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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 (0·410 mM) were added to the medium just before cultivation. Citric acid (0·050·4 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 ml1). 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 ml1. 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/hog1
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
SpeI, and (iii) the HIS1 marker in plasmid pGEM-HIS1 (Wilson et al., 1999
).
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The resultant HOG1/hog1 : : 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
/hog1
(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.
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RESULTS |
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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 16 March 2005;
revised 3 June 2005;
accepted 9 June 2005.
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