The MAP kinase Hog1p differentially regulates stress-induced production and accumulation of glycerol and D-arabitol in Candida albicans

Gerald Kayingo and Brian Wong

Dept of Internal Medicine, Section of Infectious Diseases, Yale University and VA Connecticut Healthcare System, 950 Campbell Avenue (111-I), West Haven, CT 06516, USA

Correspondence
Brian Wong
brian.wong{at}yale.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Candida albicans produces and accumulates large amounts of the polyols D-arabitol and glycerol in culture, and/or in infected mammalian tissues. However, the effects of environmental stresses on production and accumulation of these polyols, and the means by which polyol production and accumulation are regulated have not been studied. C. albicans grown in glucose at 30 °C (i) produced maximal amounts of glycerol within 6 h, (ii) produced maximal amounts of D-arabitol and ribitol within 12 h, and (iii) released most of these polyols into the extracellular environment. C. albicans responded to osmotic and citric acid stress by producing and accumulating more glycerol, and to temperature and oxidative stresses by producing more D-arabitol. The increase in intracellular glycerol was proportional to extracellular osmolarity, suggesting that glycerol functions as an osmolyte. The MAP kinase Hog1p is required for wild-type glycerol production in several fungal species subjected to osmotic stress, but it is not known if Hog1p plays a role in regulating D-arabitol production. Therefore, two C. albicans hog1 null mutants were constructed and tested for the ability to produce glycerol and D-arabitol in response to environmental stresses. The ability to grow and produce glycerol when exposed to osmotic or citric acid stresses, and to produce D-arabitol when exposed to oxidative stress, was partially dependent on Hog1p, but the ability to produce D-arabitol when exposed to temperature stress was Hog1p independent. These results imply that multiple pathways regulate glycerol and D-arabitol synthesis in C. albicans.


Abbreviations: FOA, 5-fluoroorotic acid; HOG, high-osmolarity glycerol; MAP, mitogen-activated protein


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pathogenic fungi encounter a wide variety of potentially harmful environmental challenges during their proliferation in a host. Factors such as the host's immune system, nutrient availability, temperature, pH, redox state, oxygen levels and osmolarity can vary considerably, and often cause severe stress. However, fungal cells are endowed with adaptive mechanisms that sense and respond appropriately to changes in environmental conditions. One of the most pronounced physiological responses to environmental stress is the ability to produce and accumulate protective solutes such as glycerol, D-arabitol and mannitol. These polyol metabolites can function as intracellular osmolytes (Brown, 1978; Yancey et al., 1982) and as metabolic reducing equivalents (Ansell et al., 1997; Minard & McAlister-Henn, 2001); in addition, they can quench reactive oxygen intermediates produced by host defence systems (Chaturvedi et al., 1996b; Jennings et al., 1998). There are also several reports suggesting that polyols may have important roles in host–pathogen interactions (Chaturvedi et al., 1996a; de Jong et al., 1997; Jennings et al., 1998).

The mechanisms by which a few fungi regulate glycerol production and accumulation have been studied, and include (i) transcriptional regulation of the genes involved in glycerol synthesis, (ii) modulation of membrane permeability to conserve or release intracellular glycerol, and (iii) changes in dissimilation and uptake rates depending on growth conditions (Edgley & Brown, 1983; Kayingo et al., 2001). During osmotic-stress conditions, many fungal species maintain an osmotic equilibrium by synthesizing and accumulating high amounts of glycerol. In Saccharomyces cerevisiae, osmotic stress-induced glycerol production is mediated by Hog1p, a mitogen-activated protein (MAP) kinase in the high-osmolarity glycerol (HOG) pathway (Albertyn et al., 1994; Brewster et al., 1993; Posas et al., 2000; Rep et al., 2000; O'Rourke & Herskowitz, 2004). Hog1p is a member of a conserved family of MAP kinases that transmit environmental stress signals in a variety of signalling pathways (Banuett, 1998; Gustin et al., 1998; Herskowitz, 1995). When cells are exposed to osmotic stress, Hog1p is rapidly phosphorylated and translocated into the nucleus, where it triggers a transcriptional response affecting approximately 10 % of the yeast genes, including those involved in glycerol synthesis (Hohmann, 2002). Although the HOG pathway was originally recognized as an osmosensing signal-transduction pathway in S. cerevisiae (Brewster et al., 1993), recent studies have shown that this pathway is also involved in responses to heat (Winkler et al., 2002), oxidative stress (Bilsland et al., 2004; Haghnazari & Heyer, 2004) and citric acid stress (Lawrence et al., 2004).

Candida species are important human pathogens, particularly in people undergoing chemotherapy, organ-transplant recipients and AIDS patients (Calderone, 2002; Pfaller et al., 1998). Candida albicans is the most commonly encountered species (Pfaller et al., 1998); it produces large amounts of D-arabitol and glycerol in culture, and large amounts of D-arabitol in the tissues of infected animals (Bernard et al., 1981, 1985; Kiehn et al., 1979; Wong et al., 1982). However, the physiological roles of these metabolites, as well as the environmental signals and the mechanisms regulating their production and accumulation in C. albicans, are not well understood. San José et al. (1996) suggested that C. albicans utilizes glycerol as an intracellular osmolyte, and that osmotic stress-induced glycerol accumulation and osmoadaptation are at least partially controlled by the MAP kinase Hog1p. Subsequent studies have shown that the HOG pathway has additional functions in C. albicans, which include adaptation to oxidative stress, cell wall biosynthesis, adherence, morphogenesis and virulence (Alonso-Monge et al., 1999; 2003). In addition, Smith et al. (2004) recently demonstrated that the C. albicans Hog1p is activated in response to diverse stress conditions, and plays a pivotal role in the regulation of the core stress response in C. albicans.

It is not known if Hog1p also regulates D-arabitol production and accumulation in C. albicans. Since our group is interested in understanding the relationships between polyol homeostasis, environmental stress tolerance and virulence in C. albicans, the aims of this study were (i) to determine the effect of environmental stresses, such as temperature, oxidative stress and osmotic stress, on D-arabitol and glycerol production and accumulation, and (ii) to analyse the role of Hog1p in regulating stress-induced D-arabitol and glycerol accumulation in C. albicans.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
The C. albicans strains used in this study are shown in Table 1. Strains BWP17 (Wilson et al., 1999) and DAY185 (Davis et al., 2000) were obtained from A. P. Mitchell (Columbia University, New York, USA). The hog1{Delta} mutant strains GK-Y00, GK-Y001a, GK-Y001b and GK-Y002 were constructed as described below.


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Table 1. Yeast strains

 
C. albicans strains were grown at 30 °C in defined medium [2 % glucose, 0·67 % yeast nitrogen base (YNB)] supplemented with the appropriate auxotrophic requirements and adjusted to pH 5·4. In some experiments, glucose was replaced by galactose (3 %), D-arabinose (4 %) or xylose (5 %) as the sole carbon source. Yeast-phase cells were converted to hyphal-phase cells by adding 10 % human serum to YNB/glucose, or by growing cultures in Lee's medium (Lee et al., 1975) at 37 °C.

To study the effects of various environmental stresses on polyol production and accumulation, samples were harvested from exponential-phase cultures (YNB/glucose at 30 °C), and they were exposed to several different environmental stresses that (i) are known to influence polyols, or (ii) would be expected to occur during infection of a mammalian host. For 37 °C experiments, sample preparation (growth medium, harvesting and washing) was carried out at room temperature before cells were shifted to 37 °C. Sterile human serum (10 %), and H2O2 (0·4–10 mM) were added to the medium just before cultivation. Citric acid (0·05–0·4 M) was added to the medium from a stock of 1·5 M, and the pH was adjusted to 3·5 by NaOH. The effect of osmotic stress was tested in medium containing either 1 M NaCl, 0·75 M sorbitol or 1·6 M glucose. To determine if polyols accumulated in proportion to the applied osmotic stress, cells were exposed to varying amounts of NaCl (0 to 1 M). Growth was monitored by measuring the OD600. For plate assays, an aliquot from an exponentially growing culture was serially diluted, and 5 µl from each dilution was spotted onto YNB/glucose plates with different stress conditions. All experiments were repeated at least twice.

Polyol extraction and measurement.
To determine the total amounts of polyols produced and accumulated by C. albicans, triplicate samples (1 ml) were heated at 100 °C for 10 min, and the supernatants obtained by centrifugation (1000 g, 5 min) were frozen until needed for polyol analysis. To differentiate intracellular or extracellular contents from the total polyol pools, harvested samples were filtered (Whatman GF/C filter 25 mm) or quickly centrifuged to separate the cell pellets from the supernatants. The supernatants were saved for extracellular-polyol measurements. The cell pellets on filter discs were washed three times with iso-osmolar media (this has the same osmolarity as the growth medium to avoid polyol efflux during washing), and immediately resuspended in 1 ml cold polyol extraction buffer (Van Eck et al., 1989). Intracellular polyols were then extracted by boiling the samples for 10 min. After centrifugation, the supernatant containing the extracted intracellular material was retained for further analysis. The total, intracellular and extracellular polyol contents were determined by GC. Samples (50 µl) were mixed with equal volumes of the internal standards methylmannoside and methylglucoside (80 µg ml–1). Acetone (1 ml) was added, followed by centrifugation to pellet insoluble materials. The supernatants were transferred to derivatization vials in a water bath (50 °C), and then dried completely (approximately 1 h) under a stream of nitrogen. For derivatization, 100 µl from a mixture of trimethylsilyl imidazole and dimethyl formamide was added to each dried sample and incubated for 45 min at 55 °C. After cooling to room temperature, 200 µl of hexane was added, mixed well and the sample centrifuged for 5 min at 3000 g. The supernatant was then analysed by GC. For the analysis of polyol content, analytical grade standards containing D-arabitol, ribitol, xylitol, mannitol, and sorbitol (Sigma-Aldrich) were used in varying concentrations ranging from 25 to 100 µg ml–1. Glycerol was measured with a commercial enzymic kit (Roche Diagnostics). For dry weight (biomass) measurements, Candida cells were harvested by filtration (GF/C 25 mm Whatman), and washed with an equal volume of water. The filters containing the cells were oven-dried at 80 °C overnight and then weighed. Polyol quantities were expressed in mg (g cell dry wt )–1. Student's t test was used to calculate the significance of differences between groups.

Construction and properties of C. albicans hog1 null mutants.
The homozygous C. albicans hog1{Delta}/hog1{Delta} mutants GK-Y001a and GK-Y001b were constructed in C. albicans strain BWP17 by the short-flanking-homology PCR method (Wilson et al., 1999). First, we used PCR with primers HOG1-5dr and HOG1-3dr (see Table 2 for all synthetic oligonucleotides) to add 69 bp of noncoding DNA from the 5' end, and 77 bp of noncoding DNA from the 3' end, of C. albicans HOG1 to (i) the URA3-dpl200 marker in plasmid pDDB57 (Wilson et al., 2000), (ii) the ARG4 marker in plasmid pRS-Arg4{Delta}SpeI, and (iii) the HIS1 marker in plasmid pGEM-HIS1 (Wilson et al., 1999).


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Table 2. Oligonucleotide primers

 
To delete the first chromosomal HOG1 allele, the targeting cassette that contained the URA3-dpl200 marker was introduced into C. albicans BWP17 by the lithium acetate method (Wilson et al., 1999), and the transformants were selected and purified on minimal media lacking uridine. The transformants were then tested for homologous integration of the URA3-dpl200 cassette into the HOG1 locus by gene-specific PCR using HOG1-primer 1 with HOG1-primer 2 or URA3-primer 3, and by Southern blot hybridization with a digoxigenin-labelled probe amplified from C. albicans SC5314 genomic DNA by PCR with HOG1-primer 5 and HOG1-primer 6 (Table 2).

The resultant HOG1/hog1{Delta} : : URA3-dpl200 mutant (strain GK-Y00) was transformed again with HOG1-targeting cassettes that contained either the ARG4 or the HIS1 marker; the respective transformants of interest were selected on minimal media lacking arginine or histidine, and their genotypes were determined by gene-specific PCR with HOG1-primer 1 and ARG4-primer 4, or HOG1-primer 1 and HIS1-detect (Table 2), and by Southern blot hybridization as above. To put a single copy of HOG1 back into the hog1{Delta}/hog1{Delta} (strain GK-Y001a), we first looped out the URA3 marker by cis recombination between the flanking dpl200 repeats, and then selected uridine auxotrophs with 0·1 % (w/v) 5-fluoroorotic acid (FOA). Next, a linearized construct containing the URA3 gene inserted between the coding and noncoding regions of HOG1 was generated by (i) using PCR with primers HOG1-SphI and HOG1-SacII (Table 2) to amplify a 2043 bp fragment of C. albicans SC5314 genomic DNA that contained the entire HOG1 ORF, (ii) digesting the resulting PCR product with SphI and SacII, and ligating it into the pGEM-URA3 plasmid, (iii) using PCR with primers HOG1-NotI and HOG1-SalI (Table 2) to amplify 500 bp of noncoding C. albicans SC5314 genomic DNA from the 3' end of HOG1, and (iv) digesting this PCR product with NotI and SalI, and ligating it into the pGEM-HOG1-URA3 plasmid on the 3' end of URA3. The resultant plasmid was linearized with SphI and NsiI, and the 3·9 kb fragment containing the HOG1 coding sequence, URA3 and 500 bp of noncoding DNA from the 3' end of the HOG1 locus was introduced into the FOA-resistant clone derived from C. albicans GK-Y001a by the lithium acetate method. Ura+ transformants were selected, and genomic DNA from these transformants was analysed for homologous integration of the targeting cassette into the HOG1 locus by gene-specific PCR and by Southern blot hybridization.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Polyol production and accumulation
We first analysed cultures of glucose-, galactose- or xylose-grown C. albicans cells to determine which polyols were produced, and whether these polyols were released into the environment. C. albicans cells cultured in YNB/glucose medium at 30 °C produced a large amount of D-arabitol, trace amounts of ribitol, and no detectable xylitol, mannitol, galactitol or sorbitol (Fig. 1). Enzymic analysis indicated that the cells also produced a large amount of glycerol. The pattern of polyol production was the same when the cells were cultivated in YNB/galactose or YNB/D-arabinose, but C. albicans produced large amounts of xylitol when it was cultured in YNB/xylose (data not shown).



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Fig. 1. Polyols produced by exponential phase C. albicans SC5314 cells grown at 30 °C. GC analyses of a standard mixture of multiple polyols (a) and extracts of C. albicans SC5314 cultures in YNB/glucose (b) showed that glucose-grown C. albicans produced large amounts of D-arabitol (retention time 12·49 min), smaller amounts of ribitol (12·63 min), and no detectable xylitol (12·24 min), mannitol (17·41 min), or sorbitol and galactitol (17·69 min). For quantitative analysis, {alpha}-methylmannoside (13·81 min) and {alpha}-methylglucoside (16·07 min) were added to the samples as internal standards. The peaks at 16·41 min and 18·20 min in (b) are anomers of glucose. Glycerol was quantified enzymically, data not shown.

 
Glycerol production was maximal after 6 h, whereas D-arabitol production reached maximal levels after 12 h (Fig. 2a, b). Most of the polyols produced by these cultures were released from the cells into the medium (Fig. 2c). Total polyol content declined after prolonged incubation, presumably because the polyols were catabolized after preferred substrates were exhausted.



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Fig. 2. Total and extracellular D-arabitol and glycerol production. C. albicans SC5314 ({blacktriangleup}) was cultured in YNB/glucose medium at 30 °C (a). Total (b) and extracellular (c) glycerol levels ({blacklozenge}) reached maximum levels during the first 5 h of growth, whereas total and extracellular D-arabitol levels ({blacksquare}) did not reach maximum levels until 12 h.

 
The morphological growth phase of C. albicans cells had little effect on polyol production. Glycerol and D-arabitol production by yeast-phase cells cultured in YNB/glucose was similar in value to that observed in hyphal-phase cells cultured in YNB/glucose+10 % human serum (Fig. 3a, b). Also, glycerol and D-arabitol production by hyphal-phase cells cultured in Lee's medium at 37 °C was similar in value to that observed in yeast-phase cells cultured in YNB-glucose at 37 °C (data not shown).



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Fig. 3. Effect of environmental stresses on polyol production and accumulation. (a) Total and intracellular glycerol levels increased markedly when exponentially growing C. albicans SC5314 cells were exposed for 2 h to osmotic and citric acid stresses, but not when they were exposed to increased temperatures or to H2O2. (b) In contrast, total D-arabitol levels increased markedly when C. albicans SC5314 cells were exposed for 2 h to increased temperature or to H2O2, but not when they were exposed to osmotic or citric acid stresses (data shown as means±SD, n>=3). Time-course studies showed that glycerol levels reached near-maximal levels within 2 h of exposure to osmotic stress (c), whereas D-arabitol levels did not reach near-maximal levels until 8–12 h after exposure to increased temperatures (d). Black bars, total polyol levels; hatched bars, intracellular polyol levels; {blacktriangleup}, 30 °C; {blacksquare}, 37 °C; {circ}, 30 °C/1 M NaCl; {bullet}, 37 °C/1 M NaCl.

 
Effects of environmental stresses on polyol production and accumulation
The effects of temperature, oxidative stress, osmotic stress and human serum on total polyol production, and on intracellular polyol content, are shown in Fig. 3. Glycerol production and accumulation increased markedly in cells exposed to 1 M NaCl or 0·4 M citric acid, but not in cells exposed to increased temperatures, H2O2 or serum (Fig. 3a). Increased glycerol production and accumulation were also observed when osmolar stress was induced with 0·75 M sorbitol or 1·6 M glucose (data not shown). In contrast, total D-arabitol production increased in cells exposed to elevated temperature or to H2O2, but not in cells exposed to 1 M NaCl or 0·4 M citric acid (Fig. 3b).

The increased production of D-arabitol at elevated temperatures was rapid, reaching maximum values of approximately 120 mg (g dry wt)–1 after 8 h (Fig. 3d). A similar trend was observed for ribitol production, albeit with lower concentrations. Approximately 6 mg (g dry wt)–1 ribitol was detected in the first 2 h at 37 °C, whereas only trace amounts were observed at 30 °C in the same growth period. Glycerol levels remained low throughout the entire growth period at 37 °C (Fig. 3c). However, time-course analysis indicated that when salt stress was applied, glycerol production increased to a greater extent during the first 2 h, whereas D-arabitol production did not increase until 12 h (Fig. 3d). Exposing cells to salt stress at 37 °C resulted in a rapid production of both glycerol and D-arabitol (Fig. 3c, d).

When C. albicans was grown in media containing graded amounts of NaCl for 2 h, the amount of intracellular glycerol increased directly in proportion to the extracellular osmolarity (Fig. 4a). Conversely, when osmotically stressed cells were transferred into media containing lower amounts of NaCl, the cells also released both intracellular glycerol and D-arabitol in proportion to extracellular osmolarity (Fig. 4b).



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Fig. 4. Polyol accumulation and release as functions of hyper- and hypo-osmotic stresses. The amount of intracellular glycerol increased directly in proportion to extracellular osmolarity (a) when C. albicans cells were grown in media containing graded amounts of NaCl for 2 h. Conversely, when osmotically stressed cells were transferred into media containing lower amounts of NaCl, the cells also released their intracellular polyols in proportion to extracellular osmolarity (b). Intracellular D-arabitol and glycerol content retained 5 min after hypo-osmotic shock were determined and expressed in percentages, taking the iso-osmotic values (transfer to hypo-osmotic medium containing 1 M NaCl) as 100 % (data shown as means±SD, n>=3). Black bars, intracellular D-arabitol; hatched bars, intracellular glycerol.

 
Effects of HOG1 on stress tolerance and polyol production
Since C. albicans HOG1 is known to regulate glycerol production in response to osmotic stress (San José et al., 1996), we next investigated whether this gene is required for glycerol production in response to other environmental stresses, and also if HOG1 plays a role in regulating D-arabitol production. We used homologous gene targeting to construct two independent C. albicans hog1 null mutants (strains GK-Y001a & GK-Y001b ) and to reintroduce a single copy of the C. albicans HOG1 back into its homologous locus in strain GK-Y001a, thereby generating strain GK-Y002 (hog1/hog1+HOG1). The genotypes of these strains were verified by gene-specific PCR and by genomic Southern blot hybridization (Fig. 5).



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Fig. 5. Targeted disruption of C. albicans HOG1. (a) Homologous gene targeting was used to replace the HOG1 coding sequence (bold arrow) in one chromosomal allele with URA3, and to replace the HOG1 coding sequence in the second chromosomal allele with either ARG4 or HIS1. The positions of the PCR primers, HindIII restriction sites (H), and the hybridization probe used to analyse the transformants of interest are shown. (b) HOG1-specific PCR of genomic DNA with primers 1 and 2 (see a) generated the expected 1·5 kb product from wild-type strain BWP17 (lane 1), the wild-type 1·5 kb product and a 2·2 kb URA3-containing product from strain GK-Y00 (HOG1/hog1{Delta} : : URA3-dpl200) (lane 2), and a 2·2 kb URA3-containing product and a 2·5 kb ARG4-containing product from strain GK-Y001a (hog1{Delta} : : ARG4/hog1{Delta} : : URA3-dpl200) (lane 3). PCR with primers 1 and 3 (lane 4), and 1 and 4 (lane 5) generated the expected 1·3 kb and 1·1 kb products from strain GK-Y001a, whereas PCR with primers 1 and HIS1-detect (lane 6) generated the expected 1·9 kb product from strain GK-Y001b (hog1{Delta} : : HIS1/hog1{Delta} : : URA3-dpl200). To put HOG1 back into strain GK-Y001a, we first looped out the URA3 marker, after which uridine auxotrophs were selected with 0·1 % (w/v) FOA and confirmed by PCR with HOG1-primer 1 and URA3-primer 3 (lane 7). When a 3·9 kb fragment containing URA3 and the HOG1 coding and flanking sequences was reintroduced into the FOA-resistant strain, PCR with URA3-primer 3 and HOG1-putback primer 7 generated the expected 1·85 kb product (lane 8), confirming that the URA3-HOG1 cassette had been reintegrated into its homologous locus. (c) When HindIII digests of genomic DNA from selected strains were hybridized with the probe shown in (a), the expected 5·2 kb band was found in C. albicans BWP17 (wildtype, WT), 2·1 and 3·6 kb bands were found in the hog1 null strain GK-Y001a (hh), and the 3·6 kb band and a 6·5 kb band were found in strain GK-Y002, the hog1 null strain into which a wild-type HOG1 allele had been reintroduced (h/H).

 
Since the methods and host strains we used to construct the C. albicans hog1 null mutants described above differed from those used by others (San José et al., 1996; Alonso-Monge et al., 2003), we first examined C. albicans strains GK-Y001a, GK-Y001b (hog1/hog1), GK-Y002 (hog1/hog1+HOG1) and a wild-type control, DAY185, for growth and stress phenotypes that have previously been described for C. albicans hog1 null mutants. We found that (i) C. albicans GK-Y001a and GK-Y001b were hypersensitive to osmotic and oxidative stresses, as was observed by San José et al. (1996) and Alonso-Monge et al. (2003), and (ii) these phenotypic abnormalities were corrected by reintroduction of a wild-type HOG1 allele (Fig. 6a).



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Fig. 6. Effect of hog1 null mutation on stress tolerance. When serial (10-fold) dilutions of the strains of interest were incubated under the conditions shown (a–c), we found that (i) the hog1 null mutants hog1{Delta} : : ARG4/hog1{Delta} : : URA3-dpl200 strain (hh) and hog1{Delta} : : HIS1/hog1{Delta} : : URA3-dpl200 strain (hh2) were more sensitive to osmotic, oxidative (a) and citric acid (b) stresses than was their wild-type parent (WT) or the HOG1 single allele mutant (Hh), and (ii) these phenotypic abnormalities were corrected by reintroducing one copy of the wild-type HOG1 (h/H). In contrast, the hog1 null mutation had no significant effect on growth at 37 or 40 °C (c).

 
It has recently been shown that the S. cerevisiae hog1 mutants are sensitive to citric acid stress (Lawrence et al., 2004), and that heat stress activates the yeast HOG1 pathway (Winkler et al., 2002). Therefore, we examined the effects of citric acid stress and heat stress on the C. albicans hog1 null mutants. We found that they were also sensitive to citric acid stresses at concentrations as low as 0·05 M, and this phenotype abnormality was corrected by reintroduction of a wild-type HOG1 allele (Fig. 6b). In contrast, the hog1 null mutation had no significant effect on growth at 37 or 40 °C (Fig. 6c). This last observation was in agreement with that of Smith et al. (2004), who recently showed that C. albicans hog1 null mutants were not sensitive to elevated temperatures, Hog1p phosphorylation was down-regulated upon heat stress and Hog1p did not translocate to the nucleus upon temperature upshift.

When we analysed the hog1 null mutants' polyol responses to various environmental stresses, we found that stress-induced polyol production was affected in several ways. First, the C. albicans hog1 null mutants produced much less glycerol (<30 %), when they were exposed to 1 M NaCl or 0·4 M citric acid, than did wild-type controls (Fig. 7a, c, d), and reintroduction of a single copy of wild-type HOG1 restored glycerol production almost to wild-type levels (Fig. 7a). Because of the possibility that the effects of 0·4 M citric acid were due to increased osmotic stress, we also measured glycerol production by cells exposed to lower citric acid concentrations. The effects of 0·1 and 0·2 M citric acid in wild-type C. albicans, in the hog1 null mutant strain and in the hog1/hog1+HOG1 reconstituted strain were similar to the effects of 0·4 M citric acid, except that they were of lower magnitude. Thus, glycerol induction by citric acid was not simply due to osmotic effects. Similar observations have been recently reported in S. cerevisiae (Lawrence et al., 2004). Second, the C. albicans hog1 null mutants produced much less D-arabitol when they were exposed to H2O2 than did wild-type controls (Fig. 7b, e), and reintroduction of a single wild-type HOG1 allele also restored D-arabitol production to almost wild-type levels (Fig. 7b). Third, D-arabitol production increased substantially when either wild-type C. albicans or the C. albicans hog1 null mutants were shifted from 30 to 37 °C (Fig. 7b, f) or from 30 to 42 °C (data not shown), whereas increased temperatures had no significant effects on glycerol production by either of these strains (Fig. 7a, f). Lastly, the hog1 null mutants produced significantly more D-arabitol than did wild-type controls when they were exposed to osmotic or citric acid stresses (Fig. 7b, c, d).



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Fig. 7. Effect of hog1 null mutation on polyol production and accumulation. C. albicans hog1 null mutants produced less glycerol when they were exposed to citric acid or osmotic stresses (a, c, d), and less D-arabitol when they were exposed to H2O2 stress (b, c) than did their wild-type parents. These abnormalities were corrected by reintroduction of a wild-type HOG1 allele (a, b). Production of glycerol and D-arabitol in response to increased temperature did not differ significantly in the wild-type and hog1 mutants (a, b) (data shown as means±SD, n>=3, * indicates P<0·05). Hatched bar, wild-type; white bar, hog1{Delta}; black bar, hog1{Delta}+HOG1. (c–f) Time-course studies showed that the C. albicans hog1 null mutant's inability to respond to each of the environmental stresses by producing polyols were partial rather than complete. (c) 1 M NaCl, (d) 0·4 M citric acid, (e) 10 mM H2O2 and (f) 37 °C. {bullet}, Wild-type; {blacktriangleup}, hog1{Delta}; solid line, glycerol production; broken line, D-arabitol production.

 
To verify that the effects described above were due to the presence or absence of HOG1, and not to extraneous mutations introduced inadvertently during strain construction, we also tested several additional strains for their abilities to grow and to produce polyols in the presence of selected environmental stresses. There were no significant differences in growth (Fig. 6) in the presence of NaCl, H2O2, citric acid or heat stress (i) between wild-type C. albicans strain DAY185, the heterozygous HOG1/hog1 mutant strain GK-Y00, and the hog1 null mutant into which HOG1 had been reintroduced (strain GK-Y002) or (ii) between the two independently generated C. albicans hog1 null mutants GK-Y001a and GK-Y001b (Fig. 6). Similarly, there were no significant differences in the amounts of glycerol or D-arabitol produced in response to exposure for 2 h to NaCl, H2O2 or heat stress (i) between strains DAY185, GK-Y00 and GK-Y002 or (ii) between strains GK-Y001a and GK-Y001b (Table 3).


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Table 3. Effect of hog1 mutation on total polyol production

Total glycerol and total D-arabitol at 2 h. Data shown as mg (g dry wt)–1±SD. ND, Not determined.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It was previously reported that C. albicans produces large amounts of D-arabitol in culture and in infected mammalian tissues (Kiehn et al., 1979; Wong et al., 1982), that C. albicans produces glycerol in response to osmotic stress (San José et al., 1996), and that C. albicans hog1 null mutants are intolerant to some environmental stresses (Alonso-Monge et al., 2003) and are hypovirulent in mice (Alonso-Monge et al., 1999). The present study extends those previous observations by examining how the different polyols are produced and accumulated by C. albicans under various environmental conditions that might be encountered during infection of a mammalian host, and also by examining the role of the MAP kinase Hog1p in regulating stress-induced D-arabitol and glycerol accumulation under various environmental conditions. We found that C. albicans responds to some environmental stresses by producing and accumulating glycerol, and to others by producing and accumulating D-arabitol. For example, glycerol production and accumulation increased markedly in cells exposed to NaCl and citric acid, but not in cells exposed to increased temperatures, H2O2 or serum. In contrast, D-arabitol production increased in cells exposed to elevated temperature and to H2O2, but not in cells exposed to NaCl or citric acid. These observations indicate that D-arabitol and glycerol are differentially regulated in C. albicans, which suggests that these two polyols may serve different functions. To our knowledge, it has not previously been shown that any fungus responds to different environmental stresses by producing and accumulating different acyclic polyols.

Since MAP kinases such as Hog1p are known to regulate stress responses in several fungal species, we tested C. albicans hog1 null mutants for the ability to produce polyols in response to several different environmental stresses. We found that the HOG1 gene product was required in C. albicans for wild-type glycerol production in response to osmotic or citric acid stresses, and for wild-type D-arabitol production in response to oxidative stress, but not for wild-type D-arabitol production in response to temperature stress. The observations that the hog1 null mutation did not completely abolish increased glycerol production in response to osmotic and citric acid stresses, and that it did not have any significant effect on increased D-arabitol production in response to temperature stress imply that glycerol and D-arabitol synthesis in C. albicans are regulated by both HOG1-dependent and HOG1-independent pathways. Lastly, the observation that D-arabitol production increased when C. albicans hog1 null mutants were subjected to osmotic or citric acid stress suggested that D-arabitol may function as a supplementary compatible solute when glycerol responses are attenuated, and it also showed clearly that its production is regulated at least partially via HOG1-independent pathways. Similar observations have been reported in the rice blast fungus Magnaporthe grisea, in which the MAP kinase homologue Osm1p was shown to regulate arabitol synthesis in response to osmotic stress, whereas glycerol accumulation in appressoria was independent of Osm1p (Dixon et al., 1999). Regulation of glycerol and D-arabitol production via HOG1-dependent and HOG1-independent pathways, and in response to different environmental stimuli could be accomplished via (i) separate but intersecting signal-transduction pathways that regulate the expression of single glycerol or D-arabitol biosynthetic enzyme, (ii) differential regulation of separate isoforms of key glycerol or D-arabitol biosynthetic enzymes [as in GPD1 vs GPD2, and GPP1 vs GPP2 in S. cerevisiae (Ansell et al., 1997; Norbeck et al., 1996)] or (iii) completely different biochemical pathways by which glycerol and/or D-arabitol are synthesized in response to different environment stresses. Determining if any or all of these possibilities is correct will require detailed analysis of the enzymes and genes responsible for glycerol biosynthesis and degradation, and identification and analysis of key enzymes that catalyse D-arabitol biosynthesis.

Fungi synthesize glycerol by reducing the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate with the enzyme glycerol-3-phosphate dehydrogenase (Gpd), and by dephosphorylating glycerol-3-phosphate with the enzyme glycerol-3-phosphate phosphatase (Gpp). The C. albicans genome contains homologues of S. cerevisiae GPD1, GPD2 and GPP1 (http://www.candidagenome.org). Expression of GPD2 and GPP1 is strongly induced in C. albicans by osmotic stress (Enjalbert et al., 2003; Fan et al., 2005), and the kinetics of osmotic- or oxidant-induced GPP1 expression correlates with the kinetics of Hog1p phosphorylation and nuclear localization (Smith et al., 2004). Interestingly, osmotic- or oxidative stress-induced GPP1 expression was significantly reduced in the hog1 null mutant. In contrast, GPP1 expression was equally induced by increased temperature both in the C. albicans hog1 null mutant and in its isogenic wild-type (Smith et al., 2004). Thus, it is apparent from the transcriptional profiling data that there are HOG1-dependent and HOG1-independent mechanisms regulating stress-induced gene expression in C. albicans, and that Hog1p might play different roles in the regulation of different stress-activated genes and their end products. Our results extend the transcriptional profiling results summarized above by quantifying the metabolic end products of the pathway of interest, both in wild-type C. albicans and in hog1 null mutants. Our observations that glycerol production increased markedly in response to osmotic stress in wild-type C. albicans, and that this response was markedly attenuated in hog1 mutants agreed with the observation by Smith et al. (2004) that GPP1 expression was induced in wild-type C. albicans, but not in a hog1 null mutant. However, we found no significant increases in glycerol production when C. albicans was exposed to increased temperature or oxidative stresses, whereas Smith et al. (2004) observed increased expression of GPP1 under similar conditions. These contrasting findings highlight the importance of quantifying the metabolic end product of the pathway of interest, in addition to examining patterns of gene expression. In the case of glycerol, an increase in GPP1 transcription might not result in increased glycerol production without a corresponding increase in GPD2 transcription. Furthermore, the glycerol biosynthetic rate is only one factor that may determine the amount of glycerol that the organism accumulates intracellularly. Additional processes that may participate in regulating intracellular glycerol concentrations include degradation and/or metabolism of intracellular glycerol, efflux of intracellular glycerol through the plasma membrane, and active transport of extracellular glycerol into the cell. Indeed, recent transport studies have demonstrated the presence of a constitutive active glycerol-uptake system of the proton-symport type, and a channel-mediated glycerol efflux system in C. albicans (G. Kayingo & B. Wong, unpublished data).

Although C. albicans is known to catabolize D-arabitol via an ARD1-dependent pathway (Wong et al., 1993, 1995), the pathway by which C. albicans synthesizes D-arabitol is not yet known. Therefore, we cannot comment on the mechanisms by which C. albicans regulates D-arabitol production and accumulation. Nevertheless, the results of this study suggest that D-arabitol may play a role in stress responses and pathogenesis. In the process of colonizing mucosal surfaces and/or invading deeper structures in mammalian tissues, C. albicans must adapt to a number of environmental stresses, including temperatures >=37 °C, and reactive oxidants produced by host phagocytes. It was previously reported that C. albicans hog1 null mutants underproduce glycerol and grow poorly in hyperosmotic environments, are hypersusceptible to oxidants such as H2O2, menadione and potassium superoxide, and are hypovirulent in mice (Alonso-Monge et al., 1999, 2003; San José et al., 1996). Consequently, it has generally been assumed that glycerol is the key intracellular stress protectant in C. albicans. However, the present study has shown that D-arabitol was much more abundant than glycerol in C. albicans cells grown at 37 °C. Moreover, C. albicans responded to oxidative stress by increasing D-arabitol production and not by increasing glycerol production. Lastly, increased D-arabitol production in response to oxidative stress was HOG1 dependent. Thus, it is possible that the inability of C. albicans hog1 null mutants to increase D-arabitol production in response to oxidant and other stresses may contribute to the hypovirulence of these mutants.

In summary, we have shown that D-arabitol and glycerol are the principal polyols produced by C. albicans. We also found that C. albicans responds to osmotic and citric acid stresses by producing and accumulating glycerol, and to temperature and oxidative stresses by producing D-arabitol. The MAP kinase Hog1p participates in regulating glycerol production during osmotic or citric acid stress, and D-arabitol production during oxidative stress conditions, but not during temperature stress. Because these results suggest that both glycerol and D-arabitol may play important roles in stress tolerance and pathogenesis, our future studies will focus on the downstream targets of Hog1p, enzymes in the D-arabitol and glycerol biosynthetic and catabolic pathways, and the Hog1p-dependent and -independent means by which these pathways are regulated in C. albicans.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the United States Department of Veterans Affairs, and by National Institutes of Health grant R01 AI-47442 (to B. Wong). We are grateful to V. Kalb, Y. Mao, S. Lee, A. Bartiss and Z. Zhang for useful discussions. We thank Dr A. P. Mitchell for the C. albicans strains and plasmids.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 16 March 2005; revised 3 June 2005; accepted 9 June 2005.



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