Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, E-28040 Madrid, Spain
Correspondence
Jesús Pla
jesuspla{at}farm.ucm.es
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ABSTRACT |
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INTRODUCTION |
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In fission yeast, a functional-homologue route to the HOG pathway is mediated by the Sty1 MAP kinase, which responds to several stresses, such as heat and cold shock and osmotic and oxidative stress (Samejima et al., 1997; Shiozaki & Russell, 1996
; Soto et al., 2002
). sty1 mutants are sterile and exhibit a G2 cell-cycle delay, indicating additional roles for Sty1 in meiosis and cell-cycle progression (Millar, 1999
; Shiozaki & Russell, 1995
, 1996
). The range of environmental insults that activate Sty1 is similar to the stimuli that activate the mammalian SAPKs: JNKs and p38/CSBP (Galcheva-Gargova et al., 1994
; Kyriakis & Avruch, 1996
), which control a wide variety of physiological and pathological conditions.
Candida albicans is a pathogenic yeast of great clinical interest (Fox, 1993; Odds, 1988
). Four MAP kinases have so far been identified in this organism: Mkc1, the homologue to the Slt2/Mpk1 MAP kinase from S. cerevisiae (Navarro-García et al., 1995
), Cek1, homologue to Kss1 (Csank et al., 1998
), Cek2, homologue to Fus3 (Chen et al., 2002
) and Hog1, homologue to the Hog1 MAP kinase. Hog1 has been implicated in different functions in C. albicans, such as glycerol accumulation, morphological transitions, cell-wall biogenesis and virulence (Alonso-Monge et al., 1999
; San José et al., 1996
). It has also been shown how this MAP kinase is phosphorylated when exposed to NaCl and hydrogen peroxide (Alonso-Monge et al., 2003
). Recent studies have suggested that it also exerts a regulatory role on other MAP kinases, Mkc1 and Cek1 (F. Navarro-García, B. Eisman, S. Fiuza, C. Nombela & J. Pla, unpublished data). However, the overall organization of the HOG pathway in C. albicans is not clear. Calera and co-workers reported the isolation of Ypd1 (Calera et al., 2000a
) and the Ssk1 response regulator (Calera et al., 2000b
), a member of a two-component signal-transduction pathway that was recently shown to play a role in the transmission of oxidative stress (Chauhan et al., 2003
). The role of other putative components is not clear (Chauhan et al., 2003
; Yamada-Okabe et al., 1999
), despite their role in cell-wall construction (Kruppa et al., 2003
, 2004
; Yamada-Okabe et al., 1999
).
In this work, we have identified the gene homologue to PBS2 in C. albicans and shown this protein to play a role in cell-wall construction. We also present evidence that Pbs2 mediates phosphorylation of Hog1 in response to both osmotic and oxidative stress, a phenomenon that correlates with translocation of the Hog1 MAP kinase. These results indicate that the HOG pathway is involved in the development of an adaptive response in this fungal pathogen.
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METHODS |
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Sensitivity on solid medium was tested on YPD medium supplemented with different compounds (oxidative agent, NaCl, sorbitol, Congo red or calcofluor white) at the indicated concentrations. Serially diluted (1/10) cell suspensions were spotted to examine the growth of the different strains. Plates were incubated overnight at 37 °C unless indicated otherwise.
Filamentation assays were performed by using overnight cultures in liquid YPD medium at 30 °C that were refreshed to 105106 cells ml1 in liquid YPD medium supplemented with 5 % fetal bovine serum and incubated at 24, 30 and 37 °C.
Deletion of the PBS2 gene.
A C. albicans genomic library (Navarro-García et al., 1995) was used in a screening designed to complement the osmosensitivity of a S. cerevisiae sho1 ssk2 ssk22 strain in a ura3 background. Clone 66 was identified to bear the 10·43 kb plasmid in which the PBS2 gene was identified. A 4·46 kb SnaBIClaI fragment carrying the S. cerevisiae URA3 marker gene was eliminated, generating the c66-YEP-URA plasmid. A 891 bp AccISpeI fragment from the PBS2 ORF was replaced with a 3·35 kb AccISpeI fragment from pD1 carrying the CaURA3 marker flanked by the chloramphenicol acetyltransferase (cat) gene from Escherichia coli (B. Eisman, personal communication). A 5·4 kb KpnIXbaI fragment carrying the deletion construction was used to force homologous recombination and deletion of the PBS2 gene in the RM1000 and CNC15 strains. Strains BDR18 were obtained following the procedure described previously for genetic transformation (Köhler et al., 1997
). Correct disruptions were confirmed by PCR using primers o-PBSRV (5'-CTCGGTTGACCATCTTGATC-3') and o-URA3CR (5'-GTGTTACGAATCAATGGCACTACAGC-3') for Ura+ strains or o-PBSRV and o-CF (5'-GATGTGGCGTGTCACGGTCAAA-3') for Ura strains, as well as by Southern blot. In this case, genomic DNA was digested with PvuII and the probe was obtained by PCR using the primers o-NPBSXHO (5'-CTCGAGTCTAGATTATTTATAAATATGTTTGG-3') and o-NPBSCLA (5'-ATATCGATATCTTCAACCATTCTTAATTGG-3').
HOG1-GFP and PBS2-GFP integration.
The HOG1-GFP chimaera was constructed by PCR amplification of the ORF using the primers o-5'HHOG1 (5'-CCAAAGCTTATGTCTGCAGATGGAGAATTTACA-3') and o-HOG1fusion2 (5'-GGAAAGCTTCCACCACCAGCTCCGTTGGCGGAATCCAA-3'). After incorporation of this fragment into the pGEM-T vector (Promega), it was excised as a 1138 bp fragment that was subsequently introduced in the large HindIII fragment of the pGFP-URA3 plasmid (Gerami-Nejad et al., 2001). This construction was integrated at the HOG1 locus after digestion with SalI to render the construction linear. Correct integration was detected by PCR with primers o-CH1 (5'-TCGCGAAGATCTGAAAATGTCTGCAGATGGAG-3') and o-GFPlo (5'-CCAGTAGTACAAATAAATTTTAAGGTC-3'). ACT1p-HOG1-GFP was constructed by amplifying the HOG1-GFP fusion using the primers o-5'-ACTHOG (5'-CCTCTAGAATGTCTGCAGATGGAGAATTTAC-3') and o-3-TADH (5'-CCTCTAGATTGTTTCCGTTTATACCATCC-3') and cloning it into the XbaI site of the plasmid PIR4 (I. Rios, personal communication) derived from pRM1 (Pla et al., 1995
), where the ACT1 promoter was inserted. The construction wasintegrated at the LEU2 locus after digestion with KpnI to render the plasmid linear.
The PBS2-GFP fusion was constructed by amplifying the PBS2 ORF by PCR using the primers o-PBS2N-upper (5'-GCGGCCGCTTACCAATTAAGAATGGTTG-3') and o-PBS2N-lower (5'-GCGGCCGCGGCGGATGATTATTAAGAAAGCTTCT-3'); the fragment was inserted into the NotI site of pACT1-GFP (E. Román, personal communication), a plasmid derived from pRM1, where the ACT1 promoter and the GFP gene (Cormack et al., 1997) were first inserted. The construction was then integrated at the LEU2 locus of the C. albicans genome after digestion with KpnI. Similarly, the PBS2-GFP
N fusion was obtained by amplifying an N-terminally truncated version of the PBS2 ORF by using the primers o-PBS2-NSH3 (5'-GCGGCCGCAGGGATTCTGCTCAACACTAC-3') and o-PBS2N-lower. The PCR product was also inserted at the NotI site of pACT1-GFP and integrated in the LEU2 locus after digestion with KpnI.
Nikkomycin Z assay.
MICs were determined by the microdilution method in 96-well plates as described previously (Navarro-García et al., 1998; NCCLS, 1992
). Cells (103 per well) were inoculated into SD minimal medium without uridine.
-1,3-Glucanase-sensitivity assay.
To measure the inhibition of growth caused by Zymolyase, cell cultures from an exponentially growing culture were inoculated to an OD620 of 0·025 in YPD medium supplemented with different amounts of Zymolyase 100T (ICN Biomedicals). Zymolyase was suspended in Tris/HCl (pH 7·5), 5 % glucose.
Oxidative-stress assays.
Hydrogen peroxide and menadione sodium bisulfite (MD) were obtained from Sigma. Dilutions were performed by using sterile, double-distilled H2O. Susceptibility to hydrogen peroxide was measured by using exponential- or stationary-phase growing cells in YPD medium at 37 °C. Cells (107) were then transferred to an Eppendorf tube and hydrogen peroxide was added to a final concentration of 50 mM. Tubes were incubated at 37 °C and 5 µl samples were collected at different times and spotted onto YPD plates, which were then incubated for 24 h at 37 °C and photographed. Susceptibility to MD and other oxidants was quantified in a similar way by using different concentrations (indicated in the figure legends).
Protein extracts and immunoblot analysis.
Yeast strains were grown to an OD600 of 1 at 37 °C in YPD medium (or SD medium in the case of Hog1GFP detection). NaCl or hydrogen peroxide was added to the medium at the final concentration indicated. Samples were taken 10 min after the challenge or at different time points, as indicated in the figures. Cell extracts were obtained as described previously (Martín et al., 2000). Equal amounts of proteins were loaded onto gels, as assessed by A280 measurement of the samples and Ponceau Red staining of the membranes prior to blocking and detection. Blots were probed with a phospho-p38 MAP kinase (Thr180/Tyr182) 28B10 mAb (Cell Signaling Technology) (Ab-p38P in the figures), an ScHog1 polyclonal antibody (Santa Cruz Biotechnology) (Ab-Hog1 in the figures) and phospho-p42/44 MAP kinase (Thr202/Tyr204) (Cell Signaling Technology) (Ab-p42-44P) and Ab-GFP (JL-8) (Clontech) mAbs, and developed according to the manufacturer's conditions by using a Hybond ECL kit (Amersham Biosciences).
Fluorescence microscopy.
Yeast strains were grown at 37 °C in SD medium to an OD600 of 0·8. In the case of treated cells, NaCl or hydrogen peroxide was added to the concentration specified in the figure legend and incubated for 5 min unless indicated otherwise. Samples were centrifuged and washed twice with PBS. Cells were fixed with 70 % ice-cold ethanol for 1 min, centrifuged and washed twice with PBS. DAPI (4',6-diamidino-2-phenylindole) was added to a final concentration of 2 µg ml1 to stain the DNA, thereby marking the nucleus. Viable cells were observed directly under a microscope and challenged with osmotic or oxidative shock. Fluorescence microscopy was carried out on a Nikon Eclipse TE2000-U microscope at 100x magnification. Images were captured by a Hamamatsu ORCA-ER CCD camera using AquaCosmos 1.3 software. All images were processed identically and mounted by using Adobe Photoshop 7.0.
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RESULTS |
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C. albicans PBS2 has been annotated in the Institute Pasteur Database (Fungal Galail Group) as IPF3329. This gene is 1638 bp long and encodes a 545 aa protein that shares high identity (63 %) and similarity (77 %) within the tyrosine kinase domain with the S. cerevisiae orthologue. Interestingly, differences appear in the ends, as the CaPbs2 C-terminal domain is 37 aa longer than the S. cerevisiae homologue. Although the N-terminal domain of ScPbs2 is longer than that of the C. albicans protein, both kinases conserve the proline-rich domain through which Pbs2 binds the transmembrane protein Sho1 in S. cerevisiae (Maeda et al., 1995; Posas & Saito, 1997
). In this organism, the N-terminal domain also contains the docking site for Ssk2 and Ssk22 and the nuclear export signal (NES), whilst the nuclear localization sequence (NLS) is located in the C-terminal domain of the protein (Tatebayashi et al., 2003
). Similar domains can be identified in the protein sequence of the C. albicans Pbs2, suggesting a functional conservation between both proteins (the putative NES domain can be located between aa 8 and 10, whilst the potential NLS domain is located between aa 76 and 83).
The Pbs2 proline-rich domain is dispensable to transmit the signal to the Hog1 MAP kinase
In S. cerevisiae, the N-terminal proline-rich domain is responsible for the interaction with the Sho1 adaptor protein and transmission of the signal (Maeda et al., 1995) towards the MAP kinase cascade. In order to check the functionality of this region in CaPBS2, we constructed a mutant allele of PBS2 in which this domain was deleted (aa 151). As described above, this fragment of CaPbs2 also includes the putative NES domain; deletion of the NES domain in S. cerevisiae confines the protein permanently to the nucleus (Tatebayashi et al., 2003
). When wild-type and mutant alleles were introduced in a pbs2 strain under the control of the ACT1 promoter, they complemented all phenotypes attributed to pbs2 and restored signalling in response to hydrogen peroxide and sodium chloride, as with the wild-type allele (Figs 1, 4
and to be described through the present work). From these observations, we conclude that the proline-rich domain is dispensable to transmit the signal towards the downstream MAP kinases.
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As expected, the absence of the PBS2 gene generated cells that were defective in growth under hyperosmotic conditions. As shown in Fig. 1, no differences were found in osmosensitivity between the hog1, pbs2 and hog1 pbs2 mutant strains. This phenotype was not restricted to sodium chloride, but was also evident with sorbitol, an osmostressing agent of a non-ionic nature. Such defects were also observed when cells were grown in liquid YPD medium supplemented with 1 M sorbitol. After 24 h growth in this medium, pbs2 mutants reached an OD600 of 4·83 (9 in YPD medium), in contrast to the values observed for wild-type strains (6·89 and 9·5, respectively). These values were 1·21 for wild-type and 0·43 for the pbs2 mutant when 2 M sorbitol was used and 1·77 and 0·94, respectively, when 1·5 M NaCl was used. Under these restrictive conditions, cells displayed an altered morphology, remaining attached to their mother cell after budding, a phenomenon that resulted in clumped cells. This phenotype closely resembles the one previously observed for hog1 mutants (Alonso-Monge et al., 1999
), indicating a defect in cell separation (Fig. 1b
). The pbs2, hog1 and pbs2 hog1 mutants displayed a similar degree of osmosensitivity when assayed by using growth on both solid (Fig. 1a
) and liquid (data not shown) media, suggesting that Hog1 is the only target of Pbs2 in response to osmotic stress. These defects were reversed in pbs2 and pbs2 hog1 strains in which functional Pbs2GFP or even Pbs2
NGFP protein was reintroduced (Fig. 1a
). pbs2 cells were also unable to accumulate glycerol in response to osmotic stress when exponentially growing cells were challenged with 1 M sodium chloride (data not shown), indicating that this pathway mediates glycerol accumulation and suggesting that glycerol is a major compatible solute in this organism.
In order to define the role of PBS2 within the MAP kinase network, we performed Western blot assays to measure the phosphorylation state of the Hog1 MAP kinase by using antibodies against the TGY motif that is characteristic of stress kinases. No phosphorylated Hog1 could be detected in pbs2 mutants under either basal or activating conditions (1 M NaCl) (Fig. 2d), indicating that deletion of this kinase impairs signalling of osmotic stress towards the Hog1 MAP kinase.
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Hog1 is translocated to the nucleus upon saline shock
In order to investigate the intracellular localization of the MAP kinase Hog1 under different environmental conditions, we fused the ORF of HOG1 to the N terminus of the green fluorescent protein (GFP). When it was expressed under its own promoter, the HOG1-GFP fusion was able to rescue the osmosensitivity of the hog1 mutant cells. Western blot assays demonstrated that the protein fusion was expressed correctly, as it generated a polypeptide of the expected molecular mass (69·7 kDa) that was wholly functional (as it was able to complement hog1 mutant phenotypes similarly to a wild-type Hog1 protein) (data not shown). Nevertheless, the GFP signal was not intense enough to visualize the protein intracellularly. However, expression of the HOG1-GFP fusion under the ACT1 promoter, a constitutively strongly expressed promoter, revealed that the chimaera was localized throughout the cytoplasm and nucleus (being excluded from the vacuole) in non-stressed cells. When cells were exposed to an osmotic shock (1 M NaCl), the fluorescent signal concentrated in the nucleus (Fig. 2b). Translocation of Hog1 after osmotic challenge was rapid and could be observed in both fixed and viable cells (Figs 2 and 3
). As shown in Fig. 3
, both the phosphorylation and nuclear localization of Hog1 took place within the first minute after the shock and remained for approximately 15 min. These results suggest that these phenomena are temporally linked. The translocation of the Hog1GFP chimaera did not take place in a hog1 pbs2 mutant in response to osmotic stress, and neither activation (Fig. 2d
) nor nuclear accumulation (Fig. 2c
) was observed. These data indicate that PBS2 is required for both events to occur.
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hog1 and pbs2 mutant strains displayed different behaviour under oxidative stress
One of the most important challenges that C. albicans must face during progression of an infection is oxidative stress. Previous work from our group has shown that the Hog1 MAP kinase plays a crucial role in the response and adaptation to oxidative stress (Alonso-Monge et al., 2003). Not only is Hog1 phosphorylated when cells are exposed to hydrogen peroxide, but this MAP kinase is also needed to survive under oxidative-stress conditions. Is this role dependent exclusively on Hog1? To answer this question, we performed different experiments.
First, cells were spotted onto YPD plates supplemented with hydrogen peroxide or MD, the latter being a generator of superoxide ions. As can be observed in Fig. 4(a), hog1, pbs2 and hog1 pbs2 cells were sensitive to these compounds. The mutant strains were hardly able to grow on 300 µM MD or 6 mM hydrogen peroxide plates.
A slight but reproducible increase in sensitivity was observed in the pbs2 mutant, compared with hog1 and hog1 pbs2 mutant strains. To further characterize this different phenotype, we performed experiments in which the kinetics of viability were measured among the different strains. Exponential- and stationary-phase cultures were exposed to a lethal concentration of oxidant agents in liquid medium and samples were taken at different times and spotted onto YPD plates. As shown in Fig. 4(b and c), the pbs2 mutant lost viability faster than the wild-type and, again, faster than the hog1 mutant. Remarkably, the double mutant hog1 pbs2 displayed an intermediate phenotype. Western blot assays showed that Hog1 phosphorylation is also Pbs2-dependent in response to hydrogen peroxide (Figs 4d and 5
bc
), as well as in response to osmotic stress (NaCl). These results suggest that, although Pbs2 and Hog1 function within the same pathway, both proteins have additional and separate roles in mediating oxidative stress.
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The HOG pathway mediates adaptation to stress
When cells are exposed to sub-lethal stress, they mount an adaptive response that enables toleration to subsequent, more severe stresses. In order to determine the role of Pbs2 in this process, exponentially growing cells were exposed to a high concentration of hydrogen peroxide (50 mM) after a previous pre-treatment with a sub-lethal concentration of hydrogen peroxide (5 mM). Samples were taken at different times and spotted onto YPD plates at 37 °C. Only in the wild-type strain was an increase in tolerance observed when cells were pre-treated (Fig. 5a). Mutants in the MAP kinase and MAPK kinase (hog1, pbs2 and hog1 pbs2) lost viability even faster than samples that were not previously exposed to oxidative stress. As shown, hog1, pbs2 and hog1 pbs2 mutants died within seconds after addition of 50 mM hydrogen peroxide, a result that can be explained because of the failure of these strains to respond to the previous hydrogen peroxide treatment. Similar results were obtained when the assay was performed at 30 °C (data not shown). This observation suggests that hog1, pbs2 and hog1 pbs2 mutants are not only intrinsically more sensitive to oxidative agents, but also defective in the development of an adaptive response. One interpretation of this result is that oxidative challenge causes a high concentration of ROS in the mutant cells, which are unable to restore the basal/physiological levels of these reactive species because of the absence of Pbs2 and/or Hog1.
We also monitored the phosphorylation of MAP kinases by Western blot during this adaptation process. Cells were challenged with a low dose of hydrogen peroxide (5 mM) and afterwards were treated with a 50 mM hydrogen peroxide shock. As observed in Fig. 5(b), phosphorylation of Mkc1 is detected in response to the first oxidative shock, whereas a subsequent exposure fails to activate this MAP kinase. The Hog1 protein became phosphorylated after the first and second shocks, although with different kinetics, as this was delayed and less prolonged (only detectable within 5 min) after the second addition of hydrogen peroxide. Untreated cells activated Mkc1 and Hog1 at 50 mM hydrogen peroxide (data not shown).
The adaptive signal is relatively dose-independent: when using a lower concentration (5 mM hydrogen peroxide) as the second challenge, we observed similar results (Fig. 5c). Mkc1 became activated after the first oxidative shock, but not the second, whereas Hog1 was phosphorylated after both oxidative challenges. No activation of Hog1 was detected in pbs2 cells and, obviously, not in hog1 or hog1 pbs2 mutants. Phosphorylation of Mkc1 was significantly lower in these mutant strains than in the wild-type strain, as described above. These results imply that the cells adapted to oxidative stress in a relatively dose-independent manner.
Pbs2 and Hog1 play different roles in cell-wall biogenesis
The HOG pathway has been suggested to be involved in construction of the cell wall in both S. cerevisiae and C. albicans (Alonso-Monge et al., 1999; Jiang et al., 1995
). In S. cerevisiae, pbs2 and hog1 mutants have been reported to be resistant to calcofluor white (García-Rodriguez et al., 2000
) and sensitive to cell wall-degrading enzymes (Zymolyase and Quantazyme) (Alonso-Monge et al., 2001
; Kapteyn et al., 2001
). In C. albicans, disruption of the Hog1 MAP kinase results in cells that are resistant to certain cell-wall biogenesis inhibitors (Alonso-Monge et al., 1999
). To determine whether this phenotype is exclusive to the Hog1 MAP kinase, we checked the growth of pbs2 cells in the presence of Congo red and calcofluor white, two compounds that interfere with cell-wall assembly. We checked different concentrations of Congo red (150 and 200 µg ml1) and calcofluor white (12, 24 and 50 µg ml1). Perhaps unexpectedly, only those mutants where the HOG1 gene was absent showed resistance to Congo red (Fig. 6a
), whilst the pbs2 mutant displayed an intermediate resistance, observed only at the lowest Congo red concentration tested (150 µg ml1). Reintroduction of the PBS2 gene into the pbs2 mutant strain restored the wild-type phenotype. In the case of calcofluor white, the mutant strains behaved similarly, although differences were more subtle but still reproducible (Fig. 5b
). The arrangement of strains according to decreasing sensitivity to calcofluor white was: wild-type, pbs2, hog1 pbs2, hog1. Transformation of pbs2 mutant with the PBS2-GFP construct restored the wild-type phenotype (Fig. 5
). These data indicate a significant difference between hog1 and pbs2 mutants in C. albicans, in contrast to the situation in S. cerevisiae. It is noteworthy that no phosphorylation of Hog1 was detected in response to Congo red after 1 h incubation in YPD medium supplemented with 15 µg Congo red ml1 (data not shown).
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We also checked the susceptibility of the different strains to the -1,3-glucanase-enriched preparation Zymolyase (which also contains additional
-1,6-glucanase and proteinase activities). Cells were grown in YPD medium supplemented with different amounts of Zymolyase and incubated overnight. In this case, strains defective in PBS2 and HOG1 genes were significantly more sensitive to Zymolyase than wild-type and control strains in which the corresponding genes were reintroduced. hog1, pbs2 and hog1 pbs2 mutant strains lysed at 50 U Zymolyase ml1, whereas control strains were able to grow at concentrations higher than 200 U ml1.
Pbs2 represses hyphal formation in C. albicans
Morphological studies revealed that pbs2 mutants display an enhanced ability to develop hyphae under different conditions. Serum was added at limiting concentrations (1, 5, 10 and 20 %) to YPD liquid medium and cultures were incubated at different temperatures (24, 30 and 37 °C). Deletion of PBS2 increased hyphal growth under all conditions tested; pbs2 and hog1 pbs2 cells exhibited an enhanced ability to form filaments, even greater than that of the hog1 mutant. This phenotype is depicted in Fig. 7, in which the microscopic appearance of the cultures in YPD medium supplemented with 5 % fetal bovine serum is shown. HOG-pathway mutants filament at any temperature (24, 30 or 37 °C). The frequency of branched hyphae was similar between all strains and calcofluor staining of these filaments showed no differences compared with wild-type (data not shown), indicating that correct localization of chitin deposition in these structures also takes place in HOG-pathway mutants. They are, at least by using these criteria, similar to wild-type filaments.
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DISCUSSION |
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We previously demonstrated the involvement of Hog1 in the morphological transition, a physiologically relevant aspect in C. albicans (Alonso-Monge et al., 1999). pbs2 cells, as well as hog1 mutant cells, display a hyperfilamentous behaviour in limiting concentrations of serum. This result, together with the altered colony morphology exhibited by the mutants, reinforces the repressor role of the HOG pathway in the morphological transition (Alonso-Monge et al., 1999
).
In C. albicans, the Hog1 MAPK has been implicated in the response to oxidative stress (Alonso-Monge et al., 2003) and recent experiments demonstrate that hydrogen peroxide signalling to Hog1 is dependent on the Ssk1 branch (Chauhan et al., 2003
). The fact that Hog1 became phosphorylated when exposed to hydrogen peroxide in a Pbs2-dependent manner demonstrates the implication of this route in the response to oxidative stress. However, the differential sensitivity to oxidative agents displayed by the pbs2 and hog1 mutants suggests that Pbs2 may play an additional role that is independent of Hog1. This role could be mediated by Mkc1, as the level of phospho-Mkc1 in response to hydrogen peroxide is reduced significantly in pbs2, hog1 and pbs2 hog1 mutant strains. Another possible mechanism could be through Hog1. Hog1 could repress gene transcription in its non-phosphorylated state and, consequently, lack of the protein could cause a phenotype different from the inactive (not phosphorylated) Hog1, similar to that observed for other MAP kinases (Madhani et al., 1997
). Genes not repressed by Hog1 could then be expressed and, consequently, protect cells from a certain degree of oxidative stress. The oxidative-stress responses appear to be regulated, at least in part, at the transcription level, with Yap1 and Skn7 being two important oxidative-stress regulators in S. cerevisiae (Costa et al., 2002
). The Yap1 homologue in C. albicans, Cap1, does not appear to be Hog1-dependent (Alonso-Monge et al., 2003
), whilst the epistatic relationship between Hog1 and Skn7 has not been analysed in this pathogen. CaHog1 may control transcription through other transcription factors, such as Msn2 and Msn4 or Sko1.
An alternative possibility is that either Pbs2 or Hog1 could phosphorylate another kinase involved in response to oxidative damage. In Schizosaccharomyces pombe, Sty1, the homologue of Hog1, interacts with and phosphorylates the protein kinase Cmk2 under oxidative insult (Sánchez-Piris et al., 2002). Although the role of Cmk2 in oxidative stress is not clear, it has been suggested that Sty1 may regulate translation through Cmk2 to display oxidative stress-induced responses in a similar way to that in S. cerevisiae. ScHog1 activates the Rck2 kinase (Bilsland-Marchesan et al., 2000
; Teige et al., 2001
), which, in turn, inhibits protein biosynthesis. Although the role of Rck2 was initially defined in the context of osmostress, current studies show that the major role of Rck2 may be to deal with oxidative stress (Bilsland et al., 2004
). In S. cerevisiae, the signalling cascade is Pbs2Hog1Rck2 (Bilsland-Marchesan et al., 2000
; Jiang et al., 2004
), although Rck2 phosphorylation is not absolutely HOG1-dependent. In C. albicans, there are two homologue proteins to Rck2 and two to Cmk2 that could be controlled by Hog1 or Pbs2, generating a specific response to the stimulus. Both NaCl and hydrogen peroxide cause growth arrest in C. albicans (R. Alonso-Monge, unpublished data), possibly through some of these Ser/Thr protein kinases. Although both signals converge at the Pbs2 kinase and therefore to Hog1, the mechanisms implicated in the responses generated are not the same and additional elements must be involved to discriminate between stimuli. Moreover, the localization pattern observed for Hog1 depends on the insult. Clearly, CaHog1 is translocated to the nucleus when cells are exposed to saline stress, whereas no nuclear accumulation was observed under oxidative shock. This result disagrees with that observed by Smith et al. (2004)
. This group showed CaHog1 translocation upon different stress conditions, including hydrogen peroxide, CdSO4 and KCl. Different explanations for the discrepancy between our results and those reported previously could be the genetic approach used or simply the sensitivity of the system. In any case, the translocation of Hog1 upon oxidative stress has been reported recently in S. cerevisiae as being less pronounced and slower than that upon osmotic stress (Bilsland et al., 2004
; Smith et al., 2004
).
Our work suggests that the HOG pathway is also important for developing tolerance to oxidative stress. Hydrogen peroxide induces a specific protein oxidation in yeast cells; oxidative proteins accumulate in the cells, even at low adaptive levels. Treatment of C. albicans with low concentrations of either hydrogen peroxide or MD induces an adaptive response that protects cells from the lethal effects of a subsequent challenge with higher concentrations of these oxidants (Jamieson et al., 1996). This protection was not evident in cells lacking the HOG1 and/or PBS2 genes. Furthermore, although two MAPKs, Hog1 and Mkc1, are implicated in the oxidative-stress response in C. albicans, the activation pattern after a second oxidative challenge is different. Mkc1 is phosphorylated after the first oxidative insult alone, whilst Hog1 activation is observed after the first and second hydrogen peroxide challenges. This observation may suggest that the response generated by Mkc1 confers tolerance to the stress, although disruption of MKC1 does not lead to hypersensitivity to oxidative agents (F. Navarro-García, B. Eisman, S. Fiuza, C. Nombela & J. Pla, unpublished data).
Another interesting observation is the differential phenotype related to the cell wall that was detected in hog1 and pbs2 mutants. The link between the HOG pathway and cell-wall architecture has been reported in both S. cerevisiae (Alonso-Monge et al., 2001; García-Rodriguez et al., 2000
; Jiang et al., 1995
; Kapteyn et al., 2001
) and C. albicans (Alonso-Monge et al., 1999
). We describe growth differences between hog1 and pbs2 in the presence of Congo red and calcofluor white. No other significant differences between hog1 and pbs2 mutants were observed when susceptibility to Nikkomycin Z or Zymolyase was assessed. Nevertheless, it is remarkable to note the resistance to the chitinase inhibitor Nikkomycin Z that was displayed by mutants in the HOG pathway compared with the wild-type strain and, in contrast, the higher sensitivity to the
-1,3-glucanase Zymolyase. The lack of HOG1 and/or PBS2 genes may cause alterations in the cell-wall composition or architecture and these changes in the distribution and proportion of the polymers that form the cell wall may lead to this phenotype. For example, the S. cerevisiae phr1 mutant exhibits a higher proportion of chitin and sensitivity to Nikkomycin Z and calcofluor white (Popolo & Vai, 1998
), whereas mutants in chitin synthases chs2 and chs8 have less chitin synthase activity and are therefore hypersensitive to calcofluor white (Munro et al., 2003
). In S. cerevisiae, mutants defective in the HOG pathway are resistant to calcofluor white, although no differences were detected in chitin synthesis during vegetative growth or during calcofluor treatment (García-Rodriguez et al., 2000
). Additionally, overexpression of the PBS2 gene leads to higher
-1,3-glucan synthase activity and resistance to
-1,3-glucanases (Lai et al., 1997
). In C. albicans, therefore, the HOG pathway may contribute to maintenance of the cell-wall architecture under non-inducing conditions, probably through interaction with other MAP kinase routes.
In summary, our results reveal that Pbs2 and Hog1 play different roles in cell-wall construction and in the oxidative-stress response. Further work will be aimed at characterizing their roles during pathogenesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alonso-Monge, R., Navarro-García, F., Molero, G., Diez-Orejas, R., Gustin, M., Pla, J., Sánchez, M. & Nombela, C. (1999). Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 181, 30583068.
Alonso-Monge, R., Real, E., Wojda, I., Bebelman, J.-P., Mager, W. H. & Siderius, M. (2001). Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects. Mol Microbiol 41, 717730.[CrossRef][Medline]
Alonso-Monge, R., Navarro-García, F., Román, E., Negredo, A. I., Eisman, B., Nombela, C. & Pla, J. (2003). The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans. Eukaryot Cell 2, 351361.
Banuett, F. (1998). Signalling in the yeasts: an informational cascade with links to the filamentous fungi. Microbiol Mol Biol Rev 62, 249274.
Bilsland, E., Molin, C., Swaminathan, S., Ramne, A. & Sunnerhagen, P. (2004). Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance. Mol Microbiol 53, 17431756.[CrossRef][Medline]
Bilsland-Marchesan, E., Ariño, J., Saito, H., Sunnerhagen, P. & Posas, F. (2000). Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1. Mol Cell Biol 20, 38873895.
Brewster, J. L. & Gustin, M. C. (1994). Positioning of cell growth and division after osmotic stress requires a MAP kinase pathway. Yeast 10, 425439.[Medline]
Brewster, J. L., de Valoir, T., Dwyer, N. D., Winter, E. & Gustin, M. C. (1993). An osmosensing signal transduction pathway in yeast. Science 259, 17601763.[Medline]
Calera, J. A., Herman, D. & Calderone, R. (2000a). Identification of YPD1, a gene of Candida albicans which encodes a two-component phosphohistidine intermediate protein. Yeast 16, 10531059.[CrossRef][Medline]
Calera, J. A., Zhao, X.-J. & Calderone, R. (2000b). Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans. Infect Immun 68, 518525.
Chauhan, N., Inglis, D., Roman, E., Pla, J., Li, D., Calera, J. A. & Calderone, R. (2003). Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynthesis and adaptation to oxidative stress. Eukaryot Cell 2, 10181024.
Chen, J., Chen, J., Lane, S. & Liu, H. (2002). A conserved mitogen-activated protein kinase pathway is required for mating in Candida albicans. Mol Microbiol 46, 13351344.[CrossRef][Medline]
Cormack, B. P., Bertram, G., Egerton, M., Gow, N. A. R., Falkow, S. & Brown, A. J. P. (1997). Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology 143, 303311.[Medline]
Costa, V. M. V., Amorim, M. A., Quintanilha, A. & Moradas-Ferreira, P. (2002). Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7. Free Radic Biol Med 33, 15071515.[CrossRef][Medline]
Csank, C., Schröppel, K., Leberer, E., Harcus, D., Mohamed, O., Meloche, S., Thomas, D. Y. & Whiteway, M. (1998). Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis. Infect Immun 66, 27132721.
Davenport, K. R., Sohaskey, M., Kamada, Y., Levin, D. E. & Gustin, M. C. (1995). A second osmosensing signal transduction pathway in yeast: hypotonic shock activates the PKC1 protein kinase-regulated cell integrity pathway. J Biol Chem 270, 3015730161.
Fox, J. L. (1993). Fungal infection rates are increasing. ASM News 10, 515518.
Galcheva-Gargova, Z., Dérijard, B., Wu, I.-H. & Davis, R. J. (1994). An osmosensing signal transduction pathway in mammalian cells. Science 265, 806808.[Medline]
García-Rodriguez, L. J., Durán, A. & Roncero, C. (2000). Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions. J Bacteriol 182, 24282437.
Gerami-Nejad, M., Berman, J. & Gale, C. A. (2001). Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans. Yeast 18, 859864.[CrossRef][Medline]
Gimeno, C. J., Ljungdahl, P. O., Styles, C. A. & Fink, G. R. (1992). Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68, 10771090.[CrossRef][Medline]
Gustin, M. C., Albertyn, J., Alexander, M. & Davenport, K. (1998). MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62, 12641300.
Haghnazari, E. & Heyer, W.-D. (2004). The Hog1 MAP kinase pathway and the Mec1 DNA damage checkpoint pathway independently control the cellular responses to hydrogen peroxide. DNA Repair (Amst) 3, 769776.[CrossRef][Medline]
Hoefen, R. J. & Berk, B. C. (2002). The role of MAP kinases in endothelial activation. Vascul Pharmacol 38, 271273.[CrossRef][Medline]
Jamieson, D. J., Stephen, D. W. S. & Terrière, E. C. (1996). Analysis of the adaptive oxidative stress response of Candida albicans. FEMS Microbiol Lett 138, 8388.[CrossRef][Medline]
Jiang, B., Ram, A. F. J., Sheraton, J., Klis, F. M. & Bussey, H. (1995). Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action in a pathway that includes modulation of EXG1 transcription. Mol Gen Genet 248, 260269.[Medline]
Jiang, L., Niu, S., Clines, K. L., Burke, D. J. & Sturgill, T. W. (2004). Analyses of the effects of Rck2p mutants on Pbs2pDD-induced toxicity in Saccharomyces cerevisiae identify a MAP kinase docking motif, and unexpected functional inactivation due to acidic substitution of T379. Mol Genet Genomics 271, 208219.[CrossRef][Medline]
Kamada, Y., Jung, U. S., Piotrowski, J. & Levin, D. E. (1995). The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev 9, 15591571.[Abstract]
Kapteyn, J. C., Ter Riet, B., Vink, E., Blad, S., De Nobel, H., Van Den Ende, H. & Klis, F. M. (2001). Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall. Mol Microbiol 39, 469480.[CrossRef][Medline]
Kirsch, D. R. & Whitney, R. R. (1991). Pathogenicity of Candida albicans auxotrophic mutants in experimental infections. Infect Immun 59, 32973300.[Medline]
Köhler, G. A., White, T. C. & Agabian, N. (1997). Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid. J Bacteriol 179, 23312338.
Kruppa, M., Goins, T., Cutler, J. E. & 7 other authors (2003). The role of the Candida albicans histidine kinase (CHK1) gene in the regulation of cell wall mannan and glucan biosynthesis. FEMS Yeast Res 3, 289299.[CrossRef][Medline]
Kruppa, M., Jabra-Rizk, M. A., Meiller, T. F. & Calderone, R. (2004). The histidine kinases of Candida albicans: regulation of cell wall mannan biosynthesis. FEMS Yeast Res 4, 409416.[CrossRef][Medline]
Kültz, D. & Burg, M. (1998). Evolution of osmotic stress signaling via MAP kinase cascades. J Exp Biol 201, 30153021.
Kyriakis, J. M. & Avruch, J. (1996). Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18, 567577.[CrossRef][Medline]
Lai, M. H., Silverman, S. J., Gaughran, J. P. & Kirsch, D. R. (1997). Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance and other cell wall effects in Saccharomyces cerevisiae. Yeast 13, 199213.[CrossRef][Medline]
Liu, H., Köhler, J. & Fink, G. R. (1994). Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266, 17231726.[Medline]
Madhani, H. D., Styles, C. A. & Fink, G. R. (1997). MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation. Cell 91, 673684.[CrossRef][Medline]
Maeda, T., Takekawa, M. & Saito, H. (1995). Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. Science 269, 554558.[Medline]
Martín, H., Rodríguez-Pachón, J. M., Ruiz, C., Nombela, C. & Molina, M. (2000). Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J Biol Chem 275, 15111519.
Millar, J. B. A. (1999). Stress-activated MAP kinase (mitogen-activated protein kinase) pathways of budding and fission yeasts. Biochem Soc Symp 64, 4962.[Medline]
Millar, J. B. A., Buck, V. & Wilkinson, M. G. (1995). Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev 9, 21172130.[Abstract]
Munro, C. A., Whitton, R. K., Hughes, H. B., Rella, M., Selvaggini, S. & Gow, N. A. R. (2003). CHS8 a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth. Fungal Genet Biol 40, 146158.[CrossRef][Medline]
Navarro-García, F., Sánchez, M., Pla, J. & Nombela, C. (1995). Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity. Mol Cell Biol 15, 21972206.[Abstract]
Navarro-García, F., Alonso-Monge, R., Rico, H., Pla, J., Sentandreu, R. & Nombela, C. (1998). A role for the MAP kinase gene MKC1 in cell wall construction and morphological transitions in Candida albicans. Microbiology 144, 411424.[Medline]
NCCLS (1992). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast: Proposed Standard M27-P. Villanova, PA: National Committee for Clinical Laboratory Standards.
Negredo, A., Monteoliva, L., Gil, C., Pla, J. & Nombela, C. (1997). Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans. Microbiology 143, 297302.[Medline]
Odds, F. C. (1988). Candida and Candidosis. London: Baillière Tindall.
O'Rourke, S. M. & Herskowitz, I. (1998). The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev 12, 28742886.
Pla, J., Pérez-Díaz, R. M., Navarro-García, F., Sánchez, M. & Nombela, C. (1995). Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector. Gene 165, 115120.[CrossRef][Medline]
Popolo, L. & Vai, M. (1998). Defects in assembly of the extracellular matrix are responsible for altered morphogenesis of a Candida albicans phr1 mutant. J Bacteriol 180, 163166.
Posas, F. & Saito, H. (1997). Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276, 17021705.
Posas, F., Takekawa, M. & Saito, H. (1998). Signal transduction by MAP kinase cascades in budding yeast. Curr Opin Microbiol 1, 175182.[CrossRef][Medline]
Samejima, I., Mackie, S. & Fantes, P. A. (1997). Multiple modes of activation of the stress-responsive MAP kinase pathway in fission yeast. EMBO J 16, 61626170.
Sánchez-Piris, M., Posas, F., Alemany, V., Winge, I., Hidalgo, E., Bachs, O. & Aligue, R. (2002). The serine/threonine kinase Cmk2 is required for oxidative stress response in fission yeast. J Biol Chem 277, 1772217727.
San José, C., Alonso-Monge, R., Pérez-Díaz, R., Pla, J. & Nombela, C. (1996). The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans. J Bacteriol 178, 58505852.
Shiozaki, K. & Russell, P. (1995). Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature 378, 739743.[CrossRef][Medline]
Shiozaki, K. & Russell, P. (1996). Conjugation, meiosis, and the osmotic stress response are regulated by Spc1 kinase through Atf1 transcription factor in fission yeast. Genes Dev 10, 22762288.[Abstract]
Singh, K. K. (2000). The Saccharomyces cerevisiae Sln1p-Ssk1p two-component system mediates response to oxidative stress and in an oxidant-specific fashion. Free Radic Biol Med 29, 10431050.[CrossRef][Medline]
Smith, D. A., Nicholls, S., Morgan, B. A., Brown, A. J. P. & Quinn, J. (2004). A conserved stress-activated protein kinase regulates a core stress response in the human pathogen Candida albicans. Mol Biol Cell 15, 41794190.
Soto, T., Beltrán, F. F., Paredes, V., Madrid, M., Millar, J. B. A., Vicente-Soler, J., Cansado, J. & Gacto, M. (2002). Cold induces stress-activated protein kinase-mediated response in the fission yeast Schizosaccharomyces pombe. Eur J Biochem 269, 50565065.
Tatebayashi, K., Takekawa, M. & Saito, H. (2003). A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway. EMBO J 22, 36243634.
Teige, M., Scheikl, E., Reiser, V., Ruis, H. & Ammerer, G. (2001). Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast. Proc Natl Acad Sci U S A 98, 56255630.
Vilella, F., Herrero, E., Torres, J. & de la Torre-Ruiz, M. A. (2005). Pkc1 and the upstream elements of the cell integrity pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, are required for cellular responses to oxidative stress. J Biol Chem (in press).
Yamada-Okabe, T., Mio, T., Ono, N., Kashima, Y., Matsui, M., Arisawa, M. & Yamada-Okabe, H. (1999). Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans. J Bacteriol 181, 72437247.
Received 22 October 2004;
revised 22 December 2004;
accepted 6 January 2005.
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