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Eukaryotic Cell, September 2007, p. 1635-1645, Vol. 6, No. 9
1535-9778/07/$08.00+0     doi:10.1128/EC.00106-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The High-Osmolarity Glycerol Response Pathway in the Human Fungal Pathogen Candida glabrata Strain ATCC 2001 Lacks a Signaling Branch That Operates in Baker's Yeast

Christa Gregori,¹ Christoph Schüller,² Andreas Roetzer,² Tobias Schwarzmüller,¹ Gustav Ammerer,² and Karl Kuchler^1*

Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University Vienna,¹ Department of Biochemistry and Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria²

Received 4 April 2007/ Accepted 26 June 2007

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The high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway mediates adaptation to high-osmolarity stress in the yeast Saccharomyces cerevisiae. Here we investigate the function of HOG in the human opportunistic fungal pathogen Candida glabrata. C. glabrata sho1 (Cgsho1) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1 mutants. However, deletion of CgSHO1 in other genetic backgrounds fails to cause osmostress hypersensitivity, whereas cells lacking the downstream MAP kinase Pbs2 remain osmosensitive. Notably, ATCC 2001 Cgsho1 cells also display methylglyoxal hypersensitivity, implying the inactivity of the Sln1 branch in ATCC 2001. Genomic sequencing of CgSSK2 in different C. glabrata backgrounds demonstrates that ATCC 2001 harbors a truncated and mutated Cgssk2-1 allele, the only orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC 2001 is caused by a point mutation in Cgssk2-1, which debilitates the second HOG pathway branch. Functional complementation experiments unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in osmostress adaptation. In contrast to yeast, however, Cgsho1 mutants display hypersensitivity to weak organic acids such as sorbate and benzoate. Hence, CgSho1 is also implicated in modulating weak acid tolerance, suggesting that HOG signaling in C. glabrata mediates the response to multiple stress conditions.

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Yeast cells have developed the ability to counteract changing osmolarity conditions by inducing appropriate physiological responses and adaptive adjustments (4). Osmoadaptation pathways have been studied primarily in Saccharomyces cerevisiae but lately also in pathogenic fungi with clinical relevance, such as Candida albicans (2, 37, 42). The haploid Candida glabrata is the second most prevalent fungal pathogen in humans after C. albicans (15, 35), accounting for about 20% of all systemic diseases caused by Candida spp. C. glabrata populates mucosal surfaces and the guts of healthy individuals, but it also causes life-threatening systemic infections in immunocompromised patients (29).

Candida spp. must be able to adapt to osmotic changes in the microenvironment during host invasion and systemic spreading, as well as to evade host defense strategies (2). However, the underlying mechanisms are not well understood. In yeast, mitogen-activated protein kinase (MAPK) cascades drive important signaling pathways, thus allowing cells to adapt to changing environmental conditions. S. cerevisiae harbors at least five MAPK signaling pathways mediating stress response, including the protein kinase C (PKC) cell integrity pathway (22), the protein kinase A growth control pathway (36), the mating pheromone response pathway (30, 40), and the spore wall assembly pathway (19, 39). Finally, the well-characterized high-osmolarity glycerol (HOG) pathway is essential for yeast survival under-high osmolarity conditions, since it triggers adaptation through intracellular accumulation of glycerol as the adaptive osmolyte (4, 10). Two cell surface membrane sensors, Sho1 and Sln1, constitute two functionally redundant signaling branches that sense and transduce signals through the downstream HOG MAPK pathway (23, 44). Sho1 and Sln1 have been termed osmosensors, but recent studies indicate that Sho1 transduces the stress signal rather than sensing changes in osmolarity (33, 43). Experiments based on systems biology analysis have demonstrated that Sho1 is phosphorylated in a Hog1-dependent manner, indicating a negative-feedback loop acting on the transmembrane protein (11). The key MAPK, Hog1, is activated by phosphorylation through the upstream MAPK kinase (MAPKK) Pbs2. Whereas pbs2 and hog1 deletion strains are osmosensitive (4), mutations affecting only one of two upstream branches do not cause osmophenotypes, since Sln1 and Sho1 can each independently trigger Hog1 activation (27, 44).

Orthologues of Hog1 are present in other fungi as well as in animals (27). In S. cerevisiae, the HOG pathway responds to a limited range of stress conditions, mainly high osmolarity, heat stress (46), intracellular methylglyoxal accumulation (1, 24), citric and acetic acid stresses (21, 25), and oxidative stress (3). Interestingly, Hog1 orthologues play a more general role in regulating a core stress response in C. albicans and Schizosaccharomyces pombe (7, 42). Furthermore, a lack of C. albicans Hog1 (CaHog1) leads to impaired virulence (2). Recent studies demonstrated that CaSho1 plays only a minor role in osmostress adaptation. Nevertheless, CaSho1 is important for growth under oxidative stress conditions, and it mediates phosphorylation of the Cek1 MAPK in exponentially growing cells (37).

Because all components of the yeast HOG pathway are present in C. glabrata, we constructed strains lacking C. glabrata SHO1 (CgSHO1) and CgPBS2 from strain ATCC 2001, which was sequenced by the Génolevures Consortium (8). Unexpectedly, a Cgsho1 strain displays severe osmosensitivity, a phenotype not observed for the corresponding S. cerevisiae sho1 (Scsho1) mutant. However, the Cgsho1 phenotype is restricted to ATCC 2001. Genomic sequencing demonstrates the inactivity of the Sln1 branch in ATCC 2001, since this strain harbors a truncated nonfunctional Cgssk2-1 allele, causing the removal of the CgSsk2 kinase domain. Importantly, analysis of ATCC 2001 and other strain backgrounds revealed that the physiology of the C. glabrata HOG pathway is closely related to that of S. cerevisiae. Interestingly, it also seems distinct from the yeast HOG pathway, since it performs additional functions, such as involvement in modulating resistance to certain weak organic acids in C. glabrata.

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Strains, plasmids, and growth conditions. All strains used in this study are listed in Table 1. S. cerevisiae strain YCG9A was obtained by crossing W303-1A ssk2 ssk22 with W303-1B sho1. BY4741 sho1 ssk1 was obtained by crossing strains BY4741 sho1 and BY4742 ssk1. For gene disruption in the ATCC 2001, BG2, and Cg2633 backgrounds, we used the dominant SAT1 marker cassette amplified from plasmid pSFS2 (34) with primers SAT1-P2 and SAT1-P5 (Table 2). For construction of the CgSSK2 genomic deletion cassette, the nourseothricin marker gene NAT1 was amplified from plasmid pJK863 (41) using primers NAT1-P2 and NAT1-P5. Disruption cassettes were generated by fusion PCRs as described previously (26) and transformed into C. glabrata strains via electroporation as described elsewhere (34). For gene disruption using the HIS3 marker, we amplified the marker gene from plasmid pTW23 (14) using primers HIS3-P2 and HIS3-P5. Sequences of all primers used for PCR amplification of disruption cassettes are listed in Table 2.

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TABLE 1. Fungal strains used in this study

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TABLE 2. Oligonucleotides used in this study

For genomic integration of SSK2, a 6,970-bp PCR fragment containing the entire SSK2 open reading frame (ORF) including flanking 5' and 3' untranslated regions was amplified from genomic DNA of strain BG2 and transformed into strain ATCC 2001 sho1. Transformants were selected on yeast extract-peptone-dextrose (YPD) supplemented with 1.2 M NaCl. Correct genomic replacement of ssk2-1 by SSK2 was verified by sequence analysis of the relevant genomic region. Of 10 transformants tested, all encoded the wild-type Cys1668, demonstrating restoration of the mutated ssk2-1 allele. Clones C1, C2, and C3 were selected for further experiments to test for pathway activation.

Rich medium (YPD) and synthetic medium (SC) for yeast cultures were prepared essentially as described elsewhere (13). Unless otherwise indicated, all strains were grown routinely at 30°C. For the selection of nourseothricin-resistant transformants, 200 µg/ml of nourseothricin (Werner Bioagents, Jena, Germany) was added to YPD agar plates. For reintroducing CgSHO1 into the deletion strain, a 2,330-bp fragment containing the entire SHO1 ORF, including the 600-bp 5' promoter and the 700-bp 3' untranslated region, was PCR amplified from ATCC 2001 genomic DNA und ligated into the pGEM-T easy vector (Promega). Primers used for PCR were 5-ExSHO1 (5'-GCAATTGTGGGAGCCACAGGATC-3') and 3-ExSHO1 (5'-GAGAAAGAAGGTTATGCCAGC-3'). The ARS-CEN-TRP cassette was isolated from plasmid pCgACT14 (17) via partial digestion with AatII and ligated into the corresponding restriction site of pGEM-T Easy harboring CgSHO1. For the empty-vector control, the SHO1 insert was removed from the vector with EcoRI restriction sites, followed by religation. The same EcoRI-digested fragment was used for cloning into EcoRI-digested pRS316 and YEp352, yielding two S. cerevisiae plasmids named pRS-CgSHO1 and YEp-CgSHO1, respectively.

Growth inhibition assays. To determine susceptibilities to osmostress, methylglyoxal or high temperatures, exponentially growing cultures were adjusted to an optical density at 600 nm (OD₆₀₀) of 0.1 and diluted 1:10, 1:100, and 1:1,000. Equal volumes of serial dilutions were spotted onto YPD plates containing various concentrations of NaCl, sorbitol, KCl, or methylglyoxal. Plates were incubated at 30°C or 42°C for 24 h to 48 h. For acetate plates, YPD (pH 4.5) (adjusted with HCl) was supplemented with acetate from an 8.7 M acetic acid stock solution adjusted to pH 4.5 with NaOH. Plates containing other weak acids were prepared exactly as previously described (18).

Preparation of cell extracts and immunoblotting. To investigate phosphorylation of Hog1 using self-made antibodies, cultures of the Cgsho1 strain transformed either with a plasmid expressing CgSHO1 or with the empty-vector control were grown to the exponential-growth phase before addition of 0.5 M NaCl. Cells were harvested from 40-ml samples taken at different time points and were washed with H₂O. Cells were then lysed with glass beads in 300 µl of buffer A [50 mM HEPES (pH 8.0), 0.4 M (NH₄)₂SO₄, 2 mM EDTA, 5% (vol/vol) glycerol, 50 mM sodium fluoride, 20 mM tetra-natrium-diphosphate, 1 mM sodium orthovanadate, 10 mM beta-glycerophosphate, protease inhibitor] using the Fast Prep machine (Qbiogene). Extracts were cleared by centrifugation steps at 3,000 x g and 20,000 x g. Aliquots corresponding to 50 µg of total extract per lane were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to nitrocellulose membranes. Immunoblotting was carried out using phosphospecific polyclonal antibodies recognizing activated Hog1 isoforms. For detection of phosphorylated Hog1 isoforms using anti-phospho-p38 MAPK antibodies (Cell Signaling Technologies), trichloroacetic acid extracts were prepared as described elsewhere (9), and cell lysates equivalent to 0.4 OD₆₀₀ unit were resolved by SDS-PAGE. Anti-ScPgk1 antibodies recognizing CgPgk1 were used to detect the loading control (20).

Preparation of antibodies. Polyclonal anti-phospho-Hog1 antibodies were raised in rabbits against a 16-residue Hog1-specific peptide conjugated to the keyhole limpet hemocyanin carrier protein. The crude Hog1 phosphopeptide NH₂-CARIQDPQMTGYVSTR-COOH (phosphorylated residues are boldfaced) was purified by high-performance liquid chromatography through a Phenomix Jupiter column with a linear acetonitrile gradient, and fractions were analyzed by mass spectrometry. Fractions containing peptides with the predicted mass of the Hog1 phosphopeptide were lyophilized and reconstituted in Tris-buffered saline. About 1 mg of purified keyhole limpet hemocyanin-coupled peptide antigen was used for immunization of rabbits, carried out by Gramsch Laboratories (Schwabhausen, Germany). The specificity of the antiserum obtained was tested using appropriate cell extracts of wild-type S. cerevisiae and mutants lacking Hog1.

Southern blotting and DNA sequencing. For identification of mutations and for filling the unsequenced genomic gap, CgSSK2 fragments up to 4 kb were amplified from genomic DNA and directly subjected to DNA sequencing using the BigDye Terminator cycle sequencing kit (version 3.0) and ABI PRISM sequencing system 310 (Applied Biosystems, Germany) according to the manufacturer's instructions.

Southern blotting was performed as described elsewhere (2a). Genomic DNA was isolated by phenol-chloroform-isoamyl alcohol (PCI) extraction and digested with HindIII for 3 h, followed by overnight digestion with NotI (5 U/µg). Probes were obtained by PCR amplification using primers SHO1-SAT1-P1 and SHO1-SAT1-P2 (probe A) as well as SHO1-SAT1-P4 and SHO1-SAT1-P6 (probe B). [-³²P]CTP radiolabeling was carried out with the Megaprime DNA labeling system (Amersham) according to the manufacturer's protocol.

Microscopy. Microscopy was done on an Axioplan 2 (Zeiss) microscope. Pictures were captured with a Spot Pursuit (Sony) camera. Time lapse microscopy was controlled by MetaFluor software. Cells were grown to the early-exponential-growth phase in synthetic medium, made to adhere to a coverslip by using concanavalin A, mounted, and fixed on microscopy slides with silicon vacuum grease.

Nucleotide sequence accession numbers. The genomic sequences for the CgSSK2 genes in various C. glabrata strain backgrounds have been deposited in GenBank under accession numbers EF193044 and EF193045.

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C. glabrata strains display different osmostress sensitivities. To investigate the role of the HOG pathway in the pathogenic fungus C. glabrata, we generated deletion mutants lacking crucial genes of the HOG pathway. In S. cerevisiae, the HOG pathway bifurcates upstream of the MAPKK Pbs2 into two branches, the Sho1 and the Sln1 branch. Each of these branches can independently mediate the osmostress response (30). In strain ATCC 2001, for which a genomic sequence has been determined (8), the Sln1 branch seems to be truncated at the level of the MAPKK kinase (MAPKKK) gene SSK2. The database entry indicates that the genomic CgSSK2 region is disrupted by an unresolved sequence gap. More importantly, the kinase domain of the CgSSK2 gene is disrupted by a stop codon, suggesting a pseudogene, or at least a nonfunctional Ssk2 protein encoded by this locus. These facts suggested to us a possible difference between the C. glabrata and S. cerevisiae HOG pathways.

Therefore, we chose the CgSHO1 gene to inactivate the remaining branch in ATCC 2001, as well as the downstream CgPBS2 gene to achieve complete inactivation of the HOG pathway. Furthermore, we generated disruptions of SHO1 and PBS2 in two other C. glabrata strains, BG2 (6) and the clinical isolate Cg2633 (kindly provided by Helena Bujdakova). We took advantage of the dominant nourseothricin resistance marker SAT1, since no auxotrophic marker is available in these clinical strains. SHO1 and PBS2 were disrupted using the three-way PCR method in BG2, Cg2633, and ATCC 2001. Correct deletion of the SHO1 locus was tested by Southern blot analysis (Fig. 1B). The Cgsho1 deletion strains obtained were then tested for growth under high-osmolarity conditions (Fig. 1A). Only the ATCC 2001 Cgsho1 strain showed severely impaired growth on high salt concentrations, whereas a lack of SHO1 in BG2 and Cg2633 did not result in increased osmosensitivity (Fig. 1A). As expected, however, deletion of PBS2 led to osmostress hypersensitivity in all three strains (Fig. 1A). Thus, deletion of the MAPKK Pbs2 causes a severe phenotype on high NaCl concentrations that is independent of the genetic background. The observed salt stress sensitivity of a C. glabrata Cgsho1 strain is hence restricted to ATCC 2001, supporting the idea of an inactive Sln1 branch in strain ATCC 2001. Disruption of SHO1 in this strain could therefore lead to complete inactivation of the HOG pathway.

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FIG. 1. Osmostress sensitivities of C. glabrata pbs2 and sho1 strains. (A) Cultures of the C. glabrata strains ATCC 2001, Cg2633, and BG2, as well as their isogenic Cgpbs2 and Cgsho1 deletion strains, were grown to an OD600 of 1, diluted to an OD600 of 0.1, 0.01, or 0.001, and spotted onto YPD and YPD agar plates containing the indicated amounts of NaCl. Plates were incubated at 30°C for 2 days. WT, wild type. (B) Genomic DNA was isolated from ATCC 2001, BG2, Cg2633, and their isogenic Cgsho1 deletion strains, followed by digestion with HindIII and NotI. After agarose separation of DNA fragments and transfer to nitrocellulose membranes, radiolabeled probes A and B were used for detection of the SHO1 WT gene and the disrupted allele. Restriction sites and expected fragment sizes for the WT and Cgsho1 disruption strains are illustrated schematically (not drawn to scale).

Deletion of CgSHO1 in ATCC 2001 causes general osmostress sensitivity and thermosensitivity. To investigates the role of Sho1 and the HOG pathway in the ATCC 2001 background, we constructed further SHO1 deletion strains. We chose strains HT6 and HTU (16, 45), since auxotrophic markers are available in these strains. Genomic disruption cassettes were generated by three-way PCR using the CgHIS3 gene as a selectable marker and were used for transformation. Several independent transformants were obtained, and homologous recombination leading to gene disruption was confirmed by PCR and Southern blot analysis (data not shown).

Transformants of HT6 and HTU derivatives in which SHO1 was correctly replaced were then tested for growth under high-osmolarity conditions. Strain HTU Cgsho1 showed impaired growth on plates containing 0.7 M NaCl and completely failed to grow on 1 M NaCl, whereas the wild-type strain tolerated concentrations as high as 1.2 M NaCl (Fig. 2). Deletion of CgSHO1 also led to reduced growth on plates supplemented with other types of osmolytes, namely, sorbitol and KCl. Interestingly, we observed an increased temperature sensitivity of HTU Cgsho1 at 42°C, although at 37°C no altered sensitivity was observed (data not shown). Reintroducing SHO1 on a plasmid could restore growth at high osmolarity and elevated temperatures (Fig. 2). Similar phenotypes were also observed for HT6 Cgsho1 strains (data not shown). In S. cerevisiae, the HOG pathway is also involved in the response to increasing concentrations of methylglyoxal (1, 24), a toxic intermediate of carbon metabolism. Similarly, the HTU Cgsho1 strain displayed slightly increased sensitivity to 40 mM and 50 mM methylglyoxal relative to that of wild-type strains (Fig. 2).

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FIG. 2. Sensitivity of the HTU Cgsho1 strain to osmostress, high temperatures, and methylglyoxal. Cultures of the wild-type (WT) strain (HTU), HTU Cgsho1, and HTU Cgsho1 transformed either with a vector expressing CgSHO1 or with the empty vector were diluted to an OD600 of 0.1, 0.01, or 0.001 and spotted onto YPD and YPD agar plates containing the indicated amounts of NaCl, KCl, sorbitol (Sorb), or methylglyoxal (MG). Plates were incubated at 30°C for 2 days or at 42°C for 1 day.

Lack of CgSHO1 in the ATCC 2001 genetic background inactivates the HOG pathway. In S. cerevisiae, HOG pathway activation leads to dual phosphorylation of Hog1 to ultimately activate the MAPK cascade. We therefore investigated the phosphorylation status of Hog1 in C. glabrata in response to osmostress. Cultures of the Cgsho1 strain transformed either with a plasmid expressing CgSHO1 or with the empty vector were grown to the exponential-growth phase before addition of 0.5 M NaCl. Samples were taken at several time points and extracts separated by SDS-PAGE. Activated Hog1 isoforms were detected using phosphospecific polyclonal antibodies recognizing phosphorylated Hog1 (P-Hog1). Whereas in the strain harboring wild-type CgSHO1 a transient phosphorylation signal in response to osmostress was detectable, this signal was absent in the Cgsho1 strain (Fig. 3A). This finding supports a functional block of the signaling cascade upstream of CgHog1. Even increasing the NaCl concentration to 1 M failed to trigger phosphorylation and activation of CgHog1 in the absence of Sho1 (data not shown).

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FIG. 3. Loss of Hog1 phosphorylation and altered morphology of HTU Cgsho1 under salt stress. (A) Cultures of HTU Cgsho1 transformed either with a plasmid expressing CgSHO1 or with the empty vector were grown to an OD600 of 1 before addition of 0.5 M NaCl. Samples were taken at the indicated time points (minutes). Glass bead extracts were prepared from these samples, and 50 µg of total-cell extract per lane was separated on a 10% SDS-PAGE gel. Immunoblotting was carried out using phosphospecific polyclonal antibodies detecting activated Hog1 isoforms. c.r., cross-reactions. (B) Cultures of HTU Cgsho1 transformed either with a vector expressing CgSHO1 or with the empty vector were grown to an OD600 of 0.6 and then either treated with 0.5 M NaCl or left unstressed for 2 h before pictures were taken. (C) HTU Cgsho1 was grown to an OD600 of 0.6 before cells were immobilized onto a slide with concanavalin A. Growth was then inspected under the microscope under stress and nonstress conditions for 4 h, and pictures were taken at the indicated time points.

In C. albicans, deletion of components of the HOG pathway influences cell morphology and filamentation (37). Likewise, in S. cerevisiae, cells lacking HOG1 exhibit a cell morphology phenotype (5). Therefore, we microscopically inspected Cgsho1 cells with respect to their morphology under osmostress. We took pictures of exponentially growing cells 2 h after incubation with or without 0.5 M NaCl (Fig. 3B). Cells containing wild-type SHO1 looked normal under both conditions, whereas growth of the Cgsho1 strain caused abnormal cell morphology under high-osmolarity conditions (Fig. 3B). About 50 to 80% of the cells were elongated or swollen. We then immobilized cells on a microscope slide and inspected the growth patterns of the same cells under stress and nonstress conditions for as long as 4 h, covering several cell divisions. In response to osmotic stress, the Cgsho1 strain seemed to display severe budding defects (Fig. 3C). Therefore, Sho1 must play a major role in mediating the osmostress response and Hog1 phosphorylation in C. glabrata strains HT6 and HTU, demonstrating an inactive Sln1 branch in the ATCC 2001 background.

C. glabrata Sho1 can functionally complement the lack of S. cerevisiae Sho1. Next, we investigated whether the function of Sho1 within the signaling cascade of the HOG pathway is conserved between C. glabrata and S. cerevisiae. To check the functionality of the CgSHO1 gene in S. cerevisiae, we cloned the CgSHO1 ORF, including the 600-bp promoter and 700-bp 3' untranslated regions, into the CEN-based yeast vector pRS316 and the multicopy vector YEp352. Plasmids pRS-CgSHO1 and YEp-CgSHO1 were used for transformation of the S. cerevisiae recipient strain YCG9A (sho1 ssk2 ssk22). Positive transformants were then analyzed for growth on plates containing 1 M or 1.2 M NaCl, concentrations that severely impaired the growth of the control strain YCG9A (Fig. 4A). Introducing CgSHO1 either on a multicopy plasmid or on a CEN-based plasmid fully restored growth of the S. cerevisiae sho1 ssk2 ssk22 strain YCG9A (Fig. 4A). These data suggest that we have indeed identified CgSho1 as the orthologue of Sho1 from S. cerevisiae.

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FIG. 4. Expression of CgSho1 in yeast can complement the loss of ScSho1. (A) S. cerevisiae strains W303-1B, YCG9A (sho1 ssk2 ssk22), and YCG9A transformed with either pRS-CgSHO1 or pYEp-CgSHO1 were diluted to an OD600 of 0.1, 0.01, 0.001, or 0.0001 and spotted onto YPD and YPD agar plates containing NaCl. Plates were incubated at 30°C for 2 days. (B) Alignment of the protein kinase domains of C. glabrata (ATCC 2001) and S. cerevisiae Ssk2. CgSSK2 encodes a translational stop within a region highly conserved between S. cerevisiae and C. glabrata (indicated). Below the alignment are diagrams of the proteins encoded by the SSK2 ORFs in the two distinct C. glabrata strains, ATCC 2001 and BG2, as well as S. cerevisiae Ssk2. PKD, protein kinase domain.

CgSSK2 carries a point mutation in ATCC 2001 but not in other C. glabrata strains. We initially found that only ATCC 2001 sho1 is hypersensitive to osmostress. In other backgrounds, the same deletion did not influence resistance to high concentrations of NaCl. Hence, we assumed that the Sln1 branch in ATCC 2001 either is not functional or is absent, perhaps due to inactivity of one of the proteins of this branch. The annotated genome sequence of ATCC 2001 published by the Génolevures Consortium contains two ORFs highly similar to the 5' and 3' halves of the S. cerevisiae SSK2 gene. These are separated by an unsequenced gap, implying the possibility of a pseudogene. To fill this sequence gap completely, we sequenced this region by standard primer walking, covering a sequence of 1,680 bp. The deduced amino acid sequence is identical for all three strains, ATCC 2001, BG2, and Cg2633. This newly sequenced region combines both previously identified ORFs into a single ORF encoding a 1,667-residue protein. However, the genome sequence of the ATCC 2001 CgSSK2 locus carries a translational stop codon within the predicted protein kinase domain, which is otherwise highly conserved between S. cerevisiae and C. glabrata (Fig. 4B). Sequencing of the equivalent genomic region showed that BG2 and Cg2633 did encode the conserved Cys1668 at the identical position. Furthermore, we sequenced the relevant genomic region in the SSK2 genes of seven C. glabrata clinical isolates (kindly provided by Helena Budjakova). However, none of the clinical isolates carried this stop mutation present in the ATCC 2001 strain (data not shown). Moreover, we sequenced the entire SSK2 gene in the BG2 wild-type strain, potentially encoding a 1,755-residue CgSsk2 protein containing the entire protein kinase domain (Fig. 4B).

Because methylglyoxal sensitivity and acetic acid hypersensitivity in S. cerevisiae are caused mainly by an inactive Sln1 branch (24, 25), we performed additional spot tests using these compounds. These experiments revealed that wild-type ATCC 2001 was also significantly more sensitive to methylglyoxal and acetate than other strains investigated (Fig. 5A). On methylglyoxal, ATCC 2001 showed growth defects similar to those of a BG2 Cgpbs2 deletion strain (Fig. 5A); disruption of CgSHO1 further increased sensitivities, as described above. The Cgsho1 and Cgpbs2 deletions in ATCC 2001 did not further increase acetate sensitivity. For complementation analysis, we introduced a functional SSK2 allele into the genome of the ATCC 2001 sho1 strain. Since all attempts to clone CgSSK2 into a C. glabrata vector failed, we integrated the CgSSK2 allele encoded in the BG2 genome into the corresponding genomic locus of ATCC 2001 sho1. Cells carrying the replaced SSK2 gene were selected on plates containing 1.2 M NaCl and verified by genomic sequencing. Growth inhibition analysis with three independent transformants (C1 to C3) revealed that introduction of the gene encoding full-length CgSSK2 into ATCC 2001 sho1 restored growth at high osmolarity and elevated temperatures. Moreover, all clones grew on higher acetate and methylglyoxal concentrations than the corresponding wild-type strain (Fig. 5B).

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FIG. 5. The ATCC 2001 genome carries a nonfunctional truncated ssk2-1 allele. (A) Cultures of the C. glabrata strains ATCC 2001 and BG2 as well as their isogenic Cgpbs2 and Cgsho1 deletion strains were diluted to an OD600 of 0.1, 0.01, or 0.001 and spotted onto YPD and YPD agar plates containing the indicated amounts of methylglyoxal (MG) or onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing the indicated amounts of acetate. Plates were incubated at 30°C for 2 days. (B) Cultures of the C. glabrata strain ATCC 2001 and the isogenic Cgpbs2 and Cgsho1 deletion strains, as well as three independent clones of ATCC 2001 sho1 SSK2 (C1 to C3), were grown to the exponential-growth phase, diluted to an OD600 of 0.1, and spotted along with serial 1:10 dilutions onto plates containing the indicated amounts of NaCl, MG, or acetate. Plates were incubated at 30°C for 2 days or at 42°C for 1 day. (C) Cultures of ATCC 2001, BG2, and BG2 Cgssk2 were diluted to an OD600 of 0.1, 0.01, or 0.001; serial dilutions were spotted onto YPD and YPD agar plates containing 50 mM MG or onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing 80 mM acetate. Plates were incubated at 30°C for 2 days. (D) Cultures of BG2 and ATCC 2001 and the indicated isogenic deletion strains, as well as three clones of ATCC 2001 sho1 complemented for SSK2 (C1 to C3), were grown to the early-exponential-growth phase before 0.5 M NaCl was added. Samples were taken at the indicated time points and crude trichloroacetic acid extracts prepared. Aliquots corresponding to 0.4 OD600 equivalent per lane were fractionated through a 10% SDS-PAGE gel. Immunoblotting was carried out using polyclonal anti-phospho-p38 MAPK or anti-Pgk1 antibodies. Extracts of ATCC 2001 sho1 SSK2 C2 and C3 were detected on different immunoblots, as indicated by the separation of these gels.

Furthermore, we deleted parts of the CgSSK2 ORF (bp 2063 to 5005) in wild-type BG2. As expected, the mutant strain obtained, BG2 Cgssk2, displayed higher sensitivity on acetate and, to a lesser extent, on methylglyoxal than wild-type BG2 but growth similar to that of wild-type ATCC 2001 (Fig. 5C). Furthermore, we performed immunoblot analysis to determine the phosphorylation status of CgHog1 in strains lacking SSK2 or cells expressing restored CgSsk2. In strain BG2 with CgSSK2 deleted, the Hog1 phosphorylation signal decreased more rapidly after osmostress than that in the wild type. Strikingly, reintegration of the functional CgSSK2 allele in the ATCC 2001 background with CgSHO1 deleted led to a phosphorylation signal in the unstressed situation, indicating constitutive Hog1 phosphorylation. This hyperphosphorylation phenotype was observed for all three independent clones tested (Fig. 5D). Taken together, our results show that the truncated allele of CgSSK2 in ATCC 2001 (Cgssk2-1) completely debilitates the Sln1 branch. Moreover, our data also demonstrate that the HOG pathway plays similar physiological roles in the osmostress response in baker's yeast and the human fungal pathogen C. glabrata (Fig. 6).

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FIG. 6. HOG pathways operating in S. cerevisiae and C. glabrata. Activation of the MAPKK Pbs2 can occur through at least two distinct upstream osmosensing mechanisms. One branch links the osmosensing protein Sln1 via Ypd1, Ssk1, and the MAPKKKs Ssk2 and Ssk22 to Pbs2. In the other branch, Sho1 functions to link an as yet unidentified osmosensor to the downstream components Cdc42, Ste20, Ste50, and the MAPKKK Ste11. Activated Pbs2 phosphorylates the MAPK Hog1, which in turn activates a variety of transcription factors. As indicated, Ssk22 does not exist in C. glabrata, and ATCC 2001 contains the ssk2-1 allele, encoding a truncated and nonfunctional Ssk2 version.

Mutants of the C. glabrata HOG pathway display sensitivity to weak organic acids. Recent studies in yeast revealed that Hog1 plays a role in resistance to acetic acid and that the response to acetic acid stress is mediated through the Sln1 branch of the HOG pathway (25). Our results indicate similar roles of the HOG pathway in C. glabrata and baker's yeast. A Cgpbs2 mutant in the BG2 background showed defects in growth on plates containing 100 mM acetate relative to the wild type, whereas the Cgsho1 mutant in the same background did not show altered sensitivities (Fig. 5A). However, as already observed for methylglyoxal, ATCC 2001 is clearly more sensitive to acetic acid than BG2. The Cgsho1 and Cgpbs2 deletions did not further increase sensitivities in ATCC 2001. Since we observed acetate hypersensitivity in HOG pathway mutants, we tested the growth of the same set of C. glabrata strains on plates containing the weak organic acids sorbate, propionate, and benzoate. Surprisingly, we also observed increased sensitivities of mutant strains under these conditions, a phenotype that was not observed in S. cerevisiae. Disruption of CgPBS2 in BG2 led to slightly impaired growth on 5 mM benzoate and 2.5 mM sorbate, whereas deletion of CgSHO1 did not cause hypersensitivity in a strain background with a functional Sln1 branch (Fig. 7A). Similar sorbate and benzoate hypersensitivities were observed for Cgpbs2 in ATCC 2001. Notably, a Cgsho1 mutant was even more sensitive to sorbate, benzoate, and propionate in this genetic background. In contrast, neither blockage of one or both upstream branches nor a lack of the downstream MAPKK Pbs2 caused any detectable weak-acid sensitivities in S. cerevisiae (Fig. 7B). In summary, our data suggest an additional role of the C. glabrata HOG pathway in weak-acid response, which is quite distinct from its osmosensing function in S. cerevisiae. However, blockage of the signaling of both upstream branches caused a more severe phenotype than disruption of the downstream MAPKK Pbs2, perhaps implying yet another signal transfer bypassing Pbs2 or involvement in another parallel and yet undisclosed pathway in stress sensing.

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FIG. 7. C. glabrata HOG pathway mutants display sensitivity to weak acids. (A) Cultures of the C. glabrata strains ATCC 2001 and BG2, as well as their isogenic Cgpbs2 and Cgsho1 deletion strains, were diluted to an OD600 of 0.1, 0.01, or 0.001 and spotted onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing the indicated amounts of sorbate, propionate, or benzoate. Plates were incubated at 30°C for 2 days. WT, wild type. (B) Cultures of WT S. cerevisiae W303-1A and its isogenic sho1, ssk1, pbs2, and ssk1 sho1 deletion strains were grown to the exponential-growth phase, and serial dilutions were spotted onto YPD (pH 4.5) and YPD (pH 4.5) agar plates containing the indicated amounts of sorbate, propionate, or benzoate or onto YPD containing 1.2 M NaCl.

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Adaptation to changes in the microenvironment during disease progression is an essential mechanism for the survival of fungal pathogens. MAPK cascades are important components in such cellular adaptation programs. This work aimed to define the role of orthologues of the S. cerevisiae HOG MAPK pathway in the pathogenic yeast C. glabrata. The analysis of physiological roles for the HOG pathway in the well-studied human fungal pathogen C. albicans revealed a strong phenotype, quite distinct from corresponding single mutations in S. cerevisiae. For example, Hog1 appears to be implicated in more-diverse stress conditions than its S. cerevisiae orthologue (42). Likewise, the role of Sho1 in C. albicans seems different from its osmosensing function in baker's yeast (37).

C. glabrata is a close relative of S. cerevisiae, considering genome evolution (8). Therefore, most adaptive functions of the HOG pathway are most likely also conserved. However, we show here that the genome of C. glabrata ATCC 2001 carries a putative orthologue of yeast SSK2 in a mutated form. Moreover, the SSK2 homologue SSK22 is absent from the C. glabrata genome. This fact hinted at possibly distinct functions of the MAPK pathways in these fungi, since one of the two major upstream signaling branches is debilitated. Here we show that (i) the overall functions of HOG signaling in C. glabrata are closely related to its functional counterpart in baker's yeast; (ii) CgSho1 can complement the corresponding S. cerevisiae mutant; and (iii) the Sln1 branch is inactive in the ATCC 2001 strain background due to a point mutation in the CgSSK2 gene but active in all other C. glabrata strain backgrounds tested.

Our analysis of HOG pathway mutants revealed a spectrum of phenotypes very similar to the respective mutants in baker's yeast. In S. cerevisiae, two HOG branches drive Hog1 activation; one branch uses Sln1, Ypd1, Ssk1, and the MAPKKKs Ssk2 and Ssk22, which then activate downstream Pbs2 (4, 32). The second branch senses through Sho1 and transduces via Cdc42 and Ste20, Ste50, and Ste11 to converge with the Sln1 branch in the activation of Pbs2 and the key kinase Hog1 further downstream (31). It is known that S. cerevisiae Sln1 carries an intrinsic histidine kinase activity and is able to control Ssk1 activity using a phosphorelay system involving Ypd1. Ssk1 then interacts with the MAPKKKs Ssk2 and Ssk22, which in turn activate Pbs2. Even though the two upstream branches of the HOG pathway seem highly specialized in detecting slightly different concentrations of salt (28), only mutations that block both branches cause severe osmosensitivities.

Our deletion analysis demonstrates that loss of C. glabrata SHO1 alone can cause osmostress hypersensitivity as a function of the genetic background. Experiments with different C. glabrata strains demonstrated that osmosensitivity of Cgsho1 mutants is restricted to ATCC 2001-derived "wild-type" strains. Our results are in line with the conclusion that the Sln1 branch is inactivated in the ATCC 2001 background. This was not observed for other clinical isolates and is therefore not a general peculiarity of C. glabrata. Although we did not detect an enhanced sensitivity of this strain to osmotic stress, methylglyoxal and acetate were markedly less well tolerated.

Sho1, the transmembrane protein of one branch of the HOG pathway, was previously thought to be a sensor of osmostress. However, recent studies also reveal an important role of Sho1 downstream of Ste11 by binding to both the activated Ste11-Ste50 complex and Pbs2, perhaps tethering a "signaling complex" at the cell surface (43). Bringing together Ste11 and Pbs2 is required for activation of Pbs2 and therefore of Hog1. The C. glabrata orthologue of yeast SHO1 can functionally complement S. cerevisiae sho1 mutants. Studies of C. albicans report that CaSHO1 plays only a negligible role in osmostress resistance, whereas Casho1 mutants are sensitive to oxidative stress (37). In our work, we failed to detect hypersensitivity of the C. glabrata Cgsho1 mutant on H₂O₂ or menadione (data not shown). Furthermore, CgHog1 phosphorylation was undetectable in response to oxidative stress (data not shown), indicating a negligible role of the C. glabrata HOG pathway in the oxidative-stress response.

It is unclear when during evolution the CgSSK2 mutation was acquired by the ATCC 2001 strain. Since inactivity of the Sln1 branch might decrease fitness in the host environment, and because other isolates do not carry the Cgssk2-1 allele, it appears likely that the Cgssk2-1 mutation was acquired during or after the isolation of the strain from its human source. Interestingly, ATCC 2001 strains carrying restored SSK2 showed increased levels of phosphorylated Hog1. Hence, acquisition of the mutated ssk2-1 allele might be a natural suppressor mutation of constitutively active Hog1 in order to bypass associated toxicities, a mechanism similar to dominant-negative activities observed for a constitutive Pbs2 kinase in baker's yeast (12). Nevertheless, this work provides highly relevant information for the fungal pathogen community, since one should keep in mind that all mutations or deletions generated in the ATCC 2001-derived genetic backgrounds are in fact double mutants also carrying a Cgssk2-1 allele. Thus, any stress-related phenotypes and molecular cross talk between different signaling pathways may be caused by synthetic genetic interactions due to the presence of the mutated Cgssk2-1 allele and the lack of the second gene. This is particularly relevant and should be considered for genome-wide knockout approaches on C. glabrata or studies addressing the molecular cross talk between several MAPK pathways in stress response and adaptation. Determining possible differences in the virulence of HOG pathway mutants would indeed be interesting, since this may relate to the in vivo host situation. However, available mouse models for Candida glabrata are suboptimal and difficult to establish. Therefore, we are in the process of establishing a novel Drosophila melanogaster insect model with mutations in the toll pathway to be employed for Candida glabrata virulence studies (D. Ferrandon et al., unpublished data).

Response to weak organic acids, apart from acetic acid, fails to trigger Hog1 signaling in yeast. By contrast, we show that C. glabrata double mutants in the Ssk2 and Sho1 branch are highly sensitive against weak acids of medium chain length such as sorbic acid. No similar phenotype is observed for yeast. The observation that Cgpbs2 mutants are less sensitive than the Cgssk2 Cgsho1 double mutant perhaps points to a bypass to compensate for the lack of CgPbs2, although indirect genetic effects cannot be ruled out. However, the hypersensitivity of the Cgssk2 Cgsho1 double mutant implies a compensatory mechanism that is operative in S. cerevisiae but missing or nonfunctional in C. glabrata. In conclusion, our data demonstrate that several characteristics of HOG signaling are conserved between baker's yeast and the human pathogen C. glabrata, although fundamental differences exist with regard to the activating cues. Exploring these differences will also shed further light on networks and cross talk between MAPK pathways in yeast.

 
We thank our colleagues Ken Haynes, Janet Quinn, Helena Budjakova, and Steffen Rupp for the gifts of reagents, strains, and materials as well as for stimulating discussions. We thank Wolfgang Reiter for providing the phosphospecific antibodies and Walter Glaser for help with designing oligonucleotides and with DNA sequence deposition. We thank Dominique Ferrandon for fruitful suggestions about the use of the fly model to study the virulence of C. glabrata.

This work was supported in part by the EC project "EURESFUN" (STREP-2004-CT-PL518199), the EC Marie Curie Training Network "CanTrain" (CT-2004-512481), and a project of the "Wiener Wissenschafts & Technologie Fonds" WWTF (HOPI-LS133) (to K.K. and G.A.). Additional funds came from the Herzfelder Foundation (to C.S.).

 
* Corresponding author. Mailing address: Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria. Phone: 43-1-4277-61807. Fax: 43-1-4277-9618. E-mail: karl.kuchler{at}meduniwien.ac.at

Published ahead of print on 6 July 2007.

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Eukaryotic Cell, September 2007, p. 1635-1645, Vol. 6, No. 9
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