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Eukaryot Cell. Aug 2009; 8(8): 1197–1217.
Published online Jun 19, 2009. doi:  10.1128/EC.00120-09
PMCID: PMC2725552

Remodeling of Global Transcription Patterns of Cryptococcus neoformans Genes Mediated by the Stress-Activated HOG Signaling Pathways [down-pointing small open triangle]

Abstract

The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.

Whether an organism is able to survive and proliferate in certain environmental niches is mainly determined by the ability to sense and adapt to diverse environmental stresses and maintain cellular homeostasis. Cells achieve homeostasis by deploying a series of complex signaling networks. Among these, the p38/Hog1 mitogen-activated protein kinase (MAPK)-dependent signaling pathway plays a pivotal role in regulating a plethora of stress responses in eukaryotic organisms ranging from yeasts to humans (5). The mammalian stress-activated p38 MAPK transduces myriad stress-related signals, governing adaptation to osmotic changes and UV irradiation, programmed cell death, and immune responses by controlling cytokine production and inflammation (10, 32). Comparable stress-sensing signaling cascades have been also uncovered in many fungal species (5, 9). Fungi contain p38-like MAPKs, mostly known as Hog1 MAPKs, to modulate a range of stress responses (5).

The regulatory mechanism of the p38/Hog1 MAPK pathway is widely conserved in many eukaryotic cells. Under unperturbed normal conditions, the p38/Hog1 MAPK remains unphosphorylated, but in response to certain environmental stresses, it is activated by dual phosphorylation of Thr and Tyr residues in the TGY motif via a MAPK kinase (MAPKK) that is activated through phosphorylation by its upstream MAPKK kinase (MAPKKK) (5). Subsequently, the phosphorylated p38/Hog1 MAPKs dimerize and are translocated into the nucleus to trigger activation of transcription factors and induce a plethora of stress defense genes to counteract external stress conditions (see reviews in references 5, 27, 28, 32, and 36).

In spite of the conserved regulatory mechanism of the p38/Hog1 MAPK, fungi and mammals have unique upstream regulatory systems. In particular, fungi employ a two-component-like phosphorelay system, which has been discovered only in bacteria, fungi and plants, but not in mammals. The fungal phosphorelay system consists of three components, including hybrid sensor kinases, a histidine-containing phosphotransfer protein, and response regulators, all of which are absent in mammals and therefore considered as candidate antifungal targets (5, 9).

The basidiomycete Cryptococcus neoformans, an opportunistic human-pathogenic fungus causing meningoencephalitis, also utilizes the Hog1 MAPK pathway for adaptation to a wide range of environmental stresses, including osmotic shock, UV irradiation, heat shock, oxidative damage, toxic metabolites, and antifungal drugs (5-8, 35). Compared to other fungal Hog1 MAPK systems, however, the C. neoformans Hog1 MAPK pathway is uniquely specialized not only to respond to diverse environmental stresses but also to control production of two virulence factors, the antiphagocytic capsule and antioxidant melanin, and sexual differentiation. Hence, the Hog1 MAPK may play a pivotal role as a key signaling regulator in C. neoformans that modulates cross talk with other signaling pathways (5-8, 35). Recently, we reported that the Hog1 MAPKs in a number of C. neoformans strains are constitutively phosphorylated under unstressed conditions and in response to osmotic shock rapidly dephosphorylated for activation (6-8, 35), which is in stark contrast to other fungal Hog1 MAPK systems. Dual phosphorylation of the TGY motif in Hog1 requires the Pbs2 MAPKK (8).

Upstream of the Pbs2-Hog1 pathway, a fungus-specific phosphorelay system has also been discovered in C. neoformans (7). The C. neoformans phosphorelay system comprises seven different sensor hybrid histidine kinases (Tco1 to Tco7), the Ypd1 phosphotransfer protein, and two response regulators (Ssk1 and Skn7) (7). The Pbs2-Hog1 pathway is mainly regulated by Ssk1, but not by Skn7 (7). Among seven Tco proteins, Tco1 and Tco2 play discrete and redundant roles in activating Ssk1 and the Pbs2-Hog1 MAPK pathway (7). However, since Tco1 and Tco2 regulate only a subset of Ssk1- and Hog1-dependent phenotypes, other upstream receptors or sensor proteins remain to be elucidated. More recently, we identified Ssk2 as an interfacing MAPKKK between the phosphorelay system and the Pbs2-Hog1 MAPK pathway, through comparative analysis of meiotic maps between the serotype D f1 sibling strains B-3501 and B-3502, which show differential Hog1 phosphorylation patterns (6). Most notably, interchange of SSK2 alleles between the two C. neoformans strains showing differential Hog1 phosphorylation patterns exchanged the phenotypes governed by constitutive Hog1 phosphorylation (6). Unlike Saccharomyces cerevisiae and Schizosaccharomyces pombe, C. neoformans harbors a single MAPKKK, Ssk2, which is necessary and sufficient to control the Hog1 MAPK (6). Nevertheless the downstream signaling network of the Hog1 MAPK pathway in C. neoformans was unknown. Identification and characterization of the downstream signaling network of the Hog1 MAPK are important to further understand the complex phosphorelay system and the Hog1 MAPK signaling network.

Here we investigated the downstream signaling network of the HOG pathway by performing genome-wide comparative transcriptome analysis through DNA microarray analysis with the C. neoformans wild-type (WT) strain H99 and hog1Δ, ssk1Δ, and skn7Δ mutant strains responding to high osmotic shock, fludioxonil treatment, and oxidative stress. In this study, we not only gained important insight into global transcriptional remodeling patterns of cryptococcus genes for counteracting external stresses but also elucidated a number of novel characteristics of the HOG pathway and stress-related genes, as well as the Hog1-, Ssk1-, and/or Skn7-dependent genes. Hence this study provides an excellent opportunity to develop a novel therapeutic approach to treat the life-threatening fungal meningitis caused by C. neoformans.

MATERIALS AND METHODS

Strains and growth conditions.

The C. neoformans strains used in this study are listed in Table S1 in the supplemental material and were cultured in YPD (yeast extract-peptone-dextrose) medium unless indicated separately. The sch9Δ (CNAG_06301.2; with the H99 gene identification [ID] no., “CNAG_XXXXX.2,” indicated as by a five-digit number hereafter), ena1Δ (00531), ubc6-2Δ (02214), ubc8Δ (04611), pdr5Δ (00869), pdr5-2Δ (04098), pdr5-3Δ (06348), and yor1Δ (03503) mutants were obtained from the C. neoformans deletion mutant library (Fungal Genetics Stock Center; http://www.fgsc.net/), which was constructed by the Madhani laboratory (38). As a control WT strain for phenotypic analysis of these mutants we used the H99 isolate CMO18, which was used for construction of Madhani's C. neoformans deletion mutant library. To verify each mutant recovered from the deletion mutant library, diagnostic PCR was performed with primers listed in Table S1 in the supplemental material to check whether the corresponding genes were disrupted. In addition, the ena1Δ mutant (AI167) and its complemented strains (AI173) were also kindly provided by Alex Idnurm (University of Missouri) (31).

For total RNA isolation used in DNA microarray analysis, the WT H99 strain and hog1Δ (YSB64), ssk1Δ (YSB261), and skn7Δ (YSB349) mutant strains were grown in 50 ml YPD medium at 30°C for 16 h. Then 5 ml of the overnight culture was inoculated into 100 ml of fresh YPD medium and further incubated at 30°C until it approximately reaches an optical density at 600 nm (OD600) of 1.0. For time zero samples, 50 ml of the 100-ml culture was sampled and rapidly frozen in liquid nitrogen. To the remaining 50-ml culture, 50 ml of YPD containing 2 M NaCl, 40 μg/ml fludioxonil (Pestanal; Sigma), or 5 mM H2O2 was added. During incubation, 50 ml of the culture was sampled at 30 and 60 min, pelleted in a tabletop centrifuge, frozen in liquid nitrogen, and lyophilized overnight. The lyophilized cells were subsequently used for total RNA isolation. As biological replicates for DNA microarrays, three to four independent cultures for each strain and growth condition were prepared for total RNA isolation.

Total RNA preparation.

For total RNA isolation, the lyophilized cell pellets were added to a 3-ml volume of sterile 3-mm glass beads, homogenized by shaking, added to 4 ml of TRIzol reagent (Molecular Research Center), and allowed to incubate at room temperature for 5 min. Then 800 μl of chloroform was added, incubated for 3 min at room temperature, transferred to 15-ml round-bottom tubes (SPL), and centrifuged at 10,000 rpm at 4°C for 15 min in a Sorvall SS-34 rotor. Two milliliters of the supernatant was transferred to a new round-bottom tube, 2 ml isopropanol was added, the tube was inverted several times, and the mixture was allowed to incubate for 10 min at room temperature. Then the mixture was recentrifuged at 10,000 rpm at 4°C for 10 min, and the pellet was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC)-treated water and centrifuged at 8,000 rpm at 4°C for 5 min. The pellet was dried and resuspended with 500 μl DEPC-treated water. The concentration and purity of total RNA samples were calculated by measuring OD260 and gel electrophoresis, respectively. For control total RNA, all total RNAs prepared from WT and hog1Δ, ssk1Δ, and skn7Δ mutant cells grown under the conditions described above were pooled as reference RNAs.

cDNA synthesis and Cy3 and Cy5 labeling.

For cDNA synthesis, the total RNA concentration was adjusted to 1 μg/μl with DEPC-treated water, and 15 μl of the total RNA was added to 1 μl of 5 μg/μl oligo(dT) (5′-TTTTTTTTTTTTTTTTTTTTV-3′)-pdN6 (Amersham) (1:1 mixture of 10 μg/μl, respectively), incubated at 70°C for 10 min, and place on ice for 10 min. Then 15 μl of the following cDNA synthesis mixture was added and incubated at 42°C for 2 h: 3 μl 0.1 M dithiothreitol, 0.5 μl RNasin (Promega), 0.6 μl aa-dUTP [5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate]-dNTPs (a mixture of 6 μl dTTP, 4 μl aa-dUTP, 10 μl dATP, 10 μl dCTP, and 10 μl dGTP at 100 mM each), 1.5 μl AffinityScript reverse transcriptase (Stratagene), 3 μl AffinityScript buffer, and 7 μl water. Then 10 μl of 1 N NaOH and 10 μl of 0.5 M EDTA (pH 8.0) were added and incubated at 65°C for 15 min. After incubation, 25 μl of 1 M HEPES buffer (pH 8.0) and 450 μl of DEPE-treated water were added, and the whole mixture was concentrated through a Microcon30 filter (Millipore) and vacuum dried for 1 h.

For Cy3 and Cy5 (Amersham) labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 μl dimethyl sulfoxide, and 1.25 μl of each dye was aliquoted into separate tubes. The cDNAs prepared as described above were added to 9 μl of 0.05 M Na-bicarbonate (pH 8.0) and incubated at room temperature for 15 min. The cDNAs prepared from pooled reference RNAs were mixed with Cy3 as a control, and the cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy5. For a dye-swap experiment, control and test RNAs were labeled oppositely. Each mixture was further incubated at room temperature for 1 h in the dark and purified with the QIAquick PCR purification kit (Qiagen).

Microarray hybridization and washing.

A C. neoformans serotype D 70-mer microarray slide containing 7,936 probes (Duke University) was prehybridized at 42°C in 60 ml of prehybridization buffer (42.4 ml sterile distilled water, 2 ml 30% bovine serum albumin, 600 μl 10% sodium dodecyl sulfate [SDS], 15 ml 20× SSC (saline-sodium citrate, 3 M NaCl, 0.3 M sodium citrate [pH 7.0]), washed with distilled water and isopropanol, and dried by brief centrifugation (110 × g for 2 min). The Cy3- and Cy5-labeled cDNA samples were combined, concentrated through a Microcon30 filter, and vacuum dried. The dried cDNA samples were resuspended with 24 μl of 1× hybridization buffer (250 μl 50% formamide, 125 μl 20× SSC, 5 μl 10% SDS, 120 μl distilled water [dH2O], for a total of 500 μl), added with 1 μl poly(A) tail DNA (Sigma), further incubated at 100°C for 3 min, and allowed to cool for 5 min at room temperature. The microarray slides were aligned into the hybridization chamber (DieTech), any dust was removed, and the slides were covered by Lifterslips (Erie Scientific). The Cy3- and Cy5-labeled cDNA samples were applied between Lifterslips and slides. To prevent slides from drying, 10 μl of 3× SSC buffer was applied to the slides, which were subsequently incubated for 16 h at 42°C. After incubation, the microarray slides were washed with the following three different washing buffers for 2, 5, and 5 min, respectively, on an orbital shaker: wash buffer 1, 10 ml 20× SSC, 600 μl 10% SDS, 189.4 ml dH2O, preheated at 42°C; wash buffer 2, 3.5 ml 20× SSC, 346.5 ml dH2O; and wash buffer 3, 0.88 ml 20× SSC, 349.12 ml dH2O. Three to four independent DNA microarrays with three to four independent biological replicates were performed, including a one-dye swap experiment.

Microarray slide scanning and data analysis.

After hybridization and washing, the microarray slides were scanned with a GenePix 4000B scanner (Axon Instrument) and the signals were analyzed with GenePix Pro (version 4.0) and gal file (http://genome.wustl.edu/activity/ma/cneoformans). Since total RNAs isolated from serotype A C. neoformans strains were hybridized on the microarray slides printed with the serotype D 70-mer oligonucleotide sequences, the serotype A gene IDs were mapped to those of the serotype D using BLASTN with cutoff E value of E−6. C. neoformans H99 gene sequences that were updated at 24 November 2008 were downloaded from the Broad Institute (http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans). The functional category of each C. neoformans H99 gene was assigned using the NCBI KOG database (http://www.ncbi.nlm.nih.gov/COG/grace/shokog.cgi). Using the serotype A gene sequence, each S. cerevisiae gene name or ID listed in the tables in the supplemental material was identified by BLASTP search (E value cutoff, E−6). For hierarchical and statistical analysis, data transported from GenePix software were analyzed with GeneSpring (Agilent) by employing Lowess normalization, reliable gene filtering, hierarchical clustering (standard correlation and average linkage) and zero transformation, and analysis of variance (ANOVA) analysis, as well as Microsoft Excel software (Microsoft).

Northern hybridization.

Northern blot analysis was performed with 10 μg of total RNA from each strain that was used for DNA microarray analysis. Electrophoresis and hybridization were carried out by following the standard protocols previously described (4). Probes for each gene were prepared by PCR amplification with primers listed in Table S1 in the supplemental material, gel extracted, and radiolabeled with the Rediprime II random prime labeling system (Amersham).

Quantitative real-time RT-PCR.

Real-time reverse transcription-PCR (RT-PCR) for quantitatively measuring relative expression levels of ERG11 was performed with primers listed in Table S1 in the supplemental material and cDNAs that were generated using the SuperScript II reverse transcriptase system with total RNAs used in DNA microarray analysis. Relative gene expression was calculated by the threshold cycle (2−ΔΔCT) method (39). ACT1 was used for normalization of gene expression.

Comparison of stress response genes between C. neoformans and other fungi.

Protein sequences from C. neoformans H99, S. cerevisiae, S. pombe, and C. albicans were used to perform the BLASTP search against each other. S. cerevisiae sequences were downloaded from Saccharomyces Genome Database (http://www.yeastgenome.org/). S. pombe sequences were downloaded from Schizosaccharomyces pombe GeneDB (http://www.sanger.ac.uk/Projects/S_pombe/). C. albicans sequences were downloaded from the Candida Genome Database (http://www.candidagenome.org/). Orthologs were selected on the basis of best reciprocal BLAST hit above a cutoff E value of E−6 (see Table S2 in the supplemental material). To compare the expression of stress response genes in four fungi, we used the transcriptome data set from C. neoformans H99 (this study), S. cerevisiae (25), S. pombe (20), and C. albicans (22, 23).

Ergosterol assay.

Ergosterol content was measured as previously described (3), but with slight modification. Briefly, each C. neoformans strain was grown in 100 ml YPD medium for 24 h at 30°C. The 100-ml culture was divided into two 50-ml cultures for duplicate measurement, pelleted, and washed with sterile water. The cell pellet was frozen in liquid nitrogen and lyophilized overnight. The dried cell pellet was weighed for normalization of ergosterol content, 5 ml of 25% alcoholic potassium hydroxide was added, and the sample was transferred to a sterile borosilicated glass screw-cap tube. Subsequently, the cells were incubated at 80°C for 1 h and allowed to cool to room temperature. Then 1 ml of sterile water and 3 ml of heptane were added, and the mixture was vortexed for 3 min. Then 200 μl of the heptane layer was sampled and mixed with 800 μl of 100% ethanol, and its OD was measured at both 281.5 nm and 230 nm. Ergosterol content was calculated as follows: % ergosterol = [(OD281.5/290) × F]/pellet weight − [(OD230/518) × F]/pellet weight, where F is the ethanol dilution factor and 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24(28)dehydroergosterol, respectively (3).

Stress and antifungal drug sensitivity tests.

Each strain was incubated overnight at 30°C in YPD medium, washed, serially diluted (1 to 104 dilutions) in dH2O, and spotted (3 μl) onto solid YPD medium containing the indicated concentrations of stress-inducing agents and antifungal drugs as previously described (7, 8). For the osmotic stress sensitivity test, a 0.5 to 1.5 M range of KCl or NaCl was added to YPD or YP agar medium. For the oxidative stress sensitivity test, a range of 2 to 3 mM H2O2 was added to liquefied YPD agar medium prewarmed at 55°C. To examine antifungal drug sensitivity, the cells were spotted onto agar-solid YPD media containing fludioxonil (1 to 100 μg/ml), amphotericin B (0.05 to 1.0 μg/ml) (Sigma), fluconazole (16 to 18 μg/ml) (Sigma), itraconazole (0.04 to 0.05 μg/ml) (Sigma), and ketoconazole (0.2 μg/ml) (Sigma). To test sensitivity to heavy metals, cells were spotted onto solid YPD medium containing cadmium (15 to 30 μM). To measure sensitivity to UV irradiation, each strain was spotted onto YPD agar medium first and then placed in a UV crosslinker (UVP CX-2000) at energy levels between 200 and 400 J/m2. Then spotted cells were incubated at 30°C for 2 to 4 days and photographed.

Microarray data accession number.

The whole microarray data generated by this study have been submitted to the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE16692.

RESULTS

DNA microarray analysis of C. neoformans hog1Δ, ssk1Δ, and skn7Δ mutants.

To investigate the target genes and downstream signaling network of the Skn7-, Ssk1-, and Hog1-dependent signaling pathway in C. neoformans, we performed comparative transcriptome analysis of the serotype A WT strain (H99) and hog1Δ, ssk1Δ, and skn7Δ mutants under both normal growth conditions and stressed conditions as described in Materials and Methods. For basic validation of our array quality, we monitored expression levels of the HOG1, SSK1, and SKN7 genes and known Hog1-regulated genes, such as GPP1 (glycerol-3-phosphatase) and GPD1 (glycerol-3-phosphate dehydrogenase), in our array data. As expected, the relative expression levels of the HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to the WT strain were very low (0.06-, 0.09-, and 0.22-fold changes, respectively) (Fig. (Fig.1A).1A). In addition, basal expression levels of the GPD1 (glycerol-3-phosphate dehydrogenases; 01745 and 00121) and GPP1 (dl-glycerol-3-phosphatase; 01744) homologous genes, which are well-known Hog1-regulated stress defense genes in other fungi, were more than twofold reduced in hog1Δ and ssk1Δ mutants compared to the WT (see Table S2 in the supplemental material). The GPD1 and GPP1 genes were more than twofold induced in the WT in response to osmotic shock, whereas their expression levels were substantially lower than those in hog1Δ or ssk1Δ mutants during osmotic shock, further supporting the quality of our array data (see Table S2 in the supplemental material).

FIG. 1.
Genome-wide identification of C. neoformans genes whose expression is controlled by Hog, Ssk1, and Skn7 under unstressed, normal growth conditions. The change (fold) is illustrated by color (see the color bar scale). (A) Relative expression levels of ...

Genes regulated by Hog1, Ssk1, and/or Skn7 under unstressed conditions.

First we monitored how hog1, ssk1, and skn7 mutations affect gene expression patterns in C. neoformans under unperturbed, unstressed conditions. Among 7,936 probes monitored, 3,858 probes were found to be reliable (Cy3 reference value cutoff of 10 with 100% filtering) (see Tables S3 and S4 in the supplemental material). Supporting previous findings (7, 8), the transcriptional profile of the hog1Δ mutant was markedly similar to that of the ssk1Δ mutant, based on the condition tree analysis (Fig. (Fig.1B).1B). A total of 1,697 genes exhibited significantly different expression patterns in hog1Δ, ssk1Δ, or skn7Δ mutants compared to the WT (P < 0.05, ANOVA) (Fig. (Fig.1C;1C; and see Tables S4 and S5 in the supplemental material), indicating that a significant portion of the entire C. neoformans genome could be transcriptionally affected by perturbation of the two-component system and HOG signaling pathways even under unstressed, normal conditions. Among these, 714 genes exhibited more than twofold induction (251 genes) or reduction (463 genes) in at least one of the mutants (Fig. (Fig.1D1D).

Several key findings were obtained. First, a majority of the genes (702 genes; 98.3%) were upregulated or downregulated by either Ssk1 or Hog1 under unstressed conditions, while only 86 genes (12%) were regulated by Skn7. Among the Skn7-dependent genes, only 12 genes were found to be Skn7-specific (Fig. (Fig.1D).1D). Thus hog1 and ssk1 mutations alter genome-wide transcription profiles under unstressed conditions to a greater extent than the skn7 mutation (Fig. (Fig.1D).1D). Second, there was a significantly higher overlap between Ssk1- and Hog1-dependent genes (473 out of 714 genes; 66.2%) than between Skn7- and Hog1-dependent genes (70 out of 714 genes; 9.8%), further corroborating that Ssk1 is the major upstream regulator of the Hog1 MAPK. Third, regardless of the significant overlap in genes regulated by Ssk1 and Hog1, there were a number of Ssk1-specific genes (153 genes) and Hog1-specific genes (69 genes), strongly suggesting that Ssk1 and Hog1 are not exclusively in a linear pathway and could have other targets or upstream regulators, respectively (Fig. (Fig.1D).1D). This explains why the ssk1Δ mutant exhibits slightly different phenotypes (i.e., higher and lower sensitivity to oxidative and osmotic stresses, respectively) compared to hog1Δ mutants and why Hog1 can still be phosphorylated in the absence of the Ssk1 response regulator upon exposure to NaCl (7).

Genes regulated by the two-component system and HOG pathway cover a wide variety of functional categories (see Fig. S1 to S4 in the supplemental material), indicating that active remodeling of various aspects of cellular function could occur simply by perturbation of the pathways even without external stress. When basal expression level changes of signaling components in diverse signal transduction pathways in the ssk1Δ, skn7Δ, and hog1Δ mutants were compared to the WT, several novel findings were apparent (see Fig. S1B and Table S6 in the supplemental material). First, genes required for the antiphagocytic polysaccharide capsule were significantly upregulated in ssk1Δ and hog1Δ mutants, but not in the skn7Δ mutant, compared to the WT, including the CAP10 (1.8- to ~2.2-fold), CAP59 (1.6- to ~1.8-fold), CAP60 (1.6 to ~1.9-fold), and CAP64 (1.5- to ~1.7-fold) genes. This may explain why mutation of the HOG pathway increases capsule production in C. neoformans. Second, genes required for melanin biosynthesis were significantly upregulated. The LAC1 gene required for melanin production was induced in skn7Δ (2.3-fold), ssk1Δ (2.1-fold), and hog1Δ (2.7-fold) mutants, compared to the WT, further corroborating our previous observation that melanin synthesis is enhanced by mutation of the HOG pathway and the SKN7-dependent pathway (6-8). Interestingly, expression of IPC1 (inositol-phosphorylceramide synthase 1), which catalyzes production of diacylglycerol, which activates Pkc1 for melanin biosynthesis, was also induced by mutation of the SSK1 and HOG1 genes, indicating that induction of IPC1 may contribute to increased melanin synthesis observed in the HOG mutants. Third, among genes involved in the pheromone-Cpk1 MAPK pathway for sexual differentiation, the SXI1 and GPA2 genes, encoding a homeodomain-containing transcriptional regulator and a G protein α-subunit required for the pheromone-responsive Cpk1 MAPK pathway, were highly upregulated upon ssk1Δ or hog1Δ mutation (2.5- to ~3.0-fold for SXI1 and 4.6- to ~5.1-fold for GPA2, respectively). This finding suggests that increased pheromone production and sexual reproduction found in ssk1Δ and hog1Δ mutants (7, 8) may result from enhanced expression of Gpa2 that promotes and is induced during mating of C. neoformans (29, 37).

Besides the genes involved in controlling virulence factor production and sexual differentiation, several groups of genes provided novel insights into the role of the HOG pathway in virulence regulation and stress response of C. neoformans (see Tables S3 and S4 in the supplemental material). First, a group of genes involved in iron transport and regulation, include SIT1 (00815; a siderophore transporter), CFO1-2 (06241 and 02958; encoding ferroxidases) and CFT1 (06242; an iron transporter), were found to be highly induced in the ssk1Δ and hog1Δ mutants compared to the WT strain. Second, several genes involved in oxidative stress defense, including CTA1 (00575; catalase A), SOD2 (04388; a mitochondrial manganese superoxide dismutase), TRR1 (05847; thioredoxin reductase), TSA1 (03482; thioredoxin peroxidase), GRX5 (03985; glutathione-dependent oxidoreductase), CCP1 (01138; mitochondrial cytochrome c peroxidase), and SRX1 (00654; sulfiredoxin) were differentially regulated by hog1 and ssk1 mutations, further corroborating the role of the HOG pathway in oxidative stress response.

Induction of ergosterol biosynthesis by inhibition of the HOG pathway.

Among genes upregulated by mutation of the HOG1 and SSK1 genes, a gene homologous to ERG28 (03009) was notable since it plays a key role in fungal sterol biosynthesis. Previous microarray analysis performed in S. cerevisiae revealed that expression of ERG28 is tightly correlated with other ergosterol biosynthetic genes (30). This finding led us to monitor expression patterns of other sterol biosynthetic genes in our array data without considering the twofold cutoff. Interestingly, a majority of the ergosterol biosynthetic genes were upregulated in hog1Δ and ssk1Δ mutants, but not in the skn7Δ mutant, compared to the WT strain (Fig. (Fig.2A).2A). Besides ERG28, genes such as ERG11, ERG6, ERG5, ERG25, ERG20, and ERG4, were upregulated in both the ssk1Δ and hog1Δ mutants, while genes such as ERG27, ERG13, ERG26, ERG10, IDI1, HMG2, and ERG8 were upregulated only in the ssk1Δ mutant. In contrast, none of genes was significantly upregulated in the skn7 mutant, and indeed some genes, including the ERG1 and ERG3 genes, were downregulated in the skn7Δ mutant. Northern blotting and quantitative real-time RT-PCR showed higher ERG11 expression levels in the hog1Δ and ssk1Δ strains than the WT and skn7Δ mutant strains, which is in good agreement with the DNA microarray data (Fig. 2C and D).

FIG. 2.
Induction of ergosterol biosynthesis genes and cellular ergosterol contents by perturbation of the HOG signaling pathway. (A) Relative expression profiles of ergosterol biosynthesis genes in hog1Δ, ssk1Δ, and skn7Δ mutants compared ...

To further verify the microarray data, we examined whether increased expression levels of some of the ergosterol biosynthesis genes indeed affect cellular ergosterol content in the hog1Δ and ssk1Δ mutants (Fig. (Fig.2B).2B). In accordance with the microarray data, cellular ergosterol content was significantly higher in the hog1Δ and ssk1Δ mutants than in the WT strain and the skn7Δ mutant (Fig. (Fig.2B),2B), suggesting that increased expression of ergosterol biosynthetic genes leads to enhanced production of cellular ergosterol. Supporting this finding, the ssk2Δ (MAPKKK) and pbs2Δ (MAPKK) mutants in the HOG pathway were also found to contain significantly higher levels of cellular ergosterol than the WT and the skn7Δ mutant (Fig. (Fig.2B).2B). Taken together, ergosterol biosynthesis is repressed by the HOG pathway under normal conditions.

Inhibition of the HOG signaling pathway dramatically increases antifungal activity of amphotericin B against C. neoformans.

The finding that ergosterol biosynthesis is induced by inhibition of the HOG pathway prompted us to investigate the susceptibility of the mutants in the two-component system and the HOG pathway to antifungal drugs that target the ergosterol biosynthetic genes or ergosterol itself. We hypothesized that increased ergosterol content observed in the ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants could render them hypersensitive to amphotericin B due to the increased number of drug targets. Confirming this hypothesis, the ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants exhibited dramatic hypersensitivity to amphotericin B treatment compared to the WT (Fig. (Fig.3A).3A). In contrast, the skn7Δ mutant showed WT levels of susceptibility to amphotericin B (Fig. (Fig.3A3A).

FIG. 3.
Inhibition of the HOG pathway confers synergistic antifungal effects with amphotericin B in C. neoformans. (A and B) Each C. neoformans strain, including the WT (H99) and the hog1Δ (YSB64), pbs2Δ (YSB123), ssk2Δ (YSB264), ssk1 ...

We also monitored amphotericin B susceptibility of C. neoformans strains having mutations of hybrid sensor kinases (Tco1 to Tco7, except for Tco6), which act upstream of the Ssk1 response regulator. Previously we have shown that Tco1 and Tco2 play redundant and distinct roles in controlling a subset of Hog1-dependent phenotypes. Here we found that Tco1 and Tco2 play discrete roles in sensing and responding to amphotericin B. Deletion of TCO2 conferred hypersensitivity to amphotericin B, similar to the hog1Δ mutant (Fig. (Fig.3B).3B). However, the fact that the degree of hypersensitivity observed in the tco2Δ mutant is less than that in the ssk1Δ mutant suggests that constitutively phosphorylated Hog1 may repress the ergosterol biosynthetic pathway under normal conditions regardless of the presence of receptors/sensors since Ssk1, Ssk2, and Pbs2, but not the Tco2 protein, are all involved in constitutive phosphorylation levels of Hog1 (6-8). To test this hypothesis, we examined amphotericin B sensitivity of other C. neoformans strains, such as JEC21 and B3501-A, showing differential Hog1 phosphorylation levels (6). In support of the second hypothesis, the JEC21 strain, in which Hog1 is not constitutively phosphorylated (6), was even more hypersensitive to amphotericin B than the ssk2Δ mutant in the H99 strain background (Fig. (Fig.3C).3C). In the JEC21 strain background, mutation of the SSK2, PBS2, and HOG1 genes did not affect sensitivity to amphotericin B (Fig. (Fig.3C).3C). In contrast, the B3501 strain, in which Hog1 is constitutively phosphorylated, albeit to a lesser extent than in the H99 strain, exhibited reduced susceptibility to amphotericin B than JEC21 (Fig. (Fig.3C).3C). Similar to the H99 strain, mutation of the SSK2 MAPKKK that abolishes Hog1 phosphorylation (6) increased amphotericin B sensitivity (Fig. (Fig.3C).3C). Taken together, these data strongly indicate that constitutively phosphorylated Hog1 represses the ergosterol biosynthetic pathway under normal conditions.

To further support this finding, we also examined the susceptibility of the mutants to azole compounds, including triazoles (fluconazole and itraconazole) and imidazole (ketoconazole), which inhibit the fungal cytochrome P450 enzyme 14α-demethylase and prevent conversion of lanosterol to ergosterol. We had expected that the ssk1Δ and hog1Δ mutants having increased expression of many ergosterol biosynthesis genes, particularly including ERG11, would show higher resistance to azole compounds. The ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants all exhibited increased resistance to fluconazole and ketoconazole but not to itraconazole (Fig. (Fig.4).4). Interestingly, the skn7Δ mutant also showed higher resistance to fluconazole and ketoconazole than the WT for unknown reasons (Fig. (Fig.4).4). The fact that the skn7Δ mutant exhibited WT levels of ERG11 expression (Fig. (Fig.2)2) strongly suggested that the fluconazole resistance observed in the skn7Δ mutant is an ERG11-independent phenomenon. Interestingly, none of the hybrid sensor kinases was found to be differentially involved in resistance to fluconazole and ketoconazole, further indicating that differential responses of the HOG mutants to polyene and azole drugs is not receptor or sensor mediated. In conclusion, inactivation of the HOG pathway increases ergosterol content by induction of ergosterol biosynthesis genes and therefore confers synergistic effects with amphotericin B treatment but antagonistic effects with fluconazole and ketoconazole.

FIG. 4.
Inhibition of the HOG pathway confers antagonistic antifungal effects with azole drugs in C. neoformans. Each C. neoformans strain—including the WT (H99) and hog1Δ (YSB64), pbs2Δ (YSB123), ssk2Δ (YSB264), ssk1Δ ...

The HOG pathway negatively modulates resistance to heavy metal stress.

Another key finding revealed by this array analysis is that a number of genes involved in cadmium sensitivity were differentially regulated in the hog1Δ and ssk1Δ mutants (Fig. (Fig.5A).5A). Among the 1,697 genes exhibiting different expression patterns in hog1Δ, ssk1Δ, or skn7Δ mutants, 71 genes were orthologous to genes whose mutation increases sensitivity to cadmium in either S. cerevisiae or S. pombe (Fig. (Fig.5A)5A) (33, 45). Previously it has been reported that perturbation of the HOG pathway in C. albicans, Candida lusitaniae, and S. pombe increases cadmium sensitivity (12, 19, 33). Among the 71 genes identified in our array, however, half (36 genes) were indeed induced more than 1.5-fold in either C. neoformans hog1Δ or ssk1Δ mutants compared to the WT, while only 12 genes were reduced more than 1.5-fold in the mutants (Fig. (Fig.5A).5A). This suggested the possibility that inhibition of the HOG pathway could cause cadmium tolerance in C. neoformans by activating transcription of cadmium-responsive genes. To address this model, we have examined the cadmium sensitivity of the HOG mutants in C. neoformans. Interestingly, the mutants of the HOG pathway, including the ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants, showed higher resistance to cadmium sulfate than the WT strain and the skn7Δ mutant (Fig. (Fig.5B).5B). Among hybrid sensor kinases, the tco2Δ mutant was also more resistant to cadmium, albeit to a lesser extent than the HOG pathway mutants, than the WT, indicating that Tco2 is involved in cadmium sensitivity with a positive relationship with other HOG signaling components, similar to the amphotericin B susceptibility. With the exception of Tco2, none of the Tco sensor kinases was involved in susceptibility to cadmium. Taken together, the HOG pathway negatively regulates resistance to heavy metal stress in C. neoformans.

FIG. 5.
Inhibition of the HOG pathway affects expression levels of a number of cadmium-responsive genes and increases resistance to cadmium in C. neoformans. (A) Relative expression profiles of 71 putative cadmium-responsive genes in the hog1Δ, ssk1Δ, ...

ESR and CSR genes in C. neoformans.

To investigate how the HOG pathway controls stress responses against environmental cues, genome-wide transcription patterns of the WT and hog1Δ, ssk1Δ, and skn7Δ mutant strains were monitored in response to osmotic shock, oxidative stress, and antifungal drug treatment (fludioxonil). A total of 2,218 genes in the WT were found to be more than twofold up- or downregulated at any time point (30 or 60 min) in response to at least one of the stress conditions (P < 0.05, ANOVA) and were named ESR (environmental stress regulated) genes as described previously (see Table S7 in the supplemental material) (20). Several interesting observations emerged. First, global gene expression patterns in response to H2O2 were clearly distinguishable from those in response to osmotic stress and fludioxonil treatment (Fig. (Fig.6A).6A). Second, a much greater number of genes were differentially regulated in response to H2O2 (1,719 genes) than osmotic stress (580 genes) and fludioxonil treatment (510 genes). Only a small portion of genes (125 out of 2,218 genes; 5.6%) were found to be commonly regulated in response to all stresses tested, while the majority (1,947 out of 2,218 genes; 87.8%) were stress-specifically regulated (SSR) at a twofold-change cutoff (Fig. (Fig.6B).6B). This implies that diverse signaling regulators may work to respond to each environmental cue.

FIG. 6.
ESR and CSR genes in C. neoformans. (A) ESR genes in C. neoformans. The ESR genes were defined as genes for which expression was induced or repressed by more than a twofold change in at least one time point (30 and 60 min) under any one of the following ...

Among 2,218 ESR genes, 125 genes were found to be coordinately upregulated (48 genes) or downregulated (77 genes) in response to all stresses and were named CSR (common stress regulated) genes (Fig. (Fig.6C;6C; and see Table S8 in the supplemental material). We also defined CSR extended (CSRE) genes (394 genes) as those upregulated (179 genes) or downregulated (215 genes) in response to at least two stresses (Fig. (Fig.6B;6B; and see Table S8 in the supplemental material). CSR and CSRE genes cover groups of genes involved in diverse cellular functions, indicating that the overall physiological status of C. neoformans is reorganized to adapt to any external stress and maintain normal cellular physiology (see Fig. S5 in the supplemental material). Furthermore, a significant proportion of CSR genes seemed to be modulated by Hog1 and Ssk1, but not by Skn7 (Fig. (Fig.6C),6C), indicating that the HOG signaling pathway in conjunction with the two-component system is the major controller of the common stress response in C. neoformans.

Upregulated CSR or CSRE genes were overrepresented among those involved in inorganic ion transport and metabolism and secondary metabolite biosynthesis, transport, and catabolism. Among downregulated CSR or CSRE genes, genes involved in amino acid transport/metabolism and energy production/conversion were most downregulated (9.9% each), indicating that cells lower energy production during adaptation to environmental stresses (see Fig. S5 in the supplemental material). Among upregulated CSR or CSRE genes, the ENA1 gene (00531) encoding a putative P-type ATPase sodium pump and the NHA1 (01678) gene encoding a Na+/H+ antiporter were highly upregulated (more than threefold induction) in response to exposure to 1 M NaCl. In S. cerevisiae, Nha1 and Ena1 are required for an immediate and long-term adaptation, respectively, to high-salt conditions (43). Interestingly, however, our array data showed that expression of the C. neoformans ENA1 and NHA1 genes was also highly induced in response to H2O2 in both WT and skn7Δ mutants, but not in ssk1 and hog1Δ mutants, showing that the two sodium efflux pumps appear to be controlled by the HOG pathway (Fig. (Fig.7A).7A). To address whether C. neoformans Ena1 mediates the response against osmotic and oxidative stresses, we monitored the susceptibility of the ena1Δ mutant to a variety of stresses (Fig. (Fig.7B).7B). As previously demonstrated by Idnurm et al. (31), the ena1Δ mutant was almost as resistant to osmotic shock as the WT strain (1 to 1.5 M of KCl and NaCl). Since the HOG pathway mutants showed dramatically increased sensitivity to osmotic shock under the glucose starvation condition (YP medium), we have also monitored osmotic sensitivity of the ena1Δ mutants under this condition (Fig. (Fig.7B).7B). Supporting the array data, the ena1Δ mutants exhibited highly increased osmotic sensitivity (even greater than HOG pathway mutants) to high concentrations of NaCl and KCl (Fig. (Fig.7B)7B) in YP medium, strongly suggesting that Ena1 plays a role in osmotic response under the glucose starvation condition. However, the ena1Δ mutant was as resistant to H2O2 as the WT (Fig. (Fig.7B),7B), indicating Ena1 does not play a major role in oxidative stress response. Two ena1Δ mutants independently constructed by the Madhani and Idnurm laboratories exhibited identical phenotypes (Fig. (Fig.7B7B).

FIG. 7.
Role of the Ena1 Na+/K+ efflux pump in stress response of C. neoformans. (A) Each graph illustrates induction or repression levels of ENA1 in the WT strain (H99; ○) and skn7Δ (•), ssk1Δ (□), and ...

Among other transporter genes, a gene (02455) showing the highest homology to the S. cerevisiae high-affinity choline/ethanolamine transporter Hnm1 was also upregulated in response to common stress (34). In contrast, genes involved in carbohydrate transport were significantly downregulated in response to common stresses (particularly for osmotic and fludioxonil treatment), including GAL2 (galactose permease), HXT5, HXT13, HXT5, and HXT17 (Fig. (Fig.7A;7A; and see Table S8 in the supplemental material). Furthermore, the group of genes involved in iron transport and metabolism, including CFO1 and FRE2 (06821), was commonly upregulated, indicating that these genes also play important roles in adaptation to various other stresses besides maintaining iron homeostasis.

SSR genes in C. neoformans.

As mentioned above, the majority of ESR genes were SSR in C. neoformans, suggesting that a unique set of stress defense genes is transcriptionally regulated in a stress-specific manner (Fig. (Fig.88 and and99 and see Fig. Fig.1111).

FIG. 8.
Osmotic stress-specific response genes in C. neoformans. Hierarchical clustering of the expression profiles of osmotic stress-specific response (OsSR) genes in the WT and hog1Δ, ssk1Δ, and skn7Δ mutants is illustrated. The right ...
FIG. 9.
Fludioxonil stress-specific response genes in C. neoformans. (A) Hierarchical clustering of the expression profiles of fludioxonil stress-specific response (FxSR) genes in the WT and hog1Δ, ssk1Δ, and skn7Δ mutants is illustrated. ...
FIG. 11.
Oxidative stress-specific response genes in C. neoformans. (A) Hierarchical clustering of the expression profiles of the oxidative stress-specific response (OxSR) gene group in the WT and hog1Δ, ssk1Δ, and skn7Δ mutants is illustrated. ...

Osmotic stress (NaCl SSR genes).

A total of 1,641 genes were found to be differentially regulated under osmotic stress conditions (1 M NaCl) in WT (P < 0.05, ANOVA). Among these, 580 genes (283 upregulated, 299 downregulated, with 2 genes upregulated at one time point and downregulated at another time point) were transcriptionally regulated with more than a twofold change. Half of the genes (289 genes) were osmotic SSR genes (Fig. (Fig.8),8), named as OsSR genes, and listed in Table S9 in the supplemental material, while the other half were included in the CSR and CSRE genes as described above.

Among the upregulated OsSR genes, genes involved in transport and metabolism of various metabolites, including amino acids, nucleotides, coenzymes, inorganic ions, and secondary metabolites, were most notably overrepresented (see Fig. S6 in the supplemental material), indicating that transporter and permease genes may play a role in counteracting external osmotic changes by transporting diverse osmolytes. These include DUR3 (07448; plasma membrane transporter for both urea and polyamines), MEP2/AMT2 (04758; ammonium permease), STL1 (01683; glycerol symporter), AQY1 (01742; aquaporin water channel), PHO84 (02777; high-affinity inorganic phosphate transporter), and QDR1 (02050; multidrug transporter of the major facilitator superfamily), which all belong to the group of genes showing the highest induction among OsSR genes (see Table S9 in the supplemental material). The downregulated OsSR genes include the following categories of genes, such as cytoskeleton, signal transduction mechanisms, and intracellular trafficking/secretion/vesicular transport (see Fig. S6 in the supplemental material).

Generally, expression profiles of the OsR genes were greatly affected by mutation of either the SSK1 or HOG1 gene (Fig. (Fig.8).8). Among them, the four clusters indicated in Fig. Fig.88 were notable, although the functions for a majority of the genes are unknown. In cluster II, where upregulated OsR genes were notably downregulated by mutation of the SSK1 and HOG1 genes, AQY1 is evident. In S. cerevisiae, aquaporin (Aqy1) is required for prolonged survival under rapid changes in osmolarity (13). Therefore, it is likely that C. neoformans induces expression of the AQY1 gene upon exposure to high osmotic conditions to maintain intracellular water balance. Basal and induced expression levels of AQY1 were more than 10-fold decreased in both ssk1Δ and hog1Δ mutants.

Fludioxonil stress (fludioxonil SSR genes).

C. neoformans undergoes genome-wide remodeling of transcriptional profiles by fludioxonil treatment in a similar pattern to osmotic stress (Fig. (Fig.6A).6A). A total of 1,215 genes were found to be differentially regulated under fludioxonil treatment in the WT (ANOVA; P < 0.05). Among them, 510 genes (240 upregulated and 272 downregulated, with 2 genes that were upregulated at one time point and downregulated at another time point) were transcriptionally regulated with more than twofold changes (see Table S10 in the supplemental material). Also similar to NaCl stress, 37.8% of genes (193 genes) were fludioxonil SSR (Fig. (Fig.9A)9A) and named “FxSR genes.”

Among upregulated FxSR genes, groups of genes involved in posttranslational modification, protein turnover, and lipid transport and metabolism were overrepresented (see Fig. S7 in the supplemental material). Furthermore, similar to OsSR genes, a group of genes involved in the secondary metabolite biosynthesis, transport, and metabolism were notably overrepresented in the FxSR genes. The most notable groups of genes overrepresented in downregulated FxSR genes include those involved in transport and metabolism of carbohydrates, nucleotides, lipid, and some secondary metabolites (see Fig. Fig.77 in the supplemental material).

Among the upregulated FxSR genes, several genes encoding putative membrane ATP binding cassette (ABC) transporters were most evident. In S. cerevisiae, the ABC-type multidrug transporters, including Pdr5, Pdr15, Snq2, and Yor1, play a critical role in cellular detoxification and pleiotropic drug resistance (PDR) (48). Our array data clearly showed that PDR5/15 (00869, 04098, and 06348; here named PDR5, PDR5-2, and PDR5-3, respectively), YOR1 (03503), and SNQ1 (06338) homologues were highly upregulated (up to 57-fold changes for PDR5) specifically upon exposure to fludioxonil treatment, indicating that these proteins may enhance efflux of fludioxonil (see Table S10 in the supplemental material). More interestingly, expression of these genes was even more upregulated (up to 164-fold changes for PDR5) by mutation of the HOG1 gene, which may also explain the resistance of the hog1Δ mutant to the drug treatment.

To address the role of ABC multidrug transporters, we have monitored the drug sensitivity of pdr5Δ, pdr5-2Δ, pdr5-3Δ, and yor1Δ mutants to various stress and drug treatments (Fig. 10A). All of these mutants showed WT levels of sensitivity against various stresses, such as osmotic and salt shock, UV irradiation, oxidative stress, and cadmium stress, indicating that these ABC multidrug transporters are not involved in general stress response. However, the pdr5Δ mutant, but not other pdr5-2Δ, pdr5-3Δ, and yor1Δ mutants, exhibited slightly increased sensitivity to fludioxonil and fluconazole compared to the WT, indicating that Pdr5 may be involved in efflux of antifungal drug for detoxification in agreement with our microarray data showing striking expression-level changes of PDR5 during fludioxonil exposure. Other Pdr5 homologues and Yor1 may play redundant roles in drug efflux, and therefore single mutations may not generate any discernible phenotypes. Since C. neoformans contains a number of Pdr5- or Pdr15-like ABC efflux pumps in the genome, multiple deletions of the ABC efflux pump genes may generate more readily discernible phenotypes.

FIG. 10.
Role of multidrug efflux pump genes and ubiquitin-conjugating enzymes in stress response of C. neoformans. (A and B) Each C. neoformans strain—including the WT (H99) and hog1Δ (YSB64) mutant; the control H99 WT strain CMO18 (WT-M); and ...

Expression profiles for almost half of the FxSR genes were perturbed by mutation of HOG1 and SSK1 (Fig. (Fig.9A).9A). Some of the upregulated FxSR genes (indicated as clusters I and II in Fig. Fig.9)9) were clearly downregulated in either ssk1Δ or hog1Δ mutants. In contrast, some of the downregulated FxSR genes were upregulated in the HOG mutants (indicated as cluster IV in Fig. Fig.9).9). Interestingly, the PKA1 gene (00396), encoding a cyclic AMP (cAMP)-dependent protein kinase A (PKA) catalytic subunit, was found to be upregulated in response to fludioxonil in a HOG-dependent manner (Fig. (Fig.9A;9A; and see Table S10 in the supplemental material). To address whether the cAMP/PKA signaling pathway is involved in fludioxonil sensitivity, we measured the fludioxonil sensitivity of various cAMP/PKA mutants in C. neoformans (Fig. (Fig.9B).9B). The pka1Δ mutant and other cAMP mutants (the gpa1Δ, cac1Δ, pka2Δ, and pka1Δ pka2Δ mutants), however, did not show any differential sensitivity to fludioxonil, indicating that the cAMP pathway is not directly involved in adaptation to fludioxonil. In contrast, the aca1Δ mutant was more sensitive to fludioxonil than the WT strain (Fig. (Fig.9B),9B), suggesting that AcaI is involved in response to fludioxonil independent of the cAMP pathway.

Oxidative-stress (H2O2 SSR genes).

C. neoformans remodels genome-wide expression profiles in response to H2O2 in much more unique and dramatic patterns than in response to osmotic shock and fludioxonil treatment (Fig. (Fig.6A).6A). First, the number of H2O2-regulated genes is much greater. A total of 2,700 genes were found to be differentially regulated in response to H2O2 exposure in the WT (P < 0.05, ANOVA). Among them, 1,719 genes (864 upregulated and 861 downregulated, with 5 genes that were both up- or downregulated depending on time points) were more than twofold regulated in at least one time point (Fig. 11A; and see Table S11 in the supplemental material). Second, a greater number of stress-specific genes were found in response to H2O2. Notably, 84.9% of genes (1,459 genes out of 1,719 genes) were named OxSR (oxidative stress specifically regulated) genes, indicating that C. neoformans uniquely remodels genome-wide expression profiles in response to oxidative stress.

The following categories of genes were overrepresented in upregulated OxSR genes: signal transduction, inorganic ion transport and metabolism, posttranslational modification, transcription, and amino acid transport and metabolism (see Fig. S8 in the supplemental material). Expectedly, genes encoding putative or known oxidative defense proteins were highly upregulated. These include TRR1 (05847; cytoplasmic thioredoxin reductase, 23- to ~60-fold induction), TSA1 (03482; thioredoxin peroxidase, 8- to ~18-fold induction), CCP1 (7- to ~11-fold induction), GRX3 (02950; glutathione-dependent oxidoreductase, 2-fold induction), and GPX2 (02503; phospholipid hydroperoxide glutathione peroxidase, 2.6-fold induction). Induction of TRR1, TSA1, CPP1, and GPX2 was dependent upon Skn7, Ssk1, and Hog1, further corroborating that both Skn7- and Ssk1-Hog1 signaling pathways are involved in oxidative stress response.

Among genes involved in posttranslational modification and protein turnover, a number of genes encoding ubiquitin-conjugating enzymes were notable, including UBI4 (01920; ubiquitin, ~6.0-fold), UBC4 (05696 and 01084; ubiquitin-conjugating enzyme, 2.4- to ~5.1-fold), UBC6 (02214 and 05765; ubiquitin-conjugating enzyme, ~19-fold and 2.5-fold, respectively), UBC7 (06592; ubiquitin-conjugating enzyme, ~2.1-fold), and UBC8 (04611; ubiquitin-conjugating enzyme, ~7.2-fold). (Note that 05765, named Ubc6, shows much higher homology to S. cerevisiae Ubc6 than 02214, named Ubc6-2.) A recent study shows that ubiquitin-conjugating systems required for protein degradation are one of the four group of genes that are commonly induced in response to oxidative stress in eukaryotic organisms, including humans, plants, and fission and budding yeasts (46). In fact, the ubi4Δ mutant exhibits hypersensitivity to H2O2 in S. cerevisiae (21). Furthermore, the ubiquitin-proteasome system negatively regulates the two-component system by selective degradation of Ssk1 in S. cerevisiae (44).

To address any involvement of the ubiquitin-dependent system in stress responses in C. neoformans, we monitored stress sensitivity of strains having mutation in genes encoding two ubiquitin-conjugating enzymes, including UBC6-2 and UBC8, since they showed greatest induction in response to oxidative stress (19.2- and 7.2-fold induction) (Fig. 10B). Our results demonstrated that the ubiquitin-proteasome system is involved in diverse stress responses. Although the ubc6-2Δ mutant did not exhibit any increased stress sensitivity to osmotic and oxidative stress, it showed slightly increased sensitivity to cadmium and fludioxonil. Interestingly, the ubc6-2Δ mutant showed increased sensitivity to amphotericin B but increased resistance to fluconazole, similar to the hog1Δ mutant, although to a lesser extent, indicating that Ubc6-2 may be involved in ergosterol biosynthesis. In contrast, the ubc8Δ mutants show WT levels of susceptibility to most general stresses and antifungal drugs. Interestingly, however, the ubc8Δ mutant is hypersensitive to H2O2 compared to the WT strain, indicating that Ubc8 appeared to be involved in oxidative stress response. These results indicated that the ubiquitin-dependent system appears to be involved in certain stress response of C. neoformans by employing different components of the Ubc proteins.

Two categories of genes were overrepresented in downregulated OxSR genes. One group of genes is involved in translation, ribosomal structure, and biogenesis and the other is involved in energy production and conversion. Particularly the former was most notable (23.5% versus 5.7% random occurrence) (see Fig. S8 in the supplemental material). A number of ribosomal component genes were significantly downregulated upon exposure to H2O2, including more than 90 ribosomal protein genes (see Table S11 in the supplemental material). However, the repression of ribosomal protein genes was not observed in the hog1Δ mutant, indicating that Hog1 MAPK is involved in ribosome biosynthesis. Previous genome-wide transcriptome analysis of S. cerevisiae and S. pombe also demonstrated that groups of ribosome biosynthesis genes are significantly downregulated in response to oxidative stresses (H2O2 or menadione) (14, 20, 25), indicating that inhibition of protein synthesis in response to oxidative stress is a general phenomenon in fungi.

Among the OxSR genes, a significant number of genes appear to be Hog1 dependent, as indicated as clusters I to VI in Fig. Fig.11.11. Interestingly, genes in clusters I, II, V, and VI were differentially regulated in the hog1Δ mutant, but not in the ssk1Δ mutant, further indicating that Ssk1 is not the only upstream regulator of the Hog1 MAPK particularly in oxidative stress response. Genes in OxSR clusters III and IV are both Ssk1- and Hog1-dependent genes. Interestingly, the Sch9 protein kinase (06301) in OxSR cluster III, whose expression is induced only in response to oxidative stress, was differentially regulated in the hog1Δ and ssk1Δ mutant compared to the WT (Fig. 11B). In S. cerevisiae, Sch9 kinase plays an important role in adaptation to osmotic and oxidative stresses by being recruited to promoters of osmostress-responsive genes through physical interaction with the Sko1 transcription factor and Hog1 MAPK (41). Although a Sko1-like transcription factor appears to be absent in C. neoformans, it is still possible that the Sch9 kinase could be required for adaptation to osmotic and oxidative stresses of C. neoformans in association with Hog1 and/or other unknown transcription factors. To address this possibility, we have tested the stress susceptibility of the sch9Δ mutant in C. neoformans (Fig. 11C). The sch9Δ mutant exhibited hypersensitivity to oxidative stress response compared to the WT, similar to the HOG mutants (Fig. 11C). However, Sch9 kinase appeared to be controlled by multiple signaling pathways besides the HOG pathway due to the following reasons. First, the sch9Δ mutant was as resistant to UV as the WT. Second, the sch9Δ mutant was more hypersensitive to fludioxonil and cadmium than the WT, which is in stark contrast to the hog1Δ mutant showing resistance to both agents. Third, Sch9 was not involved in susceptibility to amphotericin B and fluconazole, unlike the HOG pathway mutants (Fig. 11C). Fourth, the sch9Δ mutant showed hypersensitivity to sodium salt (Na+), but not to potassium salt (K+), whereas the hog1Δ mutant showed hypersensitivity to both salts. Taken together, Sch9 is involved in regulation of a subset of HOG-dependent phenotypes.

Comparison of stress-regulated genes between fungal species.

Finally, we have compared stress-regulated genes of C. neoformans with those of other pathogenic (C. albicans) and nonpathogenic (S. cerevisiae and S. pombe) fungi as described in Materials and Methods. We did not find any gene whose expression is commonly upregulated or downregulated under hyperosmotic conditions (OsR genes) in all four fungi (see Fig. S9A and Table S12 in the supplemental material). However, four C. neoformans OsR genes, including PRM10, STL1, ENA1, and ALD5, were also differentially regulated in at least two other fungal species, implying that these genes could play an evolutionarily conserved role in adaptation to osmotic stress.

In contrast to the OsR genes, a greater number of oxidative stress-regulated (OxR) genes were commonly regulated between all four fungal species (see Fig. S9B in the supplemental material). Among these, 13 C. neoformans OxR genes were also differentially regulated in all other fungi. These include upregulated OxR genes, such as FLR1, GPX2, RAD16, TSA1, ISU1, UBC8, and TRR1, and downregulated OxR genes, such as RLI1, UTP22, RPC40, FEN1, RPS7B, and UTP18. Furthermore, 152 (50 upregulated and 102 downregulated) C. neoformans OxR genes were also differentially regulated in at least two other fungi, indicating that regulatory mechanisms are much more shared between fungi for oxidative stress response than for osmotic stress response.

When CSR genes (oxidative and osmotic stresses) were compared between fungi, almost none of the CSR genes were commonly found in at least three out of four fungi, indicating that each fungal species contains diverse stress response and defense systems.

DISCUSSION

The major goals of this study were to characterize the genome-wide transcriptional remodeling patterns in the human pathogen C. neoformans in response to diverse environmental stresses and to elucidate the downstream network of the two-component system and HOG signaling pathway during regulation of normal growth and stress responses of C. neoformans. Through this study, we have identified novel target genes controlled by the HOG pathway and also discovered a number of unique characteristics of the HOG signaling pathway in C. neoformans, which were not apparent in our previous studies (5-8), as summarized in Fig. S10 in the supplemental material.

Generally summarizing our array data, C. neoformans expresses not only a group of genes commonly responding to diverse environmental stresses, such as osmotic shock, oxidative stress, and antifungal agents, but also a subset of genes specifically modulated by each stress named the “SSR genes.” Particularly, the remodeling of global gene expression profiles was found to be mainly controlled by the Hog1 MAPK and Ssk1 response regulator, but not by the Skn7 response regulator, further corroborating that the Ssk1-dependent Hog1 MAPK signaling pathways play central roles in stress responses. Furthermore, the Ssk1-Hog1 signaling pathway not only controls stress-induced responses but also plays important roles in maintaining a normal cellular homeostasis under unstressed conditions. A number of genes were differentially regulated by mutation of the SSK1 and HOG1 genes, but not SKN7, even under unstressed growth conditions. Under both unstressed and stressed conditions, transcriptome profiles of the hog1Δ mutant were much more similar to those of the ssk1Δ mutant than the skn7Δ mutant, further confirming that Hog1 is mostly in the linear pathway with the Ssk1 response regulator, but not with the Skn7 response regulator. However, it should be noted that a number of genes were found to be either Hog1 specific or Ssk1 specific, revealing that Hog1 and Ssk1 are not absolutely interdependent.

Among a number of novel discoveries made in this study, the findings that most of ergosterol biosynthesis genes were upregulated and the actual ergosterol content was increased by mutation of the HOG pathway were the most striking and unexpected results since these phenomena have not been observed in other fungal species reported thus far. Comparative DNA microarray analysis recently performed in C. albicans by Enjabert and coworkers revealed that the expression levels of ergosterol biosynthesis genes are indeed generally decreased in the hog1Δ mutant compared to the WT (23). Particularly, levels of expression of the ERG11 and ERG1 genes were 1.7- and 1.9-fold decreased, respectively, compared to that of the WT (23). In agreement with this result, the C. albicans hog1Δ mutant does not show any synergistic effects with most known antifungal drugs (1). In C. neoformans, however, our study clearly demonstrated that ERG11 expression levels were enhanced in both hog1Δ and ssk1Δ mutants, but not in the skn7Δ mutant, explaining why the HOG pathway mutants were highly resistant to fluconazole and ketoconazole but hypersensitive to amphotericin B. In contrast, azole drug resistance observed in the skn7Δ mutant appears to be unrelated to the ergosterol biosynthesis since the skn7Δ mutant showed WT levels of ERG11 expression and amphotericin B susceptibility. It is probable that drug efflux and/or influx systems may be altered in the C. neoformans skn7Δ mutant, as exemplified by other azole-resistant fungal strains (40).

Our discovery provides a novel antifungal therapeutic method against cryptococcosis as follows: treatment of patients by combining amphotericin B and a HOG inhibitor followed by combination therapy with azole drugs and a HOG activator. Our data strongly implicate that potent inhibitors of the HOG pathway, especially the Ssk1 response regulator or Tco2 hybrid sensor kinase, whose orthologs are not observed in humans, will have strong synergistic effects with amphotericin B to treat cryptococcosis. Our study could provide a strong case for supporting the value of genome-wide transcriptome analysis using microarray analysis by directly providing an approach for development of novel therapeutic method.

Among genes differentially regulated by the HOG pathway under normal conditions, a group of 71 genes involved in cadmium resistance were notable since involvement of the HOG pathway in heavy metal stress had not been addressed before in C. neoformans. Heavy metals, such as cadmium, affect various aspects of cellular responses, including cell cycle regulation, growth, differentiation, apoptosis, and oxidative stress response (11, 26). Recently a number of cadmium-responsive genes have been identified in both S. cerevisiae and S. pombe (33, 45). The discovery that the ssk1Δ and hog1Δ mutants exhibit increased resistance to cadmium compared to the WT and skn7Δ mutants is a somewhat unexpected result based on findings in other fungi. In S. pombe, the spc1Δ (Hog1 homolog), wis4Δ (Ssk22 homolog), and mcs4Δ (Ssk1 homolog) mutants all show hypersensitivity to both cadmium and hydrogen peroxide (33). In C. albicans, the hog1Δ mutant does not show any significant hypersensitivity to cadmium compared to the WT (2).

This study provides further insights into the downstream network of the HOG pathway for regulation of virulence factor production and sexual differentiation of C. neoformans. It has been reported that the CAP10, CAP59, CAP60, and CAP64 genes were essential for capsule biosynthesis in C. neoformans, although their biochemical properties remain to be elucidated (15-18). For melanin production, two laccase genes, LAC1 and LAC2, were found to exist in C. neoformans. Between these, Lac1 is the predominant laccase since deletion of the LAC1 gene alone, but not the LAC2 gene, abolishes melanin production in C. neoformans (49). Our array data demonstrated that all four of the capsule synthesis genes and the LAC1 gene were upregulated in the ssk1Δ and hog1Δ mutants, indicating that these genes are directly or indirectly regulated by the HOG pathway. In the skn7Δ mutant, only the LAC1 gene, but not the capsule genes, was upregulated, further corroborating that Skn7 is negatively involved in melanin, but not capsule production (7). Our array data showing upregulation of SXI1 and GPA2 by the hog1 and ssk1 mutations may provide a possible answer for the previous question of how Hog1 and Ssk1 negatively regulate pheromone production and sexual reproduction (7, 8). It has been recently reported that C. neoformans Gpa2 physically interacts with Ste3α, Gpb1, and Crg1 and therefore promotes the pheromone response MAPK pathway for mating (29, 37). As expected, overexpression of dominant active GPA2Q203L strikingly activates pheromone expression and mating (29). Therefore, our array data strongly indicate that Hog1 represses the Gpa2-mediated pheromone response pathway under normal conditions, and inactivation of the HOG pathway drastically induces GPA2 expression, which subsequently increases pheromone production and mating.

Induction of the ENA1 gene in response to osmotic stress is somewhat expected based on studies performed in other fungi. The osmoadaptation mechanism has been well characterized in S. cerevisiae. Immediately after osmotic shock, Hog1 is directly recruited to and interacts with the Nha1 Na+/H+ antiporter and the Tok1 potassium channel (to a lesser extent) to rapidly counteract increased ion concentrations in the nucleus and restore the ability of most DNA binding proteins to reassociate with the chromatin (43). After the immediate adaptation to high-salt conditions, Hog1 induces the Ena1 Na+ extrusion pump for a longer-term adaptation to high-salt conditions (43). Our phenotypic analysis of the ena1Δ mutant demonstrated that Ena1 is required for conferring resistance to osmotic stress, particularly under carbon starvation conditions (Fig. (Fig.7B).7B). Recently, Idnurm et al. identified ENA1 as a major virulence gene via signature-tagged insertional mutagenesis (31). Interestingly, the ena1Δ mutant exhibited increased sensitivity to high pH, indicating that Ena1 is required for counterbalancing the decreased H+ concentration in the environment (31). It is not known if Hog1 is similarly recruited to an Nha1 antiporter and Tok1 potassium channel for an immediate salt adaptation of C. neoformans at this point. Interestingly, however, NHA1 appears to be transcriptionally induced by osmotic stress dependent on the HOG pathway (see Table S9 in the supplemental material), which is rather unexpected since activation of Nha1 is not dependent on transcriptional activation by Hog1 but depends on a physical interaction with Hog1 in S. cerevisiae (42). The detailed mechanism of molecular interaction between Nha1 and Hog1 remains to be elucidated.

Our array study revealed novel features of the C. neoformans Sch9 protein kinase previously reported by Wang et al. (47). The prior study demonstrated that the sch9Δ mutant has increased capsule production and thermotolerance and defective mating capability (47). Regardless of the enhanced capsulation and thermotolerance that could increase pathogenicity of C. neoformans, the sch9Δ mutant is attenuated in virulence (47). Our array data and biological analysis of the sch9Δ mutant may provide an answer for its reduced virulence. The sch9Δ mutant was found to be hypersensitive to both oxidative and osmotic stress (Fig. 11B), indicating that it is unlikely to survive in the hostile host environment and would be more susceptible to host defense mechanisms. Interestingly, both basal and induced expression levels of SCH9 were significantly decreased in ssk1Δ and hog1Δ mutants, indicating that Sch9 is one of the target kinases modulated by the HOG pathway in C. neoformans. In fact, Wang et al. previously proposed that Sch9 is mainly independent of the cAMP signaling pathway, which is another major signaling pathway controlling capsule production, mating, and virulence of C. neoformans. It is possible that increased capsule production of the hog1Δ and ssk1Δ mutants (6, 8) may also result from decreased expression of SCH9 under normal conditions. The functional correlation between Sch9 and the HOG pathway has been suggested in S. cerevisiae, where mutation of the SCH9 gene also increased susceptibility to osmotic and oxidative stresses (41).

A final important discovery of our transcriptome analysis is the potential implication of the ubiquitin-proteasome system in regulation of stress responses, which was first suggested in C. neoformans. In S. cerevisiae, the pheromone-responsive MAPK pathway is tightly controlled by ubiquitin-dependent Ste11 degradation during pheromone induction (24). Furthermore, the S. cerevisiae two-component system is negatively regulated through targeted degradation of the Ssk1 response regulator by Ubc7/Qri8, an endoplasmic reticulum (ER)-associated ubiquitin-conjugating enzyme (44). Ubiquitination for targeted protein degradation by the proteasome is mediated by three classes of enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2 or Ubc), and ubiquitin-protein ligases (E3). The ubiquitin-proteasome system is involved in endoplasmic reticulum associated protein degradation (ERAD), which contributes to selective removal of misfolded proteins, or unassembled subunits of multimeric complexes. Therefore, it is conceivable that external stress, such as oxidative damage, may increase the number of misfolded or damaged proteins inside the cell, and this accumulation could be prevented by activation of the ubiquitin-proteasome system. Our study shows that the putative ubiquitin system in C. neoformans is involved not only in stress response, but also in defending against antifungal drugs (Fig. 10B). However, functions of different components of the ubiquitin-proteasome system in stress responses and their potential connection with the HOG pathway remain to be further elucidated in future studies.

In conclusion, our study highlights the importance of genome-wide comparative transcriptome analysis in human fungal pathogens for not only elucidating previously undiscovered features and target genes of the two-component system and HOG pathway but also directly suggesting a novel therapeutic approach for effective treatment of cryptococcosis. A number of features and target genes for the stress-activated two-component system and HOG pathway identified by our analysis are coincident with those obtained from other fungi, and yet several novel features uncovered by our study further confirm the unique specialization of the HOG pathway in C. neoformans. Further exploitation of the molecular mechanism between signaling components, the downstream network, and feedback regulatory mechanisms of the HOG pathway in C. neoformans will provide an unprecedented opportunity to develop a novel anticryptococcal therapy.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by the Korea Research Foundation grant funded by the Korean Government (MOEHRD; Basic Research Promotion Fund) (KRF-2008-8-0767) and in part by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (R11-2008-062-02001-0). This work was also supported in part by NIAID RO1 grant AI50438 (to J.H.).

Footnotes

[down-pointing small open triangle]Published ahead of print on 19 June 2009.

Supplemental material for this article may be found at http://ec.asm.org/.

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