A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans

The Hog1 mitogen-activated protein kinase (MAPK) plays a central role in stress responses in the human pathogen Candida albicans. Here, we have investigated the MAPK kinase kinase (MAPKKK)-dependent regulation of the pathway. In contrast to the Hog1 pathway in Saccharomyces cerevisiae, which is regulated by three MAPKKKs (Ssk2, Ssk22, and Ste11), our results demonstrate that Hog1 in C. albicans is regulated by a single MAPKKK Ssk2. Deletion of SSK2 results in comparable stress and morphological phenotypes exhibited by hog1Delta cells, and Ssk2 is required for the stress-induced phosphorylation and nuclear accumulation of Hog1, and for Hog1-dependent gene expression. Furthermore, phenotypes associated with deletion of SSK2 can be circumvented by expression of a phosphomimetic mutant of the MAPKK Pbs2, indicating that Ssk2 regulates Hog1 via activation of Pbs2. In S. cerevisiae, the Hog1 pathway is also regulated by the MAPKKK Ste11. However, we can find no connection between Ste11 and the regulation of Hog1 in C. albicans. Furthermore, expression of a chimeric Pbs2 protein containing the Ste11-dependent regulatory region of S. cerevisiae Pbs2, fails to stimulate Ste11-dependent stress signaling in C. albicans. Collectively, our data show that Ssk2 is the sole MAPKKK to relay stress signals to Hog1 in C. albicans and that the MAPK signaling network in C. albicans has diverged significantly from the corresponding network in S. cerevisiae.     

Histidine kinase two-component response regulator proteins regulate reproductive development, virulence, and stress responses of the fungal cereal pathogens Cochliobolus heterostrophus and Gibberella zeae

Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein. Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.     

Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans

The stress-activated p38/Hog1 mitogen-activated protein kinase (MAPK) pathway is structurally conserved in many diverse organisms, including fungi and mammals, and modulates myriad cellular functions. The Hog1 pathway is uniquely specialized to control differentiation and virulence factors in a majority of clinical Cryptococcus neoformans serotype A and D strains. Here, we identified and characterized the Ssk2 MAPKKK that functions upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for the difference in Hog1 phosphorylation between the serotype D f1 sibling strains B-3501 and B-3502 through comparative analysis of meiotic maps showing their meiotic segregation patterns of Hog1-dependent sensitivity to the antifungal drug fludioxonil. Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between the two strains, and the B-3501 and B-3502 SSK2 alleles were distinguished by two coding sequence changes. Supporting this finding, SSK2 allele exchange completely interchanged the Hog1-controlled signaling patterns, related phenotypes, and virulence levels of strains B-3501 and JEC21. In the serotype A strain H99, disruption of the SSK2 gene enhanced capsule and melanin biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2Δ, pbs2Δ, and hog1Δ mutants were hypersensitive to a variety of stresses and resistant to fludioxonil. In agreement with these results, Hog1 phosphorylation was abolished in the ssk2Δ mutant, similar to what occurred in the pbs2Δ mutant. Taken together, these findings indicate that Ssk2 is a critical interface connecting the two-component system and the Pbs2-Hog1 MAPK pathway in C. neoformans.     

白念珠菌SSK1突变株对氟康唑敏感性的初步研究

目的探讨氟康唑作用于白念珠菌双组分信号传导途径SSK1突变株SSK21后药物敏感性的变化。方法采用微量液体稀释法和固醇测定法测定野生株(CAF2-1)和突变株(SSK21)的最小抑菌浓度(MIC);并应用RT-PCR观察氟康唑作用前后,SSK1的表达变化。结果氟康唑对CAF2-1的MIC为16μg/mL,对SSK21为0.032μg/mL。加入氟康唑后,CAF2-1的SSK1表达明显增加,60min时达到最多。结论 SSK21对氟康唑高度敏感,SSK1基因及其相关的重要基因与药物敏感性的关系值得进一步研究,从而为新的抗真菌药物和治疗途径的研发提供参考。     

MAPKKK-independent regulation of the Hog1 stress-activated protein kinase in Candida albicans

The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2(AA)). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2(DD)) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2(DD) cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2(DD) cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2(AA) and Pbs2(DD) cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2(AA) cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.     

Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress

Genome sequencing analyses revealed that Aspergillus nidulans has orthologous genes to all those of the high-osmolarity glycerol (HOG) response mitogen-activated protein kinase (MAPK) pathway of Saccharomyces cerevisiae. A. nidulans mutant strains lacking sskA, sskB, pbsB, or hogA, encoding proteins orthologous to the yeast Ssk1p response regulator, Ssk2p/Ssk22p MAPKKKs, Pbs2p MAPKK and Hog1p MAPK, respectively, showed growth inhibition under high osmolarity, and HogA MAPK in these mutants was not phosphorylated under osmotic or oxidative stress. Thus, activation of the A. nidulans HOG (AnHOG) pathway depends solely on the two-component signalling system, and MAPKK activation mechanisms in the AnHOG pathway differ from those in the yeast HOG pathway, where Pbs2p is activated by two branches, Sln1p and Sho1p. Expression of pbsB complemented the high-osmolarity sensitivity of yeast pbs2Delta, and the complementation depended on Ssk2p/Ssk22p, but not on Sho1p. Pbs2p requires its Pro-rich motif for binding to the Src-homology3 (SH3) domain of Sho1p, but PbsB lacks a typical Pro-rich motif. However, a PbsB mutant (PbsB(Pro)) with the yeast Pro-rich motif was activated by the Sho1p branch in yeast. In contrast, HogA in sskADelta expressing PbsB(Pro) was not phosphorylated under osmotic stress, suggesting that A. nidulans ShoA, orthologous to yeast Sho1p, is not involved in osmoresponsive activation of the AnHOG pathway. We also found that besides HogA, PbsB can activate another Hog1p MAPK orthologue, MpkC, in A. nidulans, although mpkC is dispensable in osmoadaptation. In this study, we discuss the differences between the AnHOG and the yeast HOG pathways.     

Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae

In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.     

The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans.

The Candida albicans HOG1 gene (HOG1CA) was cloned by functional complementation of the osmosensitive phenotype associated with Saccharomyces cerevisiae hog1 delta mutants. HOG1CA codes for a 377-amino-acid protein, 78% identical to S. cerevisiae Hog1p. A C. albicans hog1 null mutant was found to be sensitive to osmotic stress and failed to accumulate glycerol on high-osmolarity media.     

Two-component signal transduction in human fungal pathogens

Signal transduction pathways provide mechanisms for adaptation to stress conditions. One of the most studied of these pathways is the HOG1 MAP kinase pathway that in Saccharomyces cerevisiae is used to adapt cells to osmostress. The HOG1 MAPK has also been studied in Candida albicans, and more recently observations on the Hog1p functions have been described in two other human pathogens, Aspergillus fumigatus and Cryptococcus neoformans. The important, but not surprising, concept is that this pathway is used for different yet similar functions in each of these fungi, given their need to adapt to different environmental signals. Current studies of C. albicans focus upon the identification of two-component signal proteins that, in both C. albicans and S. cerevisiae, regulate the HOG1 MAPK. In C. albicans, these proteins regulate cell wall biosynthesis (and, therefore, adherence to host cells), osmotic and oxidant adaptation, white-opaque switching, morphogenesis, and virulence of the organism.     

The Saccharomyces cerevisiae Sln1p-Ssk1p two-component system mediates response to oxidative stress and in an oxidant-specific fashion

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, environmental gamma and UV radiation, as well as several chemicals also generate reactive oxygen species, which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Oxidative stress induces the expression of several genes in yeast Saccharomyces cerevisiae. However, the primary sensor(s) that trigger the response is unknown. This study demonstrates that primary sensors of osmotic stress, the Sln1p-Ssk1p two-component proteins, are involved in sensing oxidative stress specifically induced by hydrogen peroxide and diamide, but not by other oxidants used in the study. Wild type and sln1-ssk1 mutant were treated with hydrogen peroxide, diamide, menadione, UV, and gamma-radiation. Results show that sln1-ssk1 mutant is only sensitive to hydrogen peroxide and diamide but not to other oxidants. S. cerevisiae contains an additional cell surface osmosensor, Sho1p, that targets the osmotic signal to Hog1p. Data is presented that shows Sho1 and Hog1 proteins are also involved in signaling oxidant-specific cellular damage. Furthermore, it is demonstrated that expression of the mammalian homolog of Hog1p provides protection from oxidative stress induced by hydrogen peroxide. These results suggest that Sln1p-Ssk1p and Sho1p signal transduction pathways participate in oxidative stress response. However, this response to oxidative stress is limited to specific oxidants.     

Phosphorelay-regulated degradation of the yeast Ssk1p response regulator by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, a phosphorelay signal transduction pathway composed of Sln1p, Ypd1p, and Ssk1p, which are homologous to bacterial two-component signal transducers, is involved in the osmosensing mechanism. In response to high osmolarity, the phosphorelay system is inactivated and Ssk1p remains unphosphorylated. Unphosphorylated Ssk1p binds to and activates the Ssk2p mitogen-activated protein (MAP) kinase kinase kinase, which in turn activates the downstream components of the high-osmolarity glycerol response (HOG) MAP kinase cascade. Here, we report a novel inactivation mechanism for Ssk1p involving degradation by the ubiquitin-proteasome system. Degradation is regulated by the phosphotransfer from Ypd1p to Ssk1p, insofar as unphosphorylated Ssk1p is degraded more rapidly than phosphorylated Ssk1p. Ubc7p/Qri8p, an endoplasmic reticulum-associated ubiquitin-conjugating enzyme, is involved in the phosphorelay-regulated degradation of Ssk1p. In ubc7Delta cells in which the degradation is hampered, the dephosphorylation and/or inactivation process of the Hog1p MAP kinase is delayed compared with wild-type cells after the hyperosmotic treatment. Our results indicate that unphosphorylated Ssk1p is selectively degraded by the Ubc7p-dependent ubiquitin-proteasome system and that this mechanism downregulates the HOG pathway after the completion of the osmotic adaptation.     

Plc1p is required for SAGA recruitment and derepression of Sko1p-regulated genes

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p-controlled osmoinducible genes upon osmotic shock. Although plc1Delta mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock-induced recruitment of the SAGA complex. Like plc1Delta cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p-repressed promoters in plc1Delta cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p-controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.     

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae

Las21/Gpi7 contains a heavy-metal-associated motif at its N-terminus. When this motif was disrupted by amino acid substitution, the cells acquired weak copper-resistance. We found that the previously isolated las21 mutants were strongly resistant to copper. Metallothionein is necessary for the expression of the copper-resistance of the las21 mutants. However, hyper-production of metallothionein is unlikely to be the cause of copper-resistance of the las21 mutants. Copper-sensitive mutants (collectively called Cus mutants) were isolated from the las21delta and characterized. One of the Cus genes was found to be PBS2, which encodes Hog1 MAP kinase kinase, indicating that the Hog1 MAP kinase pathway is needed for the expression of copper-resistance of the las21 mutants. As expected, the las21delta hog1delta strain was no longer copper-resistant. We found that Hog1 was constitutively activated in las21delta cells and in ssk1delta las21delta cells but not in sho1delta las21delta cells. Inactivation of either FSR2/MCD4 or MPC1/GPI13, both of which are involved in GPI anchor synthesis, like LAS21, caused a similar level of constitutive activation of Hog1 kinase and copper-resistance as found in the las21delta strain. The constitutive activation was canceled by introducing the sskl mutation, but not the sho1 mutation, in each GPI anchor mutant tested, suggesting that the defect in GPI anchor synthesis specifically affects the Slnl branch of the MAP kinase pathway. Since the wild-type cells grown in YPD containing 0.5 M NaCl do not show copper-resistance, mere activation of Hog1 is not sufficient for expression of copper-resistance. We propose that a defect in GPI anchor synthesis has multiple consequences, including activation of the Hog1 MAP kinase cascade and conferring copper-resistance.