin et al., 1999; Cheeseman et al., 2000; Hyde et al., 2001). Two ENT and three CNT functional isoforms have been identified. Human (h) and rat (r) ENT1 and ENT2 transport pyrimidine and purine nucleosides and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs, and by the ability of hENT2 and rENT2 to also transport nucleobases (Griffiths et al., 1997a, 1997b; Yao et al., 1997; Crawford et al., 1998; Yao et al., 2002a). CNT1 and CNT2 both transport uridine and adenosine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (Huang et al., 1994; Che et al., 1995, Yao et al., 1996a; Wang et al., 1997; Ritzel et al., 1997, 1998). hCNT3, its mouse (m) orthologue mCNT3 and a close relative from Eptatretus stouti, an ancient marine pre-vertebrate, transport both pyrimidine and purine nucleosides (Ritzel et al., 2001; Yao et al., 2002b). The relationships of these proteins to transport processes defined by functional studies are: ENT1 (es), ENT2 (ei), CNT1 (cit), CNT2 (cif) and CNT3 (cib).

Although ENTs are widely distributed in lower eukaryotes, they appear to be absent from prokaryotes. A number of ENT family members have recently been identified and functionally characterized from parasitic protozoa, including TgAT from Toxoplasma gondii (Chiang et al., 1999), the P1- and P2-type transporters TbNT2 and TbAT1 from Trypanosoma brucei (Maser et al., 1999; Sanchez et al., 1999), LdNT1.1 from Leishmania donovani (Vasudevan et al., 1998) and PfENT1 from Plasmodium falciparum (Carter et al., 2000; Parker et al., 2000). In contrast to their mammalian counterparts, at least some of the protozoan ENT family members appear to function as active transporters, catalysing the symport of nucleosides with protons (de Koning and Diallinas, 2000; Carter et al., 2000). PfENT1, like human and rat ENT2, also functions as a nucleobase transporter (Parker et al., 2000). Unlike ENTs, CNTs are present in both eukaryotes and prokaryotes. CNTs from lower eukaryotes and prokaryotes that have been characterized functionally include CeCNT3 from Caenorhabditis elegans (Xiao et al., 2001) and NupC from Escherichia coli (Craig et al., 1994; Loewen et al., 2003). Both use protons as the coupling cation.

In yeast, most functional studies of nucleoside transport have focused on Saccharomyces cerevisiae, and little information is available on pathogenic species such as Candida albicans (Horak, 1997). At the molecular level, two different S. cerevisiae NTs have been identified and characterized (Vickers et al., 2000). FUI1, a member the nucleobase : cation symporter-1 (NCS1) family of transporters (TC 2.A.39), exhibits high selectivity for uracil-containing ribonucleosides and imports uridine across cell-surface membranes. FUN26, a member of the ENT protein family, has a broad nucleoside selectivity and most probably functions to transport nucleosides across intracellular vacuolar membranes. FUN26 mRNA is most abundant during M phase of the cell cycle (Spellman et al., 1998), suggesting a possible role in vacuolar release of nucleosides for nucleic acid synthesis during cell division. As is also the case in parasitic protozoa, no CNTs are present in the S. cerevisiae genome.

In C. albicans, nucleoside transport is complex. Although only one C. albicans NT (NUP), a member of the NUP protein family (Detke, 1998), has been characterized so far, BLAST searches of the Stanford C. albicans genome sequence databank revealed at least four more putative NT proteins of which one (derived from Contig6-1709 and Contig6-2474) shows sequence similarity to the CNTs. In this report, we describe the molecular cloning of the cDNA encoding this C. albicans CNT, designated CaCNT, and its heterologous expression in oocytes of Xenopus laevis. CaCNT was shown to mediate H+-coupled influx of physiological purine nucleosides and uridine, as well as various cytotoxic nucleoside analogues, including cordycepin, which has potent antifungal activity in preclinical model systems when used in combination with an inhibitor of adenosine deaminase (Sugar and McCaffrey, 1998).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References

Yeast exhibit marked differences in nucleoside transport capability, e.g. S. cerevisiae transports only uridine (Horak, 1997), whereas the opportunistic pathogen C. albicans transports both pyrimidine and purine nucleosides (Rao et al., 1983; Fasoli and Kerridge, 1990). Two NT proteins, FUI1 and FUN26, have been identified in S. cerevisiae (Vickers et al., 2000) and characterized functionally by reintroduction into NT-deficient S. cerevisiae (FUI1) or production in oocytes of Xenopus laevis (FUN26). FUI1 corresponds to the uridine-specific NT present in the S. cerevisiae plasma membrane and belongs to the nucleobase : cation symporter-1 (NCS1) family. FUN26 is a member of the ENT protein family and resides mostly in intracellular membranes of S. cerevisiae. In Xenopus oocytes, sufficient recombinant FUN26 reached the plasma membrane to demonstrate that it functions as a broadly selective transporter for pyrimidine and purine nucleosides (Vickers et al., 2000). Based upon functional studies of native transporters in intact yeast (Losson et al., 1978), FUI1 may be H+-coupled, although this has not been demonstrated for the recombinant protein. Recombinant FUN26 is not H+-dependent (Vickers et al., 2000). The one NT protein characterized so far in C. albicans, NUP, is unrelated to FUI1 or FUN26 and is a member of the NUP transporter family (Detke, 1998). Characterized functionally in transformed S. cerevisiae, NUP transports purine nucleosides and perhaps thymidine, but not uridine (Detke, 1998). There is no information available on cation coupling for NUP.

We undertook BLAST searches of the C. albicans genomic database to search for other putative NT proteins. The analysis revealed C. albicans orthologues of FUI1 and FUN26, as well as a protein of 378 amino acid residues with 56% sequence identity to NUP. A fourth putative NT (derived from Contig6-1709 and Contig6-2474) showed sequence similarity to mammalian CNT proteins. Here, we describe the molecular and transport properties of this new C. albicans CNT.

C. albicans contains a mammalian/bacterial CNT homologue

The sense and antisense oligonucleotide primers described in Experimental procedures were designed to encompass the open reading frame of the full-length CNT gene in C. albicans Contig6-1709 (Stanford C. albicans genome sequence database). While no PCR product was obtained from cells from stationary cultures, PCR amplification of C. albicans cDNA from logarithmically growing cells generated a cDNA of the correct size (1827 bp). This product was subcloned into the enhanced Xenopus expression vector pGEM-HE and sequenced. The encoded 608 amino acid residue protein, designated CaCNT (GenBank™ Accession No. AY235425) (Figure 1A), contained 13 predicted transmembrane helices (TMs) and had a putative molecular weight of 67.7 kDa. CaCNT was 33% identical (44% similar) to hCNT1, 34% identical (46% similar) to hCNT2, 38% identical (49% similar) to hCNT3, and 26% identical (37% similar) to the bacterial H+/nucleoside transporter NupC, the latter protein having only 10 predicted TMs (corresponding to TMs 4–13 of the other CNTs) (Hamilton et al., 2001; Yao et al., 2002a). The 13-TM membrane architecture of CaCNT is also predicted for C. elegans CeCNT3 (Hamilton et al., 2001) and suggests that this membrane topology may be common to all eukaryotic CNTs.

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Figure 1. CaCNT is a member of the CNT family of nucleoside transporters. (A) Alignment of the predicted amino acid sequences of CaCNT (GenBank™ Accession No. AY235425), hCNT1 (human CNT1, GenBank™ Accession No. U62967), hCNT2 (human CNT2, GenBank™ Accession No. AF036109), and hCNT3 (human CNT3, GenBank™ Accession No. AF305210), and NupC (Escherichia coli, GenBank™ Accession No. X74825) using the GCG PILEUP program. Potential membrane-spanning α-helices, identified as described previously (Hamilton et al., 2001), are numbered. Putative glycosylation sites in predicted extracellular domains of hCNT1, hCNT2 and hCNT3 are shown in lowercase (n), and their positions are highlighted by an asterisk above the aligned sequences. Residues identical in CaCNT and one or more of the other transporters are indicated by black boxes. (B) Differences in deduced amino acid sequences of CaCNT and those derived from Contig6-1704 and Contig6-2474 (Stanford Genome Technology Center database). Nucleotide sequences encoding each amino acid residue are provided in parentheses. Amino acid residues and nucleotides common to CaCNT and one or both contigs are highlighted in bold. A possible strain difference or PCR-induced mutation is boxed. (C) Phylogenetic tree showing relationships between CaCNT and other functionally characterized members of the CNT transporter family. In addition to those listed in (A), these are: rCNT1 (rat CNT1, GenBank™ Accession No. U10279); pkCNT1 (pig kidney CNT1, GenBank™ Accession No. AF009673); rCNT2 (rat CNT2, GenBank™ Accession No. U25055); mCNT2 (mouse CNT2, GenBank™ Accession No. AF079853); rbCNT2 (rabbit CNT2, GenBank™ Accession No. AF161716); hfCNT (hagfish CNT, GenBank™ Accession No. AF036109), mCNT3 (mouse CNT3, GenBank™ Accession No. AF305211) and rCNT3 (rat CNT3, GenBank™ Accession No. AY059414); and CeCNT3 (also known as F27E11.2, Caenorhabditis elegans, GenBank™ Accession No. AF016413). The phylogenetic tree was constructed from a multiple alignment of the 14 CNT sequences using ClustalX, version 1.81, for Windows (Thompson et al., 1997) and KITSCH, PHYLIP, version 3.57c, software (Felsenstein, 1989)

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The nucleotide and deduced amino acid sequences of CaCNT were nearly identical to those of the open reading frame (ORF) of Contig6-1709 and to an incomplete CaCNT ORF corresponding to CaCNT amino acid residues 120–608 in Contig6-2474. Multiple sequence alignments revealed 10 single nucleotide differences between CaCNT and either Contig6-1709 or Contig6-2474, resulting in five single residue differences in predicted amino acid sequence at residues 328, 416, 418, 483 and 506 (Figure 1B). At each of the five positions, CaCNT was identical to one or other of the two contigs. One of the five positions, residue 328 in CaCNT, corresponds to a critical amino acid residue in TM 7 of mammalian CNTs, identified by chimeric and mutagenesis studies to be involved in the selectivity of human CNT1/2 for pyrimidine and purine nucleosides (Loewen et al., 1999). The presence of Gly at residue 328 in CaCNT compared to Ser319 in hCNT1 and Gly313 in hCNT2 is predictive of purine nucleoside selectivity (Loewen et al., 1999). The codon of CaCNT residue 417 contained the only nucleoside sequence difference unique to CaCNT and not found in either contig (codon GAG in CaCNT vs. codon GAA in Contig6-1709 and Contig6-2474). Both codons code for Glu. The most recent Stanford reconstruction of the C. albicans diploid genome (Assembly 19, May 2002) identifies the Contig6-1709/2474 sequence differences in Figure 1B as polymorphic in origin. Corresponding differences between these contigs and the nucleotide and deduced amino acid sequences of our protein provides further evidence of allelic heterozygosity within the CaCNT gene.

CaCNT lacked the lengthy carboxyl-terminal tail containing multiple consensus sites for N-linked glycosylation found in its human counterparts. The extracellular location of the carboxyl-terminus has been confirmed by mutagenesis of rCNT1, which is glycosylated at Asn605 and Asn643 (Hamilton et al., 2001). Despite its extracellular location, the carboxyl-terminal tail of hCNT1-3 contains multiple conserved Cys residues, suggesting possible involvement in intramolecular or intermolecular disulphide linkages. The absence of this carboxyl-terminal domain from CaCNT and NupC (Hamilton et al., 2001) indicates that such linkages, if they occur, are not required for CNT functional activity. Sequence similarity between CaCNT and human CNTs was most pronounced in TMs 4–9 and 11–12, with an average sequence identity of 52% (61% similar) within these transmembrane helices. In contrast, N-terminal domains, including TMs 1–3, were markedly more divergent. Truncated constructs of human and rat CNT1 with TMs 1–3 removed have been shown to maintain functionality, identifying the TM 4–13 region (TMs 1–10 in NupC) as the core functional unit of the CNT family of proteins (Hamilton et al., 2001).

Since we first cloned rCNT1 from rat jejunum by expression selection in Xenopus oocytes (Huang et al., 1994), more than 80 members of the CNT protein family have been identified by cDNA cloning and genome sequencing projects. At present, 14 CNT proteins have been characterized functionally, and their phylogenetic relationships are illustrated in Figure 1C. CaCNT is positioned on a separate branch distinct from E. coli NupC, C. elegans CeCNT3, E. stouti hfCNT and the CNT members from mammals. Sequence homology searches of incomplete fungi genome databases with CaCNT identified one other putative CNT nucleoside transport protein in Aspergillus fumigatus (derived from TIGR_5085, GenBank™ database). The predicted ORF of the A. fumigatus CNT exhibited 54% sequence identity (65% similarity) with CaCNT. In comparison, CaCNT shares only 32% sequence identity with CeCNT3, suggesting that yeast CNT members may comprise a separate CNT subfamily, separate from CNTs from prokaryotes and other eukaryotes.

Functional production of recombinant CaCNT in Xenopus oocytes

A representative time course of [14C]uridine uptake (20 µM, 20 °C) measured in acidified NaCl transport medium at pH 5.5 in oocytes injected with CaCNT RNA transcripts or water alone is shown in Figure 2. Uptake in CaCNT-producing oocytes was linear with time for 60 min. After 30 min, the uridine flux was 45.1 ± 5.9 pmol/oocyte, which was 52-fold higher than that of control water-injected oocytes (0.87 ± 0.15 pmol/oocyte). The latter value was similar to that observed in choline chloride transport medium at pH 5.5, consistent with CaCNT functioning as a H+/nucleoside symporter (data not shown). Subsequent kinetic experiments to determine apparent Km and Vmax values for different CaCNT permeants used a 5 min uptake interval to measure initial rates of nucleoside uptake (influx). In some experiments, radioisotope studies of CaCNT nucleoside and nucleoside drug specificity were performed using a 30 min uptake interval to maximize detection of weakly transported CaCNT permeants.

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Figure 2. Time course of uridine uptake by recombinant CaCNT produced in Xenopus oocytes. Oocytes injected with 20 nl water containing 20 ng CaCNT RNA transcript were incubated for 3 days at 18 °C in MBM (modified Barth's medium). Uptake of uridine (20 µM, 20 °C) was then measured in transport medium comprising 100 mM NaCl, pH 5.5 (solid circles), and compared with uptake in the same medium by control oocytes injected with 20 nl water alone (open circles). Each value is the mean ± S.E. of 10–12 oocytes. Error bars are not shown where S.E. values were smaller than those represented by the symbols

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Permeant selectivity of recombinant CaCNT

Figure 3A shows a representative transport experiment in Xenopus oocytes that measured CaCNT-mediated uptake of a panel of radiolabelled pyrimidine and purine nucleosides (20 µM, 20 °C) in NaCl medium at pH 5.5. CaCNT-mediated transport was calculated as uptake in RNA transcript-injected oocytes minus uptake in control water-injected oocytes. CaCNT-producing oocytes transported all purine nucleosides tested (adenosine, inosine, guanosine). Although uridine was also a good CaCNT permeant, no significant transport was detected for other pyrimidine nucleosides (cytidine, thymidine), suggesting a cif-like transport profile similar to that of hCNT2 and rCNT2 (Che et al., 1995; Yao et al., 1996a; Wang et al., 1997; Ritzel et al., 1998). The purine nucleobase hypoxanthine was also not transported, suggesting that CaCNT functions exclusively as a nucleoside transport protein. Lowering the extracellular H+ concentration by increasing the transport medium pH from 5.5 to 8.5 reduced the uridine uptake by 79%.

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Figure 3. Permeant selectivity and drug transport by CaCNT. (A) CaCNT-mediated nucleoside (uridine, adenosine, inosine, guanosine, cytidine and thymidine) and nucleobase (hypoxanthine) influx (20 µM, 20 °C, 30 min) was measured in transport medium comprising 100 mM NaCl, pH 5.5 (solid bars), or 100 mM NaCl, pH 8.5 (hatched bars). (B) CaCNT-mediated fluxes of nucleosides (uridine, inosine) (solid bars) and nucleoside drugs (5-FUrd, 5-fluorouridine; 5-F-2′dUrd, 5-fluoro-2′-deoxyuridine; zebularine; ddI; cordycepin; fludarabine; and cladribine) (shaded bars) (20 µM, 20 °C, 30 min) were measured in the transport medium, comprising 100 mM NaCl at pH 5.5. Mediated transport in (A) and (B) was calculated as uptake in RNA-injected oocytes minus uptake in oocytes injected with water alone. Each value represents mean ± S.E. of 10–12 oocytes

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Kinetic properties

Figure 4 shows representative concentration dependence curves in NaCl medium at pH 5.5 for uridine, adenosine, guanosine, and inosine influx in CaCNT-producing oocytes, compared to corresponding control fluxes in water-injected oocytes. Kinetic parameters (apparent Km and Vmax) derived from the CaCNT-mediated transport data, which were consistent with simple Michaelis–Menten kinetics, are presented in Table 1. Apparent Km values varied between 16 and 64 µM (adenosine < uridine < inosine, guanosine) and were in the same range as values obtained previously for recombinant mammalian CNT proteins (Huang et al., 1994; Che et al., 1995; Yao et al., 1996a; Ritzel et al., 1997; Wang et al., 1997; Ritzel et al., 1998, 2001). Vmax values varied between 9.6 and 43.3 pmol/oocyte/5 min (adenosine < uridine, inosine, guanosine), giving calculated Vmax : Km ratios, a measure of transport efficiency, of 1.31 (uridine), 0.61 (adenosine), 0.58 (guanosine) and 0.69 (inosine). Therefore, at low permeant concentrations below the apparent Km values, the different physiological CaCNT permeants, including adenosine, were transported at similar rates. The lower apparent Km value for adenosine transport (15.7 µM) vs. other nucleosides (33.0–64.3 µM) was consistent with adenosine having the smallest mediated flux in Figure 3A (measured at a concentration of 20 µM). Since NUP is also purine nucleoside-selective (Detke, 1998), C. albicans has at least two pathways for adenosine influx. Adenosine is a biologically important molecule in C. albicans and, through cyclic AMP and other adenosine-related metabolites, has the potential to influence the dimorphic yeast–mycelium transition in C. albicans (Sabie and Gadd, 1992). In contrast to the saturable nature of CaCNT-mediated transport of adenosine and the other nucleosides tested in Figure 4, basal nucleoside influx in water-injected oocytes exhibited a linear concentration dependence, consistent with simple diffusion across oocyte plasma membrane.

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Figure 4. Kinetic properties of CaCNT. Initial rates of (A) adenosine, (B) uridine, (C) inosine and (D) guanosine uptake (5 min fluxes, 20 °C) in oocytes injected with RNA transcripts (solid circles) or water alone (open circles) were measured in transport medium comprising 100 mM NaCl at pH 5.5. Kinetic parameters calculated from the mediated component of transport (uptake in RNA-injected oocytes minus uptake in oocytes injected with water alone) are presented in Table 1. Each value is the mean ± S.E. of 10–12 oocytes. Error bars are not shown where S.E. values were smaller than those represented by the symbols

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Table 1. Kinetic parameters of CaCNT-mediated nucleoside influx
SubstrateApparent KmaM)Vmaxa(pmol/oocyte/ 5 min)Ratio Vmax : Km
  • a

    From Figure 4.

Adenosine15.7 ± 3.29.6 ± 0.60.61
Uridine33.0 ± 5.443.3 ± 2.71.31
Inosine57.4 ± 7.639.7 ± 2.30.69
Guanosine64.3 ± 5.637.8 ± 1.50.58

Nucleoside drug transport by recombinant CaCNT

Candida albicans infections can be successfully treated with polyene antibiotics, azole derivatives or 5-fluorocytosine, but continued emergence of drug-resistant strains of pathogenic yeast has prompted the search for new drug targets in the design of novel antifungal agents (St Georgiev, 2000). Nucleoside-based therapeutics offer one such alternative, e.g. cordycepin (3′-deoxyadenosine), when combined with adenosine deaminase inhibitors (coformycin or deoxycoformycin), has potent antifungal activity against invasive candidiasis from normal and fluconazole-resistant Candida isolates in mice (Sugar and McCaffrey, 1998). Because most nucleoside analogue drugs cannot easily cross cell membranes by simple diffusion, transportability by NT-mediated processes is a potential determinant of cytotoxic efficacy of such compounds.

Previously, we have used Xenopus oocyte expression to establish that the mammalian CNT1/2/3 proteins transport a broad spectrum of antiviral and anticancer nucleoside analogues (Huang et al., 1995; Yao et al., 1996b; Ritzel et al., 1997, 1998, 2001; Mackey et al., 1999; unpublished observation). hCNT1 and rCNT1, for instance, transport the antiviral drugs 3′-azido-3′-deoxythymidine (AZT) and 2′3′-dideoxycytidine (ddC), but not 2′3′-dideoxyinosine (ddI). hCNT2 transports only ddI, while hCNT3 and mCNT3 transport all three analogues. Purine (cladribine, fludarabine) and pyrimidine (5-fluorouridine, 5-fluoro-2′-deoxyuridine, zebularine, gemcitabine) nucleoside drugs, some of which are used in anticancer therapy, have also been shown to be CNT permeants. We therefore undertook experiments in Xenopus oocytes to measure CaCNT-mediated uptake of a panel of radiolabelled pyrimidine and purine nucleoside analog drugs. Uptake (20 µM, 20 °C) was measured in NaCl medium at pH 5.5 and compared with fluxes of uridine and inosine in the same batch of CaCNT-producing oocytes. As shown in Figure 3B, CaCNT accepted purine nucleosides (cordycepin, ddI, fludarabine, cladribine) and uridine analogues (5-fluorouridine, 5-fluoro-2′-deoxyuridine, zebularine) as permeants. Fluxes were lowest for cordycepin, fludarabine and cladribine, and highest for 5-fluorouridine and 5-fluoro-2′-deoxyuridine.

With the exception of 5-fluorouridine, the measured fluxes were smaller than those for uridine and inosine, and similar to those reported previously for hCNT2-mediated transport of ddI, 5-fluorouridine, fludarabine and cladribine in oocytes and transfected mammalian cells (Ritzel et al., 1998; Lang et al., 2001). CaCNT was, therefore, relatively tolerant of substitutions at the 2′ (5-fluoro-2′-deoxyuridine, cladribine, fludarabine) and 3′ (cordycepin, ddI) positions of the sugar, with significant import at micromolar concentrations. Substitution of the 5-H group (5-fluorouridine and 5-fluoro-2′-deoxyuridine) of the pyrimidine nucleobase moiety did not significantly affect uptake, although lack of the 4-OH group (zebularine) or the presence of halogens at the 2-position (cladribine, fludarabine) of the purine nucleobase moiety may have contributed to reduced transport activity. The demonstration that cordycepin is a low-level CaCNT permeant suggests that CaCNT may play a role in cytotoxic action of this compound against C. albicans. As with the parent nucleoside adenosine and some other nucleoside drugs such as AZT (Yao et al., 1996a, 1996b), there was also substantial diffusive entry of cordycepin into oocytes. In the experiment shown in Figure 3B, the control (non-mediated) flux in water-injected oocytes was 1.7 ± 0.2 pmol/oocyte/30 min compared with 3.6 ± 0.7 pmol/oocyte/30 min in RNA transcript-injected oocytes, giving a mediated flux of 1.9 ± 0.8 pmol/oocyte/30 min. The extent of the mediated uptake of cordycepin in C. albicans will depend on: (a) the level of the cell-surface abundance of CaCNT, and (b) contributions from other C. albicans NTs.

CaCNT H+ : nucleoside cotransport

C. albicans are dimorphic fungi that can exist in either yeast or hyphal forms, with the latter implicated in the virulence of systemic yeast infections (Odds, 1994). A number of environmental factors, including ambient pH, play a part in the transition between the two forms (Odds, 1985; Gow, 1997). Alkaline pH, for instance, promotes germ tube formation, whereas acidic pH encourages yeast growth (Buchan and Gow, 1991). In yeast, protons are likely to be the preferred coupling ion for nutrient transport, since a H+ electrochemical gradient is maintained by plasma membrane H+/ATPase (Monk et al., 1991). In C. albicans, proton-pump inhibitors have been shown to inhibit germ tube formation, favouring instead a period of extended yeast growth (Biswas et al., 2001), presumably by reducing the alkalization of intracellular pH that normally precedes the yeast–hyphal transition (Kaur et al., 1988; Kaur and Mishra, 1991a). More extensive blockage of H+/ATPase activity leads to cell death (Manavathu et al., 1999). The presence of either native or environmental inwardly-directed H+ gradients are required to drive the H+-coupled nutrient transport systems that, in part, are necessary to support the accelerated growth and replication rates of C. albicans in its yeast form.

As shown in Figure 5A, external application of pyrimidine and purine nucleosides (100 µM, Na+-containing medium, pH 5.5) to CaCNT-producing oocytes generated inward currents of 7–26 nA for adenosine, guanosine, inosine and uridine, but not for thymidine or cytidine, consistent with the nucleoside-selectivity profile presented in Figure 3A. No currents were seen in water-injected oocytes in transport medium of the same composition. In addition, there were no differences between uridine-induced currents measured in Na+-containing or Na+-free choline chloride media over the pH range 5.5–8.5 (Figure 5B). Similarly, there was no effect when Li+ was substituted for Na+ (data not shown). Inward currents increased markedly as pH was lowered from 8.5 to 5.5, mirroring the pH dependence of [14C]uridine uptake seen in Figure 3A. Together, these findings established that CaCNT functions as a Na+-independent electrogenic H+/nucleoside symporter. The results of Na+ replacement and pH-dependence experiments with C. elegans CeCNT3 (Xiao et al., 2001) and E. coli NupC (Loewen et al., 2003) suggest that these transporters are also strictly H+-dependent, which may represent a common characteristic of CNT proteins found in prokaryotes, as well as in yeast and other lower eukaryotes. In contrast, mammalian CNTs function predominantly as Na+-coupled nucleoside transporters, although recent electrophysiological studies in Xenopus oocytes have found that H+ and Li+ can substitute for Na+ for CNT3, but not for CNT1 or CNT2 (unpublished observation). Therefore, the CNT family includes members that are H+-dependent (CaCNT, CeCNT3, NupC), Na+-dependent (CNT1, CNT2) and Na+/H+ (and Li+)-dependent (CNT3). It is not uncommon for cation preference to vary within a single transporter family (Reizer et al., 1994), e.g. the melibiose transporter (MelB) of E. coli mediates uphill transport of melibiose coupled to Na+ or H+ (Botfield et al., 1990), while that of Klebsiella pneumoniae couples sugar transport to H+ and Li+ (Hama et al., 1992). Similarly, mammalian SGLT1 and SGLT3 can utilize H+ or Li+ in addition to Na+ as the driving force for sugar transport, whereas SGLT2 is Na+-specific (Wright, 2001).

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Figure 5. Proton currents induced by exposure of recombinant CaCNT to nucleoside permeants. (A) Current traces of a representative voltage-clamped CaCNT-producing oocyte (upper panel) or water-injected oocyte (lower panel) perfused at room temperature with NaCl transport medium at pH 5.5 containing different pyrimidine and purine nucleosides (100 µM). A downward deflection of the current trace signifies an inward movement of positively-charged molecules. Nucleosides remain uncharged at pH 5.5. (B) Uridine-evoked maximal currents generated by CaCNT-producing oocytes in transport medium containing either 100 mM NaCl (black bars) or 100 mM choline chloride (open bars) at pH 5.5, 6.5, 7.5 or 8.5. Each value is the mean ± S.E. of six data sets produced from six individual oocytes tested in both Na+-containing and Na+-restricted media over the pH range 5.5–8.5

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A Na+/nucleoside coupling ratio of 2 : 1 has been reported for system cib in choroid plexus and microglia (Wu et al., 1992; Hong et al., 2000), whereas coupling ratios of 1 : 1 have been described for various cit and cif transport activities in different mammalian cells and tissues (reviewed in Cass, 1995). Similarly, Hill coefficients for Na+-activation of radiolabelled adenosine and uridine transport by recombinant CNTs in Xenopus oocytes were 2 for hCNT3 and mCNT3, and 1 for rCNT1 (Yao et al., 1996b; Ritzel et al., 2001). In the present study, we directly determined the H+/nucleoside coupling ratio of CaCNT by simultaneous measurement of H+ currents and [14C]uridine influx under voltage clamp conditions, as described previously for the SDCT1 rat kidney dicarboxylate transporter (Chen et al., 1998). The results of the experiments presented in Figure 6 demonstrated that CaCNT has a H+ : nucleoside coupling ratio of 1 : 1 (the slope of the regression line ± S.E. is 1.03 ± 0.02). Using the same technique, Na+ : nucleoside coupling ratios of 2 : 1 and 1 : 1 have been confirmed for hCNT3 and hCNT1, respectively (unpublished observation).

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Figure 6. Stoichiometry of H+/uridine cotransport by recombinant CaCNT. Uridine-dependent charge and [14C]-uridine uptake were simultaneously determined at Vm = −50 mV in the presence of a proton gradient for 3 min. Integration of the uridine-evoked inward current with time was used to calculate the net cation influx by converting picocoulombs to picomoles using the Faraday constant. Mediated [14C]-uridine uptake was calculated as uptake in CaCNT-producing oocytes minus uptake in water-injected oocytes. Each data point represents a single oocyte

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References

BLAST searches suggested that the C. albicans genome encodes putative NTs from at least four different protein families: (a) CaCNT from the CNT family, the topic of the present study; (b) a homologue of S. cerevisiae FUN26 from the ENT family; (c) a homologue of S. cerevisiae FUI1 from the uracil/allantoin transporter family; and (d) NUP and a NUP-related protein from the NUP transporter family. The S. cerevisiae genome, which was fully sequenced in 1996 (Goffeau et al., 1996), does not contain CNT or NUP representatives.

We obtained a PCR-amplified product corresponding to CaCNT from C. albicans logarithmically growing, but not stationary phase, cells, suggesting that expression of the CaCNT gene may be differentially regulated in the two fungal forms. The characteristics of glucose transport (Cho et al., 1994) and neutral and cationic amino acid transport (Kaur and Mishra, 1991b) also differ between the two fungal forms. Certain growth media may also contribute to differential levels of transporter activity in the plasma membrane (Horak, 1997). Unlike intracellular S. cerevisiae FUN26, which displayed weak (∼three-fold above background) nucleoside transport activity in Xenopus oocytes, recombinant CaCNT mediated large fluxes, consistent with its function as a plasma membrane nucleoside transporter.

Recombinant CaCNT produced in oocytes was electrogenic, H+-dependent and Na+- and Li+-independent. The H+ : nucleoside coupling stoichiometry was demonstrated to be 1 : 1. None of the other identified fungal NTs (NUP, FUI1 and FUN26) have been demonstrated to be cation-coupled, making CaCNT the first described active nucleoside transporter of fungi. Although CNT proteins are widely distributed in eukaryotes (mammals, fish, insects and nematodes) and prokaryotes (Gram-negative and Gram-positive bacteria), CaCNT is the first CNT family member to be identified in fungi. CaCNT mediated high-affinity transport of purine nucleosides and, unlike C. albicans NUP, also transported uridine. The transport profile of CaCNT is therefore similar to the cif-type (CNT2) transport processes characteristic of mammalian cells. Like mammalian CNTs, CaCNT also transported nucleoside analogue drugs with antiviral and anticancer activities. The present studies also demonstrated low but significant uptake of the antifungal nucleoside analogue cordycepin. While the level of drug transport was low, the results demonstrated the potential involvement of CaCNT in antifungal nucleoside drug uptake. Future structure–function studies of recombinant CaCNT produced in Xenopus laevis oocytes therefore have the potential to identify new compounds with greater transportability and hence greater activity as antifungal agents. Since CaCNT is expected to be present in the replicative form of C. albicans, CaCNT represents a potential drug target for new antifungal pharmacologic therapies, either by development of CaCNT inhibitors that might impact growth or, as illustrated by cordycepin, by its utilization as a cellular uptake mechanism for antifungal nucleoside drugs. Infectious complications due to Candida species are frequent in the clinical care of a variety of immunocompromised individuals, such as organ transplant recipients, AIDS and cancer patients and the elderly (Walsh and Groll, 1999; Garber, 2001). Some human antineoplastic and antiviral nucleoside drugs have ancillary benefits as antibacterial agents (Friedman, 1982; Keith et al., 1989; Monno et al., 1997) and the same may also be possible in the case of fungal infections.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References

Molecular cloning of CaCNT

BLAST searches of the C. albicans genome database (Stanford Genome Technology Center) found an 1827 bp sequence from Contig6-1709 with 33% and 26% identity to hCNT1 and E. coli NupC, respectively. PCR was performed on C. albicans cDNA obtained from stationary and logarithmic growth phases (Library-in-a-Tube™, BIO 101), using oligonucleotides flanking the ORF of the C. albicans genomic CNT sequence: 5′-ATGGTTTCTCCGTCCACAGATAAAGC-3′ (sense primer; corresponding to nucleotide positions 1427–1452 of Contig6-1709); and 5′-CTAGTTAATGTGGAAAGTGTTTAAATC-3′ (antisense; primer corresponding to nucleotide positions 3227–3253 of Contig6-1709). PCR-ready tubes containing C. albicans single-stranded cDNA (prepared from 0.2 µg total RNA) were used according to the manufacturer specifications, except that the PCR-ready mixture (0.2 ml) was diluted five-fold before PCR amplification. The reaction mixture (30 µl) contained 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 1 µl diluted C. albicans cDNA, 50 pmol each primer and 0.5 units of Taq : Deep Vent DNA polymerase (100 : 1) and was pipetted into a 0.5 ml centrifuge tube and layered with 30 µl mineral oil to prevent evaporation. Amplification for 1 cycle at 95 °C for 10 min, 56 °C for 1 min and 72 °C for 1 min 50 s, 35 cycles at 95 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min 50 s, and 72 °C for 10 min (RoboCycler™96 temperature cycler, Stratagene) produced a ∼1.8 kb product of the predicted size from logarithmically growing cells that was ligated into the PCR vector pGEM-T (Promega), then subcloned into the enhanced Xenopus expression vector pGEM-HE (pCaCNT-HE) (Liman et al., 1992). By providing additional 5′- and 3′-untranslated regions from a Xenopus β-globin gene flanking the multiple cloning site, the pGEM-HE construct gave greater functional activity than the pGEM-T construct and was used in the subsequent transport characterization of the yeast protein. The 1827 bp pCaCNT-HE insert was sequenced in both directions by Taq DyeDeoxyterminator cycle sequencing using an automated model 373A DNA Sequencer (Applied Biosystems). In PCR experiments under identical conditions with two separate preparations of C. albicans cDNA from stationary growth phase cells, no product was obtained.

Functional expression of CaCNT cDNA in Xenopus oocytes

Plasmid pCaCNT was digested with NheI and transcribed with T7 RNA polymerase mMESSAGE MACHINE™ in vitro transcription system (Ambion). Healthy stage VI Xenopus oocytes were injected (Inject + Matic System) with 20 nl CaCNT RNA transcript (1 ng/nl) or 20 nl water alone and incubated at 18 °C in MBM for 3 days with a daily change of medium before the assay of transport activity (Huang et al., 1994; Ritzel et al., 1997; Yao et al., 2000).

CaCNT Radioisotope Flux Studies

Transport was traced using the appropriate [3H]/[14C]-labelled nucleoside or nucleoside drug (Moravek Biochemicals, Brea, CA, or Amersham Pharmacia Biotech) at a concentration of 1 mCi/ml or 2 mCi/ml for [14C]- and [3H]-labelled compounds, respectively. Flux measurements were performed at room temperature (20 °C), as described previously (Huang et al., 1994; Ritzel et al., 1997; Yao et al., 2000) on groups of 12 oocytes in 200 µl transport medium containing either 100 mM NaCl or 100 mM choline chloride and 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES (pH 5.5, 6.5, 7.5 or 8.5). Unless otherwise indicated, the permeant concentration was 20 µM. Transport medium for adenosine uptake experiments contained 1 µM deoxycoformycin to inhibit adenosine deaminase activity. To maximize potential transmembrane H+-gradients, cells were first washed into pH 7.5 NaCl or choline chloride transport medium and only exposed to either high (pH 8.5) or low (pH 5.5 or 6.5) pH medium immediately prior to the assay of transport activity. At the end of the incubation, extracellular radioactivity was removed by six rapid washes in the appropriate ice-cold transport medium. Individual oocytes were dissolved in 0.5 ml 1% (w/v) sodium dodecyl sulphate (SDS) for quantitation of oocyte-associated 3H or 14C by liquid scintillation counting (LS 6000IC, Beckman Canada Inc.). The flux values shown are the means ± S.E. of 10–12 oocytes, and each experiment was performed at least twice on different batches of cells. Kinetic (Km and Vmax) parameters ± S.E. were determined using ENZFITTER software (Elsevier-Biosoft, Cambridge, UK). We have previously established that oocytes lack endogenous nucleoside transport activity (Yao et al., 2000).

Measurements of CaCNT-induced H+ currents

Membrane currents were measured at room temperature using the whole-cell, two-electrode voltage clamp technique (CA-1B oocyte clamp, Dagan Corp.). The microelectrodes were filled with 3 M KCl and had resistances of 0.5–1.5 MΩ. The CA-1B was interfaced to a dedicated computer via a Digidata 1200B A/D converter and controlled by Axoscope software (Axon Instruments). Current signals were filtered at 20 Hz (four-pole Bessel filter) at a sampling interval of 50 ms. For data presentation, the signals were further filtered at 10 Hz by use of pCLAMP software (Axon Instruments). Following microelectrode penetration, resting membrane potential was measured over a 15 min period prior to the start of the experiment. Oocytes exhibiting an unstable membrane potential or a low membrane potential of less than −30 mV were discarded. Individual oocytes exhibiting good resting membrane potentials were clamped at −50 mV and current measurements were sampled in transport media of the same composition used in the radiolabelled isotope transport assays. During the course of data collection, the permeant-free transport medium perfusing the oocyte was changed to one containing nucleoside at a concentration of 100 µM. After 60 s, this was exchanged with fresh medium lacking the test nucleoside.

H+ : nucleoside coupling ratios

CaCNT H+ : nucleoside stoichiometry was determined by radiotracer transport-induced current measurements under voltage-clamp conditions in transport medium containing [3H]-labelled uridine (200 µM, 2 mCi/ml). Individual oocytes were placed in a perfusion chamber and voltage-clamped at a holding potential of −50 mV in permeant-free choline chloride transport medium at pH 5.5 for a 10 min period to monitor baseline currents. The transport medium was then exchanged with medium of the same composition containing radiolabelled uridine and current was measured for 3 min followed immediately by reperfusion with permeant-free transport medium until current returned to baseline. The oocyte was recovered from the chamber and solubilized with 1% SDS for liquid scintillation counting. The total movement of charge across the plasma membrane was calculated from the current-time integral and correlated with the measured radiolabelled flux for each oocyte to calculate the charge:flux ratio. [3H]-Labelled uridine uptake in control water-injected oocytes was used to correct for basal uptake of uridine over the same incubation period. The coupling ratios (±S.E.) presented were determined from 10 individual oocytes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References

This research is funded by the National Cancer Institute of Canada, the Medical Research Council (UK) and the Alberta Cancer Board and the Alberta Heritage Foundation for Medical Research (AHFMR). S.K.L. is funded by a studentship from the AHFMR. J.D.Y. is a Heritage Medical Scientist of the AHFMR. C.E.C holds a Canada Research Chair in Oncology at the University of Alberta. Sequence data for Candida albicans were obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford.edu/group/candida. TCDB (Transport Commission Database) nomenclature was obtained from the Transport Protein database website at http://tcdb.ucsd.edu/tcdb/background.php.

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  5. Conclusions
  6. Experimental procedures
  7. Acknowledgements
  8. References
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