Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species
© Krauke and Sychrova; licensee BioMed Central Ltd. 2008
Received: 12 December 2007
Accepted: 20 May 2008
Published: 20 May 2008
The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far.
The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both Cp Cnh1 and Cd Cnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; Cp Cnh1p provided cells with a much higher salt tolerance than the Cd Cnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters.
We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.
The family of Candida species, normally a harmless human commensal of the gastrointestinal and genitourinary tract, can become a human pathogen under certain circumstances. Mainly in HIV and immunocompromised patients, Candida cause a wide range of infections and are the most prevalent pathogenic yeast. One key feature of these fungi is their ability to grow in three different morphologies: yeast, pseudohyphae and true hyphae . This reversible switching from one form to another is dependent on environmental conditions like temperature, pH, nutritional status and external/internal concentration of cations .
In general, pathogenic Candida species are osmotolerant yeasts and can grow, with the exception of Candida dubliniensis , at relatively high NaCl concentrations, although the presence of salt was shown to negatively influence several virulence traits of Candida albicans . Recent experiments also suggest a relationship between the formation of C. albicans hyphae and the intracellular concentration of potassium . Nevertheless, the regulation of intracellular potassium and sodium concentrations in Candida species has not been studied in detail.
Yeast species in general have several transport systems in their plasma membranes at their disposal to maintain homeostasis in alkali metal cations, i.e. a high ratio between potassium (which is the main intracellular cation) and toxic sodium concentrations [6–8]. Among these transport systems, Na+/H+ antiporters play an important role. Most yeasts that have so far been studied (e.g. Saccharomyces cerevisiae, Debaryomyces hansenii) possess only one type of this antiporter in their plasma membranes, which efficiently transports both sodium and potassium cations from the cells, as well as their analogues lithium and rubidium [7, 9]. A few yeast species (Yarrowia lipolytica, Schizosaccharomyces pombe) have two antiporters of this family at their disposal, one of them with a substrate preference for sodium and lithium, the other preferring potassium and rubidium [10, 11].
The Na+/H+ antiporter that has been studied the most so far is from S. cerevisiae, encoded by the NHA1 gene, and has 12 predicted trans-membrane domains and a very long hydrophilic C-terminus . Beside its function in removing toxic Na+ from cells and maintaining potassium homeostasis, it is involved in several other cellular functions such as regulating intracellular pH [12, 13], cell volume , plasma membrane potential  and the cell cycle [15, 16], and it participates in the cell response to osmotic shock [7, 17]. The Nha1p orthologs from C. albicans and Candida tropicalis Ca Cnh1p and Ct Cnh1p, respectively, were functionally characterized upon heterologous expression in S. cerevisiae. Both showed the same broad substrate specificity as Sc Nha1p [18, 19]. The deletion of CNH1 in C. albicans results in cell sensitivity to high external potassium concentrations  and under some conditions causes slight changes in cell morphology .
In this work we compared the tolerance of four different pathogenic Candida species to alkali metal cations, performed a search for Nha1/Cnh1 antiporter-encoding orthologs in their genomes, and characterized the transport properties of the Na+/H+ antiporters from the most and least tolerant species, C. parapsilosis and C. dubliniensis respectively.
Candida species differ in their halotolerance
Comparison of Candida CNH1 genes and encoded antiporters
A search in databases revealed the existence of open reading frames homologous to the CaCNH1 gene in the genomes of C. dubliniensis, C. glabrata and C. parapsilosis. In these species, just one homologous sequence was found, suggesting that their plasma-membrane Na+/H+ antiporters have a broad substrate specificity to alkali metal cations, similar to those of C. albicans and C. tropicalis. We named the identified orthologous genes according to their species of origin, CdCNH1, CgCNH1 and CpCNH1. The CdCNH1 gene is 2454 nt (818 aa) long, CgCNH1 has 2835 nt (945 aa), and the CpCNH1 gene is composed of 2955 nt (985 aa). Neither of them have any introns.
Comparison of deduced secondary structures of plasma-membrane Na+/H+ antiporters from Candida species and S. cerevisiae
Number of amino acid residues
Tms + loops
Identity (%) of Candida and S. cerevisiae alkali-metal-cation antiporters
Entire protein/tms and loops/C-terminus
46.7/ 69.5/ 19.8
Though the highest divergence from identity was found in the C-termini of all the compared antiporters, the existence of six conserved C-terminal regions described previously  was also confirmed in C.dubliniensis, C. parapsilosis and C. glabrata species (not shown). A new, approximately 25 aa-long conserved region (K/R)(L/I)SR(S/T)(L/A)SRRS(Y/F)Y(K/R)KDDP(H/N)(K/R)RKVYAHR (in Ca Cnh1p aa 639–664) preceding conserved region no. 5 ; was found in the four Candida species, except for C. glabrata.
Both the C. dubliniensis and C. parapsilosis species belong to the group of yeasts in which the CTG codon encodes a serine and not a leucine, as in other yeast species (i.e. S. cerevisiae) . One CTG codon exists at aa position 621 in CpCNH1. This serine 621 is localized in the antiporter's hydrophilic C-terminus and not in the membrane part of the protein. It is localized in a small weakly conserved area where at a similar position Cd Cnh1p (aa 568) and Ct Cnh1p (aa 644) also have a serine and Ca Cnh1p (aa 552) has an isoleucine.
Heterologous expression of Candida Cnh1ps in S. cerevisiae BW31a cells and its phenotype
In order to 1) verify whether the identified and analysed open reading frames encode functional plasma-membrane alkali-metal-cation/proton antiporters, and 2) elucidate whether the species' salt tolerance could reflect the activity of these antiporters, corresponding DNA fragments from the most and least salt tolerant species, C. parapsilosis and C. dubliniensis respectively, were expressed in a S. cerevisiae mutant lacking its own export systems for alkali metal cations (BW31a ena1-4 Δ nha1 Δ ). The lack of both Na+-ATPases, Ena1-4 and the Na+/H+ antiporter Nha1 renders these cells extremely sensitive to higher external concentrations of salts, and almost no efflux of sodium or potassium cations from them is observable. The functional expression of heterologous sodium and/or potassium exporters in BW31a cells has clear phenotypes of an increased salt tolerance and measurable alkali metal cation efflux .
pH dependence of salt tolerance of BW31a cells expressing various antiporters.
Localization of Cd Cnh1 and Cp Cnh1 antiporters in S. cerevisiae cells
Western blot analysis of antiporters' amount in cells
Efflux of Na+ and K+ from BW31a cells expressing various antiporters
Efflux of potassium and sodium cations from BW31a cells expressing various antiporters.
7.4 ± 0.7
5.0 ± 1.0
82.2 ± 0.4
55.3 ± 1.8
38.4 ± 3.0
19.5 ± 2.1
79.1 ± 2.8
32.6 ± 3.0
Discussion and Conclusion
The variable sensitivity of Candida species to NaCl has been observed and exploited previously, mainly in connection with the original niche of the species, e.g. the high NaCl tolerance of C. parapsilosis strains isolated from sea water or hypersaline brines [22, 27] or in clinical microbiological tests aiming to distinguish among Candida species (NaCl sensitivity of C. dubliniensis). In this work, we performed a series of tests to characterize the tolerance of four different pathogenic Candida species to different alkali metal cations, and addressed the question of whether the observed differences in salt tolerance could be based on the transport activity and/or specificity of Candidas' plasma-membrane Na+/H+ antiporters. Our results showed clearly that, besides differences in their tolerance to sodium, the four species tested also differ in their sensitivity to highly toxic lithium and their tolerance of high external concentrations of non-toxic potassium cations (Figure 1). C. glabrata and C. dubliniensis are the most sensitive to Li+ cations, the latter being the least alkali-metal-cation tolerant of the species tested.
To elucidate the role of Na+/H+ antiporters in Candidas' salt tolerance, we isolated and heterologously expressed genes encoding putative antiporters in the most and the least tolerant species, C. parapsilosis and C. dubliniensis. The functional characterization of these encoded proteins in an S. cerevisiae mutant lacking its own alkali-metal-cation exporters revealed that both antiporters have broad substrate specificity. They recognize at least four different cations (K+, Li+, Na+, Rb+), but transport them with varying capacities and/or affinities. C. parapsilosis and C. albicans Cnh1 proteins are very efficient transporters and their capacities exceed that of S. cerevisiae Nha1p. On the other hand, the C. dubliniensis antiporter has a much lower transport activity and its ability to recognize and transport the smallest cation, toxic lithium, is very limited compared to the other two Candida antiporters (Figure 2, Table 3). The large difference between the transport activities of the evolutionary close C. albicans and C. dubliniensis Cnh1 antiporters is worth noting, as these two proteins shared the highest level of identity (Table 2). Their core membrane section (twelve tms and connecting loops) is almost 99% identical and their N-termini, composed of 11 aa residues, are 100% identical. These two antiporters differ slightly (compared to other antiporters) in the length and composition of their hydrophilic C-termini. Whether the observed transport capacities are based on the C-terminal difference remain to be established, though previous studies showed that the C-termini of yeast Na+/H+ antiporters are not the most important factor in this activity  and that a single amino-acid exchange in one of the transmembrane domains can significantly influence both the substrate specificity and transport capacity of the antiporter [28, 29].
Though the C. parapsilosis Cnh1 antiporter mediates a high and efficient sodium efflux from S. cerevisiae cells (similarly as the Ca Cnh1p, Figure 5 and Table 4), its physiological role in C. parapsilosis remains to be established. A recent study  showed that the Cnh1 antiporter is mainly important in potassium homeostasis in C. albicans cells and its role in Na+ detoxification is rather marginal.
The differences observed in alkali-metal-cation tolerance between C. dubliniensis and C. parapsilosis/C. albicans species were also found upon testing the alkali-metal-cation tolerance of S. cerevisiae cells expressing the antiporters of these three species. Efflux measurements confirmed that the differing tolerances of S. cerevisiae cells were based on the differing transport activities of the Candida antiporters. Altogether, our results suggest that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance to high external concentrations of alkali metal cations in pathogenic Candida species.
Yeast strains, media and growth conditions
To determine the salt tolerance of various Candida species, C. albicans SC5314, C. glabrata ATCC2001, C. dubliniensis CD36 and C. parapsilosis CBS604 were used, together with S. cerevisiae S288c as a control. The CdCNH1 and CpCNH1 genes were isolated from C. dubliniensis CD36 and C. parapsilosis CBS604, and heterologously expressed in S. cerevisiae BW31a (ena1-4 Δ nha1 Δ, W303 derivative, ). Yeast cells were grown in YPD or YNB-NH4+ media supplemented with 2% glucose at 30°C. Salts were added to the media prior to and auxotrophic supplements after autoclaving.
DNA manipulations, plasmid construction and DNA sequencing
For DNA manipulations, standard protocols  were used. The CdCNH1 and CpCNH1 gene were amplified by PCR with platinum Pfx polymerase with proofreading activity (Invitrogen) using their isolated genomic DNA  as a template.
Oligonucleotides used for amplification of C. dubliniensis and C. parapsilosis CNH1 genes. Sections homologous to CNH1 genes are underlined.
Salt tolerance determination
The cell tolerance to various alkali metal cations was determined by drop test experiments. 3 μl of serial 10-fold dilutions of saturated cell cultures were spotted on solid media. For Candida species, YPD – 3% agar plates supplemented with increasing amounts of salts were used (800 – 2600 mM NaCl; 1800 – 2500 mM KCl; 20 – 1000 mM LiCl; 700 – 1800 mM RbCl). Determination of the maximum salt tolerance of S. cerevisiae BW31a cells expressing either of the antiporters was performed on solid YNB media supplemented with salts and adjusted to various pH levels as described previously . The following salt concentrations were used: 1) non-adjusted pH, 500–1500 mM NaCl; 1800–2100 mM KCl; 25–40 mM LiCl; 1000–1600 mM RbCl; 2) pH 3.5, 500–1300 mM NaCl; 1600–2200 mM KCl; 20–50 mM LiCl; 3) pH 5.5, 200–1000 mM NaCl; 1900–2200 mM KCl; 15–40 mM LiCl; 4) pH 7.0, 50–200 mM NaCl; 800–1500 mM KCl; 5–10 mM LiCl.
Cation efflux measurements
The transport activity of the antiporters was measured as the cation efflux from cells according to  with minor modifications. Cells were grown to OD600 ≈ 0.2 in YNB, harvested and then a pH 5.5 incubation buffer (20 mM MES, supplemented with 10 mM KCl or 10 mM RbCl to prevent Na+ or K+ reuptake respectively) was used. For the determination of Na+ efflux, the cells were preloaded for 60 min with 100 mM NaCl in YNB adjusted to pH 7.0 with NH4OH. The K+ efflux was measured directly in the harvested cells. Samples were taken from cell incubation cultures at regular time intervals over a period of 60 min and the cellular cation content was determined by atomic absorption spectroscopy . Each efflux experiment was repeated at least three times and representative results are shown.
Exponential phase cells (grown in YNB at 30°C, OD600 ≈ 0.15) expressing the CdCHN1 or CpCNH1 gene tagged with the GFP sequence were viewed with an Olympus AX70 microscope using a U-MWB cube with a 450–480 nm excitation filter and 515 nm barrier filter. The micrographs were recorded with a DP70 digital camera using the program DP Controller. For whole-cell pictures, Nomarski optics was used.
Exponetially growing (OD600 ≅ 0.15) BW31a cells expressing GFP-tagged antiporters were harvested and concentrated by centrifugation to OD600 = 3.0. The proteins were extracted according to  with some modifications. After resuspension of cell pellet in 150 μl freshly prepared 1.85 M NaOH with 7.5% β-mercaptoethanol and incubation for 15 min on ice, 150 μl of cold 50% trichloroacetic acid were added. After incubation on ice for 20 min the collection of precipates by centrifugation at 20,000 × g for 20 min followed. The pellet was resuspended in 190 μl of 50 mM Tris-HCl buffer (pH 6.8), containing 8 M urea, 5% sodium dodecyl sulfate (SDS), 0.1 mM EDTA and 1.5% dithiothreitol (DTT) + 10 μl 1 M Tris base. After incubation for 30 min at 37°C the samples were centrifuged at 20,000 × g for 30 min. Supernatant (7.5 μl) were directly loaded on 8% glycine gel and separated by polyacrylamid gel electrophoresis (PAGE). Separated proteins were transferred via electroblotting on nitrocellulose membrane. To detect the GFP-tagged proteins on membranes, rabbit polyclonal anti-GFP antibody (Santa Cruz Biotech., diluted 1:200), secondary goat anti-rabbit IgG antibody with conjugated peroxidase (BioRad, diluted 1:10,000) and ECL detection kit (Pierce) were used.
This work was supported by the EU grant MRTN-CT-2004-512481 CanTrain and Czech grants LC531 and AV0Z50110509. We thank Olga Zimmermannova for helpful discussions, Gary Moran, Karl Kuchler and Jozef Nosek for providing Candida strains, and Jaroslav Horak for the help with Western blots.
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