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Vol. 17, Issue 7, 3122-3135, July 2006
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Departments of *Molecular Genetics and Microbiology, Medicine, and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710
Submitted February 7, 2006;
Revised April 19, 2006;
Accepted April 21, 2006
Monitoring Editor: Peter Walter
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDISCUSSIONREFERENCES |
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; Idnurm et al., 2005).
The stress-activated mitogen-activated protein kinase (MAPK) plays an essential role in many eukaryotic systems ranging from fungi to humans (Johnson and Lapadat, 2002). In humans, two MAPKs, p38 and the c-Jun NH2-terminal kinase (JNK), transduce stress-related signals, such as radiation, osmotic shock, and heat shock, and they are involved in cell survival, programmed cell death or apoptosis, and inflammatory cytokine expression. These MAPKs are activated by several MAPK kinases (MAPKKs, i.e., MKK4 and MKK6), which in turn are activated by multiple MAPKK kinases (MAPKKKs, i.e., MEKK1 and TAK1) (Barone et al., 2001; Johnson and Lapadat, 2002). In fungi, the Hog1 homologues are the most well documented stress-activated p38-type MAPKs. Comparable with the human system, the fungal Hog1 MAPK mediates responses to a plethora of environmental cues, including osmotic shock, UV irradiation, oxidative damage, and high temperature. Activated Hog1 engages downstream effectors or transcription factors to protect cells from these eliciting stresses (Hohmann, 2002). The fungal Hog1 MAPK is also activated by a MAPKK and a MAPKKK. In particular, stress-activated MAPK signaling is important for pathogenic microorganisms because it is directly associated with successful survival and proliferation in susceptible host systems.
A defining difference between fungal and mammalian stress-activated p38/Hog1-MAPK systems lies in the upstream signaling cascade. In humans, GADD45-like genes, G protein-coupled receptors associating with Rac/CDC42 and p20-activated kinase, or tyrosine kinases such as PYK2 activate p38-MAPK pathways in response to environmental stimuli (Lee et al., 2000). In contrast, most fungi have two-component–like (Tco) systems, which function as unique upstream activators for the stress-activated MAPK module. The archetypal two-component system was first discovered in bacterial systems and is composed of two elements: 1) a sensor histidine kinase component that autophosphorylates on a histidine residue in response to an extracellular signal, and 2) a response regulator component that receives signals from the sensor kinase through phosphorylation of an aspartate residue (Nixon et al., 1986; Winans et al., 1986). The two-component system is exclusively found in prokaryotes and lower eukaryotes such as fungi and plants, but it is absent from mammals (Santos and Shiozaki, 2001; Urao et al., 2001; Catlett et al., 2003). Therefore, the two-component system is considered an appropriate target to develop antifungal or antibiotic agents. Although highly similar to the conventional bacterial two-component system, the fungal Tco system is actually composed of three elements: 1) a hybrid sensor histidine kinase containing the response regulator domain in the C terminus, 2) a histidine-containing phosphotransfer (HPt) protein, and 3) a response regulator.
The fungal two-component system has been best studied in the model yeast Saccharomyces cerevisiae. Posas et al. (1996) first reported that the S. cerevisiae HOG MAPK system is modulated by an Sln1-Ypd1-Ssk1 two-component phospho-relay system. Sln1, the only sensor kinase found in S. cerevisiae, is a transmembrane osmosensor containing both histidine kinase and response regulator domains and functions to transfer a phosphoryl group to Ypd1, which is an HPt protein. Ssk1 is a response regulator and is inactivated under normal conditions by constitutive phosphorylation from the Sln1-Ypd1 system (Posas et al., 1996). In response to hyperosmotic shock, Sln1-Ypd1–dependent phosphorylation is down-regulated and Ssk1 is dephosphorylated, allowing interaction with a downstream MAPKKK, Ssk2, to activate the HOG pathway (Posas and Saito, 1998). However, recent fungal genome sequencing projects revealed that most fungi contain a multitude of hybrid histidine kinase sensors, which is in stark contrast to S. cerevisiae (Santos and Shiozaki, 2001; Catlett et al., 2003; Dean et al., 2005). This finding strongly suggests the possibility that a microorganism expresses a unique set of sensor proteins depending on the environmental niches it encounters during evolution.
The basidiomycetous fungus Cryptococcus neoformans also uses the stress-activated Hog1 MAPK system, which is uniquely regulated compared with other fungal species (Bahnren et al., 2005). C. neoformans is an important human pathogen because it causes life-threatening fungal meningitis, mostly in immunocompromised patients (Casadevall and Perfect, 1998). As recently reported for the ongoing Vancouver Island outbreak of Cryptococcosis, a related species Cryptococcus gattii has emerged as an important primary pathogen, infecting immunocompetent individuals (Speed and Dunt, 1995; Hoang et al., 2004; Fraser et al., 2005). In a majority of C. neoformans strains, under normal in vitro growth conditions the Hog1 MAPK is constitutively phosphorylated by the Pbs2 MAPKK, and upon osmotic shock Hog1 is rapidly activated by Hog1-dependent dephosphorylation (Bahn et al., 2005). This unique regulation seems to contribute to cross-talk between the HOG pathway and other signaling cascades, including the cAMP and pheromone MAPK pathways (Bahn et al., 2005). Furthermore, the HOG pathway is involved in sensitivity to the antifungal drug fludioxonil, which affects glycerol accumulation and cell morphology (Kojima et al., 2006). However, the upstream signaling components governing the unique aspects of Hog1 regulation in C. neoformans were unknown.
Here, we have identified and characterized the Tco system in C. neoformans. Our study demonstrates that this Tco system controls the HOG pathway and regulates stress responses and antifungal sensitivity, virulence factor regulation, and sexual reproduction of C. neoformans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDISCUSSIONREFERENCES |
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; Alspaugh et al., 1997; Bahn et al., 2004; Hicks et al., 2004). For osmosensitivity assays, yeast extract-peptone (YP) media were used.
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). The 5' and 3' RACE and reverse transcription (RT)-PCR were performed according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Each RACE and cDNA product was cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced. The TCO1 and TCO2 coding sequences have been assigned GenBank accession numbers DQ383469
[GenBank]
and DQ383470
[GenBank]
, respectively. The TCO1 open reading frame (ORF) (4777 base pairs) is interrupted by 12 introns and encodes a 1383-amino acid protein. The 448-base pair 5' untranslated region of the TCO1 gene contains a single intron. The TCO2 ORF (5666-base pairs) consists of 10 exons, encoding a 1691-amino acid protein.
Response regulator encoding genes, SSK1 and SKN7, and hybrid histidine kinase genes (TCO1, TCO2, TCO3, TCO4, TCO5, and TCO7) were disrupted by biolistic transformation in the congenic C. neoformans serotype A strains H99 and/or KN99a (Nielsen et al., 2003) by overlap PCR as described previously (Davidson et al., 2002; Bahn et al., 2005). Primers for amplification of the 5' and 3' flanking regions of each gene are described in Supplemental Table 1. M13 reverse and forward primers were used to amplify the dominant selectable markers, Natr and Neor. Each gel-extracted gene disruption cassette was precipitated onto 600 µg of gold microcarrier beads (0.8 µm; BioWorld, Dublin, OH) and biolistically transformed into H99 or KN99a (Davidson et al., 2000). Stable transformants were selected on YPD medium containing nourseothricin or G418. Each mutant strain was first screened by diagnostic PCR and further confirmed by Southern blot analysis using a gene-specific probe prepared by primers listed in Supplemental Table 1 (our unpublished data).
To authenticate ssk1
mutant phenotypes, the ssk1 +SSK1 complemented strains were constructed as follows. First, H99 genomic DNA containing the full-length SSK1 gene was isolated from a C. neoformans H99 bacterial artificial chromosome (BAC) library. Using the BAC clones as templates, the 5.6-kb fragment containing the complete SSK1 gene was amplified by PCR with primers 15330 and 15331 and cloned into the pCR2.1-TOPO vector (Invitrogen), generating pCR-SSK1Ag. After confirming the DNA sequence, the insert was subcloned into pJAF12 (NEOr), generating plasmid pNEOSSK1. For the targeted reintegration of the each wild-type (WT) allele into its native locus, pNEOSSK1 was linearized by BsiWI digestion and biolistically transformed into the ssk1 strain YSB261 (Table 1).
To verify phenotypes shown for the tco1 and tco2 mutants, the tco1 +TCO1 and tco2 +TCO2 complemented strains were constructed. Using the BAC clones containing the full-length TCO1 and TCO2 genes as templates, the 5.6- and 6.4-kb fragments containing the full-length TCO1 and TCO2 genes were PCR-amplified with primers 15326/15327 and 15328/15329, respectively, and cloned into the pCR2.1-TOPO vector, generating pCR-TCO1g and pCR-TCO2g. After confirming the DNA sequence, each insert was further cloned into pJAF12 (NEOr), generating plasmid pNEOTCO1 and pNEOTCO2. For the targeted reintegration of each WT allele into its native locus, pNEOTCO1 and pNEOTCO2 were linearized by NheI and BsrGI digestion and biolistically transformed into the tco1 and tco2 strains YSB278 and YSB281, respectively (Table 1).
Heterologous Expression of C. neoformans TCO1 and TCO2 Genes in S. cerevisiae
For inducible expression of the C. neoformans TCO1 and TCO2 genes in S. cerevisiae, the full-length TCO1 and TCO2 cDNAs were amplified by RT-PCR using first strand cDNA generated from H99 total RNA (SuperScript III; Invitrogen) using primers 15318/15319 and 15320/16499, respectively, cloned into pCR2.1-TOPO vector, and sequenced. Each TCO1 and TCO2 cDNA insert was further cloned into the pESC-URA vector with the GAL10-inducible promoter system (Stratagene, La Jolla, CA), creating plasmids pESC-TCO1 and pESC-TCO2. The ura3 S. cerevisiae hog1/hog1 mutants (from the diploid homozygous deletion mutant collection) and its parental strain BY4743 (diploid generated from strains BY4741/BY4742) (Giaever et al., 2002) were then transformed with plasmid pESC-URA as a vector only control and either pESC-TCO1 or pESC-TCO2.
Assays for Capsule and Melanin Production and Mating
Qualitative visualization of capsule and melanin production was performed as described previously (Bahn et al., 2004; Hicks et al., 2004). Mating and cell fusion assays were performed as described previously (Bahn et al., 2004; Hicks et al., 2004). Images of mating and confrontation assays were captured with a Nikon Eclipse E400 microscope equipped with a Nikon DXM1200F digital camera.
Sensitivity Test for Stress Responses, Fludioxonil, and Methylglyoxal (MG)
Each strain was incubated overnight at 30°C in YPD medium, washed, serially diluted (1–104 dilutions) in distilled H2O, and spotted (3 µl) onto solid YPD medium. To examine oxidative stress, the cells were spotted on YPD containing 2, 2.5, or 3 mM H2O2. To test sensitivity to UV, cells spotted on solid YPD were exposed to UV for 0.2 min (480 J/m2) or 0.3 (720 J/m2) min using a UV Stratalinker (model 2400; Stratagene). To test temperature sensitivity, plates were incubated at 30, 37, and 40°C. To test sensitivity to fludioxonil or MG, cells were spotted on solid YPD medium containing the indicated concentration of fludioxonil (100 mg/ml stock solution in dimethyl sulfoxide; PESTANAL, Sigma-Aldrich, St. Louis, MO) or MG (Sigma-Aldrich). Each plate was incubated for 2–5 d and then photographed. To test osmosensitivity, cells grown overnight in YPD medium were spotted on solid YP medium containing 1 or 1.5 M of NaCl or KCl.
Western Blot Analysis of Hog1 Phosphorylation
Yeast cells grown to mid-logarithmic phase were mixed with an equal volume of YPD medium containing 2 M NaCl (final 1 M NaCl), 40 µg/ml fludioxonil, or 40 mM MG. A portion of culture at each time point was rapidly frozen in a dry ice/ethanol bath, resuspended in lysis buffer (Bahn et al., 2005) with 1–1.2 g of acid-washed glass beads (425–600 µm; Sigma-Aldrich), and disrupted using a bead-beater. Protein concentrations were determined with the Bio-Rad Protein Assay reagent and an equal amount of protein was loaded into a 10% Tris-glycine gel (Novex, San Diego, CA). Separated proteins were transferred to Immuno-blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and incubated overnight at 4°C with a primary antibody of rabbit p38-MAPK specific antibody (Cell Signaling Technology, Beverly, MA) to detect phosphorylated C. neoformans Hog1 and a secondary antibody of anti-rabbit IgG horseradish peroxidase-conjugated antibody. The blot was developed using the ECL Western Blotting Detection system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Subsequently, the blot was stripped and used for detection of Hog1 with a rabbit polyclonal anti-Hog1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as a loading control.
Virulence Assays
Yeast strains (wild-type [H99], tco1 [YSB278], tco1 +TCO1 complemented [YSB355], tco2 [YSB281], and tco2 +TCO2 complemented [YSB366]) were grown overnight in YPD medium at 30°C, collected by centrifugation, washed twice with sterile phosphate-buffered saline (PBS), and the final concentration was adjusted to 2 x 106 colony-forming units (CFU)/ml with sterile PBS. Female A/Jcr mice (20–24 g; NCI/Charles River Laboratories, Wilmington, MA) in each test group (10 mice per group) were inoculated with 1 x 105 CFU, in a volume of 50 µl, via nasal inhalation as described previously (Cox et al., 2000). Mice that seemed moribund (i.e., lethargic, exhibiting rapid weight loss [>15% loss], or in pain) were killed by CO2 inhalation. Survival data from the murine experiments were statistically analyzed between paired groups using the log-rank test (PRISM program 4.0; GraphPad Software, San Diego, CA). The animal protocol used for these experiments was approved by The Duke University Animal Use Committee.
RESULTS
Two-Component System in C. neoformans
To elucidate unique regulatory mechanisms impinging on the Hog1 MAPK in C. neoformans, we sought to characterize upstream signaling components of the pathway. Because a Tco system controls the stress-activated Hog1 MAPK in many fungi (for review see, Hohmann, 2002), we searched for potential two-component signaling components in the C. neoformans genome database (Loftus et al., 2005). Through BLAST searches, we identified two response regulators, an HPt protein, and seven Tco hybrid sensor kinases, each containing both a response regulator and a histidine kinase domain in a single polypeptide. The two response regulators are homologous to S. cerevisiae Ssk1 and Skn7 and were named Ssk1 and Skn7, respectively. Also the C. neoformans HPt protein Ypd1 was so named because of its high similarity to S. cerevisiae Ypd1.
Compared with the Tco system in the model yeast S. cerevisiae, C. neoformans has more divergent sensor kinase systems. Unlike the S. cerevisiae Sln1 osmosensor kinase, none of the C. neoformans histidine sensor kinases contain a transmembrane region in the N terminus, suggesting that they may all be cytosolic proteins (Figure 1). Similar to S. cerevisiae Sln1, however, most of the Tco proteins contain a single response regulator and histidine kinase domain. Tco2 is unusual in that it contains two response regulator domains and two histidine kinase domains (Figure 1). This feature has never been reported in any known hybrid histidine kinases. Thus, C. neoformans evolutionarily developed a conserved but unique set of sensor kinases compared with other fungal species.
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and pbs2 mutants are hypersensitive to osmotic shock, high temperature, UV irradiation, oxidative stress, and the antifungal drug fludioxonil (Bahn et al., 2005). C. neoformans ssk1 mutant strains displayed phenotypes similar to those observed in the pbs2 and hog1 mutants (Figure 2). In response to hyperosmolarity, the ssk1 mutant showed increased susceptibility to high concentrations of both NaCl and KCl, but to a lesser extent than hog1 and pbs2 mutants (Figure 2A). These data indicate that Ssk1 is important but not essential to mediate osmosensing signals.
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mutant, but still can be activated, albeit to a lesser extent, by high osmolarity (Figure 2C). Furthermore, this result indicates that constitutive Hog1 phosphorylation under normal conditions requires Ssk1, which seems to be constitutively active under normal conditions to activate the Hog1 MAPK module.
Unlike osmosensitivity, the ssk1 mutant exhibited other phenotypes that were completely identical to those of the pbs2 mutant. Both ssk1 and pbs2 mutants displayed hypersensitivity to H2O2, UV irradiation, and resistance to the antifungal agent fludioxonil (Figure 2B), indicating that Ssk1 is essential for responses to these signals. As might be predicted based on the analysis of Hog1 and Pbs2 (Kojima et al., 2006), the Hog1 MAPK was not phosphorylated or activated in response to fludioxonil in the ssk1 mutant (Figure 2C). Furthermore, ssk1 mutants exhibited greater resistance to high temperature than WT and complemented strains (Figure 2B).
In S. cerevisiae, MG is a metabolic by-product whose toxic action on cells can be counteracted by triggering activation of the HOG signaling pathway (Aguilera et al., 2005; Maeta et al., 2005). Here, we also found that the HOG pathway was essential to protect C. neoformans cells from MG treatment. The Hog1 MAPK was rapidly dephosphorylated in response to MG exposure (Figure 2C). Furthermore, the hog1 and pbs2 mutant strains were hypersensitive to MG (Figure 2B). Likewise, the ssk1 mutant also exhibited hypersensitivity to MG (Figure 2B). In agreement with this result, the Hog1 MAPK was not phosphorylated to any extent during MG treatment in the ssk1 mutant. Phenotypes observed were attributable to the ssk1 mutation as reintegration of the WT SSK1 allele into the disrupted ssk1 locus restored a WT phenotype (Figure 2, A and B). These data indicate that Ssk1 is an integral upstream signaling component of the Hog1 MAPK pathway in C. neoformans.
The Response Regulator Skn7 Is Independent of the Hog1 MAPK Pathway
C. neoformans contains another response regulator, Skn7, which is highly homologous to S. cerevisiae Skn7. Disruption of the SKN7 gene did not affect sensitivity to H2O2, UV irradiation, high temperature, KCl, and MG (Figure 2, A and B). Interestingly, the skn7 mutant exhibited extreme sensitivity to NaCl (even to 1 M NaCl), but not to KCl, unlike the hog1, pbs2, and ssk1 mutants (Figure 2A), indicating that Skn7 is required specifically for Na+ resistance, but not for osmoresistance in general. In further support of this conclusion, Hog1 was rapidly dephosphorylated and activated in skn7 mutants in response to high osmolarity (1 M NaCl), as similar to WT strains (Figure 2C). Furthermore, in response to MG treatment, skn7 mutants exhibited a WT Hog1 phosphorylation pattern (Figure 2C). The skn7 mutant showed intermediate resistance to fludioxonil, whereas the hog1, pbs2, and ssk1 mutants were completely resistant to fludioxonil (Figure 2B). In response to fludioxonil, the Hog1 MAPK in the skn7 mutant was regulated as in WT cells (Figure 2C), indicating that Skn7 affects fludioxonil resistance in a Hog1-independent manner. Together, these findings indicate that Skn7 functions independently from the Hog1 pathway.
Ssk1 and Skn7 Play Redundant Roles in Controlling Sexual Reproduction of C. neoformans
One unique feature of the C. neoformans Hog1 MAPK pathway is its ability to control the sexual mating processes, which is critical for generation of infectious spores (Bahn et al., 2005). Therefore, we monitored the mating ability of ssk1 and skn7 mutants. Similar to hog1 and pbs2 mutants, mating was dramatically enhanced in bilateral matings between 
ssk1 and a ssk1 mutants (Figure 3A). The enhanced mating ability of the ssk1 mutant was further evident in crosses with a mutant crippled in mating ability ( cac1 lacking adenylyl cyclase) (Figure 3B). Our previous studies demonstrate that the hog1 mutation enhances mating by derepression of mating pheromone production (Bahn et al., 2005). To address whether the ssk1 mutation also increases pheromone production, we performed confrontation assays with crg1 mutants as tester strains because these strains are known to be more sensitive to mating pheromones (Wang et al., 2004). Similar to hog1 and pbs2 mutants, ssk1 mutants were found to produce more pheromone than WT (Figure 3C). These data indicate that the Ssk1 response regulator represses pheromone production under nonmating conditions via the Pbs2-Hog1 MAPK pathway.
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mutants also showed enhanced mating in bilateral crosses (Figure 3A) and produced mating filaments when crossed with cac1 mutants (Figure 3B). In contrast to the ssk1 mutant, however, a skn7 mutants did not induce filamentation in crg1 mutants in confrontation assays, indicating WT pheromone production by the skn7 mutant (Figure 3C). Together, both response regulators negatively regulate sexual development, but each differentially controls the process.
Response Regulators Are Critical for Virulence Factor Production in C. neoformans
Another striking feature of the C. neoformans Hog1 MAPK pathway is its cross-talk with the cAMP signaling pathway to control biosynthesis of capsule and melanin, which are two major virulence factors in C. neoformans (Bahn et al., 2005). Disruption of the SSK1 gene also dramatically enhanced both capsule and melanin production, similar to the hog1 and pbs2 mutations (Figure 4), further supporting the role of the Ssk1 response regulator in the HOG pathway of C. neoformans. Strikingly, we also found that Skn7 dramatically affects melanin production, but not capsule synthesis (Figure 4). The skn7 mutation even more dramatically increased melanin production on Niger seed medium containing high glucose concentration (1–2%) than the hog1 or ssk1 mutations, suggesting that Skn7 might be involved in melanin synthesis in a Hog1-independent pathway or work independently of the phosphorylation state of Hog1.
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; Li et al., 1998). In contrast, multiple hybrid histidine kinases per genome have been identified in many other fungal species, although information about their function is very limited (Catlett et al., 2003). C. neoformans genome (Loftus et al., 2005) mining revealed that this pathogen encodes a diverse Tco sensor histidine kinase system (Idnurm et al., 2005). Considering the presence of seven diverse sensor histidine kinases in C. neoformans, we hypothesized that different sets of Tco sensors would respond to diverse environmental cues but that each signal would converge upon the Ssk1 response regulator.
To address this question, we disrupted six genes encoding potential hybrid histidine kinase sensors (tco1, tco2, tco3, tco4, tco5, and tco7; TCO6 seems to be essential for viability) and analyzed each for phenotypes compared with the hog1 and ssk1 mutants. First, we monitored the various stress responses of each tco mutant compared with the hog1 and ssk1 mutants. None of the tco mutants exhibited hypersensitivity to osmotic shock, UV irradiation, or high temperature (40°C) (Supplemental Figure 1). Only tco2 mutants displayed some hypersensitivity to 1.5 M KCl relative to WT, but to a lesser extent than the hog1 and ssk1 mutants. In agreement with these data, in response to osmotic shock (1 M NaCl), Hog1 is normally dephosphorylated in the tco1 mutant like WT (Figure 5). To monitor whether Tco1 and Tco2 play redundant roles, we also generated tco1 tco2 double mutants. In the tco2 and tco1 tco2 mutant, however, Hog1 is more highly phosphorylated for the first 30 min compared with WT cells but then efficiently dephosphorylated (Figure 6), indicating that activation of the Hog1 MAPK is delayed in the tco2 mutant and that Tco2 could play a role in Hog1 dephosphorylation. Indeed, the role of Tco2 was most obvious in response to oxidative stress (Figure 5). Both tco2 and tco1 tco2 mutants showed hypersensitivity to H2O2 (2.5–3 mM) (Figure 5). Considering that hog1 and ssk1 mutants showed higher sensitivity to H2O2 than the tco2 mutant, Tco2 is unlikely to be the sole upstream sensor component for oxidative stress.
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and tco2 mutants exhibited partial resistance to fludioxonil; however, they were still less resistant than the hog1 and ssk1 mutants (Figure 5). The tco1 tco2 double mutants displayed complete resistance to fludioxonil similar to the hog1 mutant (Figure 5), indicating that Tco1 and Tco2 independently contribute to fludioxonil resistance. Western blot analysis showed that the Hog1 MAPK was not consistently dephosphorylated in tco1 tco2 double mutants during fludioxonil exposure (Figure 6), indicating that Tco1 and Tco2 independently contribute to fludioxonil resistance via activation of the Hog1 MAPK pathway.
We also monitored MG sensitivity of tco mutants because the hog1 and ssk1 mutants showed hypersensitivity to MG. Interestingly, only tco2 mutants were highly sensitive to MG treatment (Figure 5), indicating that Tco2 could be the sensor for MG. However, the hog1 and ssk1 mutants were still more sensitive to MG than the tco2 mutant (see 15 mM MG treatment in Figure 5), indicating that another sensor, other than Tco2, could be involved in MG-specific Hog1 signaling. Similar to WT, MG treatment induces rapid dephosphorylation of Hog1 in both tco1 and tco2 mutants (Figure 6). However, Hog1 activation by dephosphorylation was significantly impaired in the tco1 tco2 double mutant (Figure 6), which indicates that both Tco1 and Tco2 play redundant roles in Hog1 dephosphorylation in response to MG. Prolonged exposure of the tco1 tco2 mutant to MG eventually resulted in dephosphorylation of Hog1, suggesting that a sensor, in addition to Tco1 and Tco2, can also mediate Hog1 activation by MG (Figure 6). Together, the sensor histidine kinases Tco1 and Tco2 mediate a subset of the Pbs2-Hog1–dependent stress response phenotypes.
Tco1 Promotes Sexual Reproduction of C. neoformans
As described above, Ssk1, Pbs2, and Hog1 are all negatively involved in differentiation of C. neoformans by repressing pheromone production. We hypothesized that the Tco proteins might also participate in the mating process. Therefore, we monitored the mating ability of each tco mutant. Most of the tco mutants, except the tco1 mutant, exhibited normal WT mating in bilateral mutant crosses ( tco x a tco) (Figure 7A). By contrast, the tco1 mutant was found to be mating defective (Figure 7A). The tco1 mutant showed reduced mating in unilateral crosses with WT and severe mating defects in bilateral crosses (Figure 7A). Reintegration of the WT TCO1 gene complemented these mating defects (Figure 7A). To address which steps in mating (i.e., cell fusion or filamentation) are affected by the tco1 mutation, we performed quantitative cell fusion assays using NAT-marked cells and NEO-marked a cells as described in Figure 7B. Interestingly, the bilateral cross between tco1 (NAT) and a tco1 (NEO) mutants produced dikaryotic cell fusion products, with only a very modest reduction in efficiency compared with WT cells (85% relative to WT). The size of the resulting dikaryotic cell colonies was heterogeneous, exhibiting either very small colonies with filaments or larger colonies with fewer filaments (Figure 7C). In contrast, WT or unilateral cell fusion products yielded colonies of homogeneous size with prolific filaments (Figure 7C). This unusual phenotype implies that Tco1 is not essential for the initial cell fusion process but that it is required for viability or stability of the dikaryon.
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mutants. All tco mutants produced WT levels of capsule (our unpublished data), in contrast to the hog1, pbs2, and ssk1 mutants that show hypercapsular phenotypes (Figure 4A). In melanin production, Tco1 was found to be a key sensor histidine kinase negatively regulating melanin synthesis, similar to Hog1, Pbs2, and Ssk1 (Figure 8). Disruption of the TCO1 gene dramatically enhanced melanin production, with the phenotype being most evident on Niger seed medium containing high glucose levels, which normally represses melanin production (Figure 8). Three independent tco1 mutants showed similar enhanced melanin production (our unpublished data), and reintegration of the TCO1 gene restored WT levels of melanin synthesis (Figure 8). As expected, tco1 tco2 mutants showed tco1 levels of enhanced melanin production (Figure 8). Therefore, Tco1 seems to be a key sensor kinase regulating melanin synthesis via the Pbs2-Hog1 pathway.
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mutant was less virulent than WT and the tco1 +TCO1 complemented strain. Mice intranasally infected with WT (H99) showed typical symptoms of fungal meningitis (cerebral protrusions, labored breathing, and rapid loss of weight) 
18 d after inoculation. In contrast, animals infected with the tco1 mutant survived significantly longer (29 d) (p < 0.001). On the other hand, the tco2 mutant was as virulent as WT (Figure 9). Animals infected with the tco2 mutants exhibited illness after 20 d, which was not statistically different from those infected with WT (p = 0.6209).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSDISCUSSIONREFERENCES |
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and tco2 mutants did not exhibit any hypersensitivity to UV irradiation or high temperature indicates either the presence of a third signaling component for the Hog1 MAPK pathway or direct internal sensing via Pbs2 or Hog1 MAPK. Alternatively, Tco1 and Tco2 could directly activate phosphatases to control Hog1 MAPK independent of Ssk1.
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), we propose that the phosphorylated version of Hog1 under normal conditions is mainly responsible for controlling virulence factors, including capsule and melanin, and sexual development (Figure 10). Phosphorylated Hog1 proteins under normal conditions are more concentrated in the nucleus (Bahn et al., 2005), where Hog1 may interact with other factors (i.e., transcription factors) to cross-talk with cAMP and pheromone MAPK signaling pathways. Under stress conditions, Hog1 is rapidly dephosphorylated by Hog1-specific phosphatases to activate downstream effector proteins counteracting eliciting stresses. Indeed, it has been demonstrated in S. cerevisiae that expression of the Hog1 target genes coincides with Hog1 dephosphorylation during transitions from severe osmolarity (1.4 M NaCl) to a lower osmolarity (0.4 M NaCl) (Van Wuytswinkel et al., 2000). Similarly, dephosphorylated Hog1 could be the active form of the MAPK to induce stress-defense genes in C. neoformans. Why C. neoformans uses this unusual mechanism of Hog1 regulation remains to be elucidated in further mechanistic detail. During evolution as a pathogen, C. neoformans may have recruited the Hog1 MAPK into the nucleus by its constitutive phosphorylation to regulate important cellular events, such as virulence factor regulation and differentiation. At the same time, phosphorylated Hog1, which is already concentrated in the nucleus under normal conditions, could be more rapidly engaged to activate target genes in response to stress.
Two pieces of evidence presented in this study indicate that Ssk1 is a key upstream response regulator for the Pbs2-Hog1 MAPK pathway. First, ssk1 mutants displayed phenotypes mostly comparable with those of hog1 and pbs2 mutants. The ssk1 mutant exhibited sensitivity to a variety of environmental stimuli and an antifungal drug, enhanced capsule and melanin production, and sexual reproduction by derepression of pheromone production. These phenotypes are also found with hog1 and pbs2 mutants. Second, in response to fludioxonil and MG, which are signaling initiators for the Hog1 pathway, Hog1 phosphorylation was completely absent in the ssk1 mutant. However, Ssk1 is not the only upstream regulator for the Hog1 pathway, because certain environmental cues, including osmotic shock, can partially bypass Ssk1 to activate the pathway.
The key finding of this study is the role of the response regulator Ssk1 in constitutive phosphorylation of the Hog1 MAPK, which is the critical difference between a majority of C. neoformans strains and other known fungal species as reported previously (Bahn et al., 2005). Disruption of the SSK1 gene abolished constitutive phosphorylation of the Hog1 MAPK under normal conditions (Figure 2C), which seemed to result in derepressed capsule and melanin production and enhanced mating in the ssk1 mutant. However, the detailed mechanism by which Ssk1 constantly triggers Hog1 phosphorylation is unclear at this point. Because Hog1 based phosphorylation was still evident under normal conditions in the tco1 tco2 mutant, another sensor kinase might be responsible for activation of Ssk1. Otherwise, certain lateral "internal signaling" events might constitutively activate Ssk1. For example, Harrison et al. (2004) showed that three distinct kinases in the S. cerevisiae cell wall integrity MAPK pathway can directly respond to different stresses, bypassing signal transduction from the upstream activator Rho1. Alternatively, a MAPKKK, uncharacterized thus far in C. neoformans, at the interface between the Pbs2-Hog1 MAPK module and the two-component system could be responsible for constitutive Hog1 phosphorylation via perturbed interactions with Ssk1. This hypothesis is currently under investigation.
The response regulator Skn7 seems to be situated in a Hog1-independent signaling pathway because none of the skn7 mutant phenotypes are congruent with those of the hog1, pbs2, and ssk1 mutants. Even in enhanced mating observed in the skn7 mutant, pheromone production was found to be normal, which is in stark contrast to hog1, pbs2, and ssk1 mutants. Skn7-related phenotypes, including sensitivity to Na+ ions, resistance to the antifungal drug fludioxonil, and hyperactive melanin production, were not uncovered in a previous study on this gene (Wormley et al., 2005). Wormley and coworkers reported that the skn7 mutation causes cell flocculation and hypersensitivity to oxidative stress caused by tert-butyl hydroperoxide and severely attenuates virulence of C. neoformans. We found that the skn7 mutants (skn7 ::URA5) constructed by Wormley et al. (2005) also displayed hypersensitivity to Na+ and partial resistance against fludioxonil, whereas its complemented strains showed WT phenotypes (our unpublished data), further corroborating our data. As expected, the skn7 mutants independently constructed by this study also showed similar flocculation phenotypes to those described by Wormley et al. (our unpublished data).
Although C. neoformans contains a phospho-relay intermediate homologue Ypd1 (29% identical to S. cerevisiae Ypd1), its function is not understood yet. C. neoformans Ypd1 seems to be essential for cell viability because deletion of the YPD1 gene was not achieved. Similarly, the TCO6 gene disruption was also not successful, indicating that the Tco6-Ypd1 system could be in the same pathway required for cellular growth. In S. cerevisiae, a transmembrane sensor histidine kinase Sln1 and Ypd1 are essential for cell viability (Maeda et al., 1994). Disruption of the SLN1 gene constitutively activates the Hog1 MAPK, which is detrimental to the cell (Maeda et al., 1994). Therefore, the cellular function of Tco6 and Ypd1 needs to be further characterized by methods other than deletion mutant analysis in a future study.
Another major finding of this study is that C. neoformans has multiple hybrid histidine kinases to sense a plethora of environmental cues, some of which converge to activate the Pbs2-Hog1 pathway, mainly through the Ssk1 response regulator (Figure 10). Here, we identified seven histidine kinases and characterized six of these kinases. With the exception of S. cerevisiae, most fungi seem to have multiple sensor histidine kinase systems. For example, multiple sensory histidine kinases are observed in several reported ascomycetes genomes, three (Mak1–3) in Schizosaccharomyces pombe, three (CaSln1, CaHK1, and CaNik1/Cos1) in Candida albicans, and 11 sensor kinases in Neurospora crassa. Catlett et al. (2003) classified these ascomycetous hybrid histidine kinases into 11 major groups, which were mainly categorized by their N-terminal variation (group I to XI). Similar to ascomycetous sensor kinases, the hybrid histidine kinases found in the basidiomycetous fungus C. neoformans exhibited extensive N-terminal variations in terms of domain structure. Based on our analysis, Tco1, with five HAMP domains, belongs to group III together with C. albicans Nik1/Cos1. Tco3, with a phytochrome domain, belongs to group VIII. Tco5 belongs to group VII. Tco6 and Tco7, with the GAF domain, belong to group I or II (Figure 1) (Catlett et al., 2003). However, C. neoformans also has unique sensor kinase systems that do not belong to any of the groups described by Catlett et al. (2003). For example, Tco2 is an unconventional sensor kinase with two histidine kinase domains and two response regulator domains within the same polypeptide (Figure 1). Furthermore, Tco3 includes a PAS domain in the C terminus (Figure 1). Therefore, the fungal histidine kinase systems seems to be even more diversified in the basidiomycetous fungus C. neoformans.
Two of the sensor kinases, Tco1 and Tco2, are responsible for a subset of Hog1 related phenotypes: Tco1 is responsible for conferring sensitivity to fludioxonil and MG, regulation of melanin biosynthesis, and sexual reproduction. Tco2 is partially responsible for response to oxidative damage, osmotic shock, and sensitivity to fludioxonil and MG. The uniqueness of Tco2 was also proven by the fact that TCO2 expression inhibits normal cell growth in S. cerevisiae, independently from Hog1 (Supplemental Figure 2). In addition to the phenotypes controlled by Tco1 and Tco2, our studies suggest that other sensors may be responsible for the remaining Hog1-related phenotypes, and these sensors need to be identified and further characterized in the future. As mentioned, Tco6 could play a role in the Pbs2-Hog1 pathway, but we were unable to explore this possibility in our current study because Tco6 seems to be essential. Otherwise, C. neoformans could contain even more distantly related sensor histidine kinases that have eluded from identification by our study. Furthermore, whether Tco1 and Tco2 controls Hog1 MAPK via the Ssk1-Pbs2 signaling cascade or by directly activating thus far unknown phosphatases remains to be elucidated.
Our study demonstrates that Tco1 and Tco2 play redundant roles in conferring fludioxonil sensitivity because only disruption of both TCO1 and TCO2 renders cells completely resistant to the drug. Previously, we have shown that fludioxonil treatment leads to dephosphorylation and activation of the Hog1 MAPK, which induces intracellular glycerol accumulation and subsequently causes cell swelling and cytokinesis defects (Kojima et al., 2006). Interestingly, cell toxicity caused by fludioxonil is synergistically enhanced by simultaneously inhibiting the cell integrity pathway, such as the calcineurin and Mpk1 MAPK pathways (Kojima et al., 2006). Monitoring of Hog1 phosphorylation in tco1 tco2 mutants during exposure to fludioxonil showed that the Hog1 MAPK is not consistently activated in the double mutant strain. It is unclear at this point whether Tco1 and Tco2 directly bind to fludioxonil to trigger Hog1 activation or instead sense fludioxonil-imposed cellular changes. We speculate that the former is the more plausible model because heterologous expression of TCO1, but not TCO2, renders fludioxonil-resistant S. cerevisiae cells sensitive to the drug in a Hog1-dependent manner (Supplemental Figure 2). Ypd1, Ssk1, the Ssk2 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK are highly conserved between S. cerevisiae and C. neoformans, and the Tco1 sensor kinase from C. neoformans triggers the conserved pathway in response to fludioxonil in S. cerevisiae. Because Tco1- and Tco2-like sensor histidine kinases are absent from humans, Tco1/2-mediated Hog1 activation, such as by fludioxonil, could be an attractive antifungal therapy.
The positive role of Tco1 in mating of C. neoformans is somewhat unexpected because the hog1, pbs2, and ssk1 mutants are derepressed in mating filament formation as a consequence of pheromone overproduction (Figure 3) (Bahn et al., 2005). However, a similar case has been reported in morphogenic transition of the pathogenic fungus C. albicans. All three sensor histidine kinases (CaSln1, CaHK2, and CaNik1) positively regulate serum-induced hyphal formation (Alex et al., 1998; Srikantha et al., 1998; Yamada-Okabe et al., 1999). However, the hog1 mutation itself derepressed hyphal development of C. albicans (Alonso-Monge et al., 1999). In particular, C. neoformans Tco1 is structurally closely correlated to CaNik1 in that both contain five HAMP repeat domains in their N termini. Whether the role of Tco1 in sexual development of C. neoformans is associated with regulation of Hog1 MAPK functions is not known at this point and needs to be further studied.
The question remains how Tco1 affects virulence of C. neoformans. Resistance to fludioxonil and defective mating would seem to be unrelated to virulence. Indeed, the ability to produce melanin even at higher glucose concentrations would be expected to increase the virulence potential of the strain. However, that tco1 mutants showed attenuated virulence, similar to hog1 and pbs2 mutants (Bahn et al., 2005), suggests that an unknown virulence factor(s) is regulated by Tco1 counteracting the hyperactive melanin production phenotype and contributing to its reduced virulence. In contrast, the finding that tco2 maintained WT virulence was rather unexpected because the mutant showed hypersensitivity to H2O2. Potentially, the tco2 mutant successfully survives and proliferates in the host because in contrast to hog1 and pbs2 mutants, it is still quite resistant to a variety of environmental stimuli, including high temperature, osmotic shock, and UV irradiation.
In conclusion, our study provides compelling evidence that the fungal two-component system governs many aspects of basic and adaptive cellular functions in the human pathogenic fungus C. neoformans. This important pathogen has maintained conserved elements and also evolved unique eukaryotic two-component systems to control stress responses, drug sensitivity, virulence regulation, and morphological differentiation. Therefore, this study not only provides an opportunity to develop a novel antifungal therapy but also provides insights to understand the general paradigms of this important signaling pathway.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Department of Bioinformatics and Life Science, Soongsil University, Seoul, 156-743 Korea.
Address correspondence to: Joseph Heitman ( heitm001{at}duke.edu)
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REFERENCES |
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