Microbial Collection and Screening Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 1176091
Author for correspondence: Yue Wang. Tel: +65 778 3207. Fax: +65 7791117. e-mail: mcbwangy{at}imcb.nus.edu.sg
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ABSTRACT |
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Keywords: Na+/H+ antiporter, Candida albicans, salt tolerance, morphogenesis, gene disruption
Abbreviations: GFP, green fluorescent protein
The GenBank accession number for the sequence reported in this work is AF128238.
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INTRODUCTION |
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How pH change leads to C. albicans growth-mode transition is largely unknown. Stewart et al. (1988) reported a dramatic transient alkalinization of cytoplasm preceding the appearance of germ tubes. Strains defective in germ-tube formation did not show such a rise of cytoplasmic pH under conditions favourable for filamentous growth (Kaur & Mishra, 1994
). Inactivation of plasma-membrane H+-extruding ATPase inhibited both cytoplasmic alkalinization and germ-tube formation (Monk et al., 1991
, 1993
). These results show that the induction of morphological transition in C. albicans is accompanied by a steep rise of cytoplasmic pH probably due to an elevated activity of plasma membrane H+-ATPase, though it is not clear whether the fluctuation of pH is the cause or consequence of morphogenesis. Na+ at above 50 mM also has an inhibitory effect on germ-tube formation (Northrop et al., 1997
). At alkaline pH, as little as 1 mM Na+ was effective in reducing acid-extrusion rate in C. albicans (Northrop et al., 1997
). Thus, high concentrations of Na+ might inhibit the germtube formation by limiting the alkalinization of cytoplasm. In addition to H+-ATPase, Na+/H+ antiporters are known to play an important role in the maintenance and regulation of intracellular H+ and Na+ homeostasis (Padan & Schuldiner, 1994
; Utsugi et al., 1998
). This antiporter carries out cross-membrane electroneutral exchange of Na+ and H+ (Jia et al., 1992
; Watanabe et al., 1995
; Hahnenberger et al., 1997
; Iwaki et al., 1998
). The observed effect of Na+ on the extrusion of H+ and germ-tube formation in C. albicans indicates the existence of a Na+/H+ antiporter(s) and their possible role in influencing the growth-mode transition.
Na+/H+ antiporter genes have been cloned and characterized from several fungi including Saccharomyces cerevisiae (NHA1; Prior et al., 1996 ), Schizosaccharomyces pombe (sod2; Jia et al., 1992
) and Zygosaccharomyces rouxii (ZSOD2 and ZSOD22; Watanabe et al., 1995
; Iwaki et al., 1998
). However, no Na+/H+ antiporter has so far been reported in C. albicans. This group of proteins are characterized by the presence of 12 putative transmembrane regions and, except for sod2p, a large cytoplasmic C terminus. Sites implicated in Na+ binding and pH sensing have been identified in Schiz. pombe sod2p and Escherichia coli NhaAp (Padan & Schuldiner, 1996
; Dibrov & Fliegel, 1998
; Dibrov et al., 1997
, 1998
). Gene deletion and heterologous-expression studies demonstrate that these proteins are involved in pH regulation of the cell and in salt tolerance.
In a screen designed to identify genes that may play a role in C. albicans growth-mode transition, we cloned a C. albicans Na+/H+ antiporter gene, CNH1. Due to the potential roles of this gene in the regulation of cellular response to pH changes and pH-induced morphogenesis, we investigated the functions of this gene in both Sacch. cerevisiae and C. albicans.
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METHODS |
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Isolation of CNH1.
The coding region of the green fluorescent protein (GFP) gene yEGFP3 (Cormack et al., 1997 ) was PCR amplified and spliced into the SmaI site of the autonomously-replicating vector pRC2312 (Cannon et al., 1992
). A clone, pRCGFP, was chosen in which the 5' end of yEGFP3 gene was proximal to the BamHI site of the vector. C. albicans genomic DNA was partially digested by Sau3AI. DNA fragments ranging from 500 bp to 1000 bp were purified from a low-melting-point agarose gel after electrophoresis and spliced into the BamHI site of pRCGFP to construct a library. The library plasmids were transformed into CAI-4 and transformants selected on GMM plates. Each transformant was grown at 30 °C in two media, GMM and GMM+5% (v/v) foetal calf serum, supporting yeast and hyphal growth, respectively. GFP expression was examined by using a fluorescence microscope. Clones showing enhanced GFP expression in the hyphal form were selected. The plasmid was recovered by lysing the transformants and transforming E. coli cells with the lysate. For the isolation of the full-length gene, a C. albicans genomic library was constructed. Genomic DNA was partially digested using Sau3AI. DNA fragments ranging from 4 to 6 kb were gel purified, ligated to pBKCMV and packaged using Stratagenes Gigapack III Goldsystem. The phage library was screened by following the manufacturers instructions. A 4 kb DNA fragment was isolated containing the full length CNH1 gene with 5' and 3' flanking sequences. DNA was sequenced by using a model 377 DNA sequencer (Applied Biosystems). The DNASTAR program was used to determine the sequence identity and hydrophilicity plots.
Southern and Northern analyses.
C. albicans genomic DNA was isolated by the method of Cannon et al. (1992) . Genomic DNA (10 µg) of each strain was cleaved by a restriction enzyme, resolved on 1% (w/v) agarose gel by electrophoresis and capillary-transferred to a Hybond-N nylon membrane (Amersham). For Northern hybridization, SC5314 was grown to exponential phase in GMM in the presence of different concentrations of NaCl, and total RNA was extracted by the method of Chirgwin et al. (1979)
. RNA (20 µg each well) was electrophoresed through a 1·2% (w/v) agarose gel containing 0·22 M formaldehyde and transferred to a Hybond-N nylon membrane. DNA probes were labelled with 32P by using the High Prime DNA Labelling Kit (Boehringer Mannheim). Hybridizations and washes of the blots were done according to standard procedures (Maniatis et al., 1982
).
Functional complementation of CNH1 in Sacch. cerevisiae B31.
The DNA fragment containing the coding region and termination signal of CNH1 was cloned into a Sacch. cerevisiae centromeric expression vector, pUS234 (gift from Uttam Surana, Institute of Molecular and Cell Biology, Singapore), under the control of the GAL1-10 promoter to give pUSCNH1 and transformed into Sacch. cerevisiae B31 cells by the method of Becker & Lundblad (1994) . Multiple colonies selected on GMM agar plates were grown separately to saturation in GMM liquid medium at 30 °C, serially diluted and spotted onto galactose MM (GalMM) agar plates containing different concentrations of NaCl.
Site-directed mutagenesis.
The Quikchange Site-Directed In Vitro Mutagenesis kit (Stratagene) was used for site-directed mutagenesis. Two aspartate residues (D310 and D311) of the CNH1 gene in pUSCNH1 were mutated to asparagines to generate a new plasmid, pUScnh1. The mutated clones were verified by sequencing and transformed into Sacch. cerevisiae B31.
Disruption of CNH1.
We used the hisGCaURA3hisG cassette to generate a gene-disruption construct for sequential deletion of both copies of CNH1 gene (see Fig. 4). The genotype of each mutant was verified by Southern analysis. A 340 bp DNA fragment immediately upstream of the coding sequence was obtained by PCR in which a PstI site was introduced into the 3' end. The PCR primers used were 5'-CTCGCCAATCAAACTCAAAT-3', -283 to -264 (the first base of the ATG start codon is +1), and 5'-GGCTGCAGCCAAGAAATTGTATCACCTG-3', +25 to +44 (PstI site is underlined). The PCR product was cloned into pGEMTeasy (Promega) and a clone, pCNH1-5', was selected in which the PstI end of the insert is proximal to the SacI site in the vector. A 470 bp DNA fragment downstream of the coding sequence was similarly obtained with a BamHI site added to the 5' end of the PCR fragment, and cloned into pGEMTeasy. The PCR primers were 5'-CCGGATCCTGTTATGCGAACCTCGGTAG-3', +2542 to +2562, and 5'-TTGGCTTGTACTTGTCGTTG-3', +3013 to +2994 (BamHI site is underlined). This fragment was recovered by BamHI/SacI digestion and spliced together with the BamHIPstI fragment of the hisGCaURA3hisG cassette into PstI/SacI-digested pCNH15' to generate pCNH1URA3. The hisGCaURA3hisG BamHIBglII fragment from pCUB-6 (Fonzi & Irwin, 1993
) was initially subcloned into pGEMTeasy to obtain the BamHIPstI fragment of the cassette. The entire disruption cassette was released from pCNH1URA3 by AatII/SacI digestion and introduced into CAI-4 by electroporation (Kohler et al., 1997
). The transformants were selected on GMM plates. The CaURA3 gene was excised by growing the transformants in YPD overnight and plating 20 µl of the culture onto GMM plates containing 1 mg 5-fluoroorotic acid ml-1 and 50 µg uridine ml-1. The ura- transformants were used for the deletion of the second copy of CNH1. We obtained two heterozygous mutants, CaTW1a and CaTW1b (CNH1/cnh1
::hisGCaURA3hisG), from independent experiments and the subsequent deletion of CaURA3 gave CaTW2a and CaTW2b (CNH1/cnh1
::hisG). From CaTW2a and CaTW2b, we obtained two homozygous mutants, CaTW3a and CaTW3b (cnh1
::hisG/cnh1
::hisGCaURA3hisG). The two independent clones of the same genotype exhibited the same phenotype.
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Growth assays.
SC5314, CaTW1 and CaTW3 cells were grown in GMM to saturation. Growth curves were obtained by seeding 1x104 cells in 50 ml Falcon tubes containing 20 ml GMM buffered at either pH 5·5 or pH 7·5 and containing 0, 25, 50 or 100 mM LiCl. The cultures were grown at 30 °C with shaking and OD600 values were determined at 1·5 h intervals. These experiments were conducted three times.
Animal experiment.
SC5314, CaTW1 and CaTW3 cells were grown at 30 °C in GMM to saturation. The cells were washed twice in PBS and resuspended in PBS to a density of 5x106 cells ml-1. For each strain, five male Balb/c mice (Jackson Labs) of 46 weeks old were injected with 1x106 cells through the lateral tail vein. Survival curves were calculated according to the KaplanMeier method using the PRISM program (version 2.01; GraphPad Software) and compared using the log-rank test. A P value less than 0·05 was considered significant. The kidneys of dead mice were removed for counting c.f.u. and microscopic examination of infected tissue. One kidney was cut into two coronal pieces and the other cut into two sagittal pieces. One piece of each kidney was put into 10% (v/v) formalin for periodic acidSchiff staining, and the other two pieces were placed in 10 ml PBS and homogenized, and 200 µl of the solution was plated onto YPD agar for colony counting.
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RESULTS |
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CNH1 null mutant cells exhibit elongated morphology under conditions for yeast growth
We observed that on YPD agar plates the homozygous cnh1 mutant CaTW3 produced wrinkled colonies, an indicator of filamentous growth (Radford et al., 1994 ). The number of wrinkled colonies increased with the time of culture, and after 10 d nearly all colonies became wrinkled. Under the same conditions, almost all colonies of SC5314 and the heterozygous mutant CaTW1 had smooth surfaces (Fig. 5
). Microscopic examination of cells from the wrinkled colonies revealed a mixture of mostly yeast cells and a significant number of highly elongated cells. This result shows that the cnh1 null mutant cells have a tendency to grow in an elongated form under a condition that normally supports yeast growth.
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Deletion of CNH1 causes retarded growth
To examine whether CaTW1 and CaTW3 were more sensitive to NaCl than SC5314, we inoculated cells onto GMM plates supplemented with NaCl at various concentrations up to 1·6 M at either pH 5·5 or pH 7·5. After 3 d incubation at 30 °C, CaTW1 and CaTW3 formed patches nearly as dense as SC5314 on all the plates. To determine the growth rates of SC5314, CaTW1 and CaTW3, we repeated the experiment in liquid GMM and monitored cell growth by measuring OD600. Under both pH conditions, CaTW1 and CaTW3 grew considerably more slowly than SC5314 (Fig. 6a, e
). In GMM, the doubling times for SC5314, CaTW1 and CaTW3 are 2·7, 3·6 and 4·9 h at pH 5·5, and 3, 3·9 and 5·3 h at pH 7·5, respectively. There is apparently a gene-dosage effect in that the heterozygous mutant CaTW1 grew at a rate intermediate between those of SC5314 and CaTW3. When 1 M NaCl was added to the medium, CaTW1 and CaTW3 did not appear to be more sensitive to the high salt concentration, because their growth rates decreased proportionately to that of SC5314 (data not shown). High concentrations of Na+ are well known to generate osmotic potential across the cell membrane. It is possible that the effect of high osmotic pressure on the cells may mask a small increase in NaCl sensitivity of CaTW1 and CaTW3. To limit the effect of high osmotic pressure of a high NaCl concentration, we redetermined the growth rates using Li+, an analogue of Na+. Li+ is much more toxic than Na+ so that it can be used at much lower concentrations without generating high osmotic potential. Li+ is often used as an analogue of Na+ in transport studies (Fleishman, 1991
; Hahnenberger et al., 1997
; Ros et al., 1998
). The effect of Li+ on the growth rates of the three Candida strains was very similar to that of NaCl (Fig. 6
). Again, CaTW1 and CaTW3 exhibited a similar level of sensitivity to an increase in LiCl concentration. These results show that the deletion of CNH1 causes a retarded growth in C. albicans but has little effect on the sensitivity of cells to high salt concentrations.
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DISCUSSION |
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Amino acid sequence alignment of all available Na+/H+ antiporters of fungi revealed a high degree of conservation, especially over the N-terminal half of the molecule, which contains all the similarly spaced transmembrane segments, indicating a conserved core structure of the ion-transport machinery. Some amino acid residues are invariable in all Na+/H+ antiporters found so far from both prokaryotic and eukaryotic organisms, such as the two aspartate residues corresponding to D310 and D311 of Cnh1p, which are believed to form the Na+-binding site (Inoue et al., 1995 ; Padan & Schuldiner, 1996
; Dibrov et al., 1998
). We demonstrated that mutation of D310 and D311 to asparagines abolished the ability of CNH1 to confer salt-tolerance upon the Sacch. cerevisiae ena1 nha1 mutant. This result is in good agreement with results obtained from Schiz. pombe sod2p carrying the same mutations (Dibrov et al., 1998
). The C-terminal half of the Na+/H+ antiporters is less conserved and varies in length, and is speculated to play a regulatory role (Aronson et al., 1982
; Balcells et al., 1997
; Dibrov & Fliegel, 1998
). Studies of the mammalian Na+/H+ antiporter family (NHE1NHE6) also revealed the existence in the C-terminal tail of consensus sites for phosphorylation by protein kinase A and/or protein kinase C as well as sites suitable for calmodulin kinases and for proline-directed Ser/Thr kinases (Paranjape et al., 1990
; Padan & Schuldiner, 1994
). Regulated phosphorylation of NHE1 and NHE3 have been experimentally demonstrated (Bianchini et al., 1997
; Wakabayashi et al., 1997
). In Cnh1p, several putative protein kinase C phosphorylation sites are found in the C-terminal region. Mutation of these sites would help to elucidate the function(s) of this region.
To better understand the physiological role of CNH1, heterozygous and homozygous gene deletion mutants were constructed. The loss of a single copy did not result in any observable changes in morphology. The wild-type and heterozygous mutants grew exclusively as yeast at pH 5·5 and~8% of the cells developed a highly elongated shape at pH 7·5 in GMM at 30 °C. In contrast, the mutants with both copies deleted grew as a mixture of yeast and elongated cells at both pH 5·5 and 7·5, the percentage of the elongated cells being 5 and 31%, respectively. These results suggest that Cnh1p activity may influence the execution of the signalling pathways for morphogenesis, presumably by participating in cytoplasmic pH regulation. However, since cross-membrane proton flux and regulation of intracellular pH need concerted actions of multiple transporters, it is premature to speculate on the molecular mechanism that links Cnh1p activity to cell morphology. Two pH-regulated genes, PHR1 and PHR2, have been shown to be involved in morphogenesis in C. albicans (Saporito-Irwin et al., 1995 ; Bernardis et al., 1998
).
The Na+/H+ antiporter has a role in salt tolerance in both fungi and bacteria (Jia et al., 1992 ; Nozaki et al., 1998
). When heterologously expressed in a Sacch. cerevisiae strain severely defective in Na+ extrusion, CNH1 confers resistance to 800 mM NaCl, indicating that CNH1 may play a similar role in C. albicans cells. However, we found that C. albicans cnh1 mutants do not exhibit higher sensitivity to an increase in either Na+ or Li+ concentration, except for a general slower growth rate in comparison with the wild-type cells. One plausible explanation for this observation is the presence of other Na+-extrusion mechanisms in C. albicans such as Na+-ATPases. In Sacch. cerevisiae there is a family of Na+-ATPases encoded by a tandem array of genes, ENA14, and their expression is induced by high-Na+ stress (Wieland et al., 1995
; Prior et al., 1996
). It has been shown that overexpression of NHA1 significantly enhanced salt tolerance of Sacch. cerevisiae cells only when the four ENA genes were deleted (Prior et al., 1996
). Clearly, the Na+-ATPases are able to compensate for the salt sensitivity resulting from the deletion of the Na+/H+ antiporter gene. Actually, Hahnenberger et al. (1997)
demonstrated that Na+/H+ antiporter and sodium pumps could be used interchangeably by Sacch. cerevisiae to regulate intracellular sodium concentration.
The animal experiment shows that the deletion of both copies of CNH1 led to a considerable delay in the killing of infected mice. Since the numbers of viable C. albicans cells in kidney at the time of animal death were comparable, the delayed death of mice infected by the null mutant is probably a consequence of its slow growth. The reason why the animals infected by the heterozygous mutant did not exhibit delayed death is not clear. It could be that its rate of growth is not sufficiently slow to have an effect on the whole process of pathogenesis. Though the relevance of CNH1 function to the virulence of the pathogen is not conclusive at the current stage of investigation, the growth defect and delayed pathogenesis as a result of CNH1 deletion render the gene a potential target for developing anti-Candida drugs.
In summary, we have isolated and functionally characterized a C. albicans Na+/H+ antiporter gene, CNH1. It confers sodium tolerance when heterologously expressed in a Sacch. cerevisiae strain defective in sodium extrusion. Deletion of CNH1 leads to slower growth rates in a gene-dosage dependent manner. Though deletion of the gene has no detectable effects on the response of cells to standard hypha-inducing conditions, a considerable fraction of the null mutant cells develop a highly elongated morphology under conditions that normally support yeast growth, suggesting that CNH1 may influence morphogenesis through different pathways.
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ACKNOWLEDGEMENTS |
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Received 21 October 1999;
revised 6 January 2000;
accepted 11 February 2000.