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.     

A third osmosensing branch in Saccharomyces cerevisiae requires the Msb2 protein and functions in parallel with the Sho1 branch

Two Saccharomyces cerevisiae plasma membrane-spanning proteins, Sho1 and Sln1, function during increased osmolarity to activate a mitogen-activated protein (MAP) kinase cascade. One of these proteins, Sho1, utilizes the MAP kinase kinase kinase Ste11 to activate Pbs2. We previously used the FUS1 gene of the pheromone response pathway as a reporter to monitor cross talk in hog1 mutants. Cross talk requires the Sho1-Ste11 branch of the HOG pathway, but some residual signaling, which is STE11 dependent, still occurs in the absence of Sho1. These observations led us to propose the existence of another osmosensor upstream of Ste11. To identify such an osmosensor, we screened for mutants in which the residual signaling in a hog1 sho1 mutant was further reduced. We identified the MSB2 gene, which encodes a protein with a single membrane-spanning domain and a large presumptive extracellular domain. Assay of the FUS1-lacZ reporter (in a hog1 mutant background) showed that sho1 and msb2 mutations both reduced the expression of the reporter partially and that the hog1 sho1 msb2 mutant was severely defective in the expression of the reporter. The use of DNA microarrays to monitor gene expression revealed that Sho1 and Msb2 regulate identical gene sets in hog1 mutants. A role for MSB2 in HOG1 strains was also seen in strains defective in the two known branches that activate Pbs2: an ssk1 sho1 msb2 strain was more osmosensitive than an ssk1 sho1 MSB2 strain. These observations indicate that Msb2 is partially redundant with the Sho1 osmosensing branch for the activation of Ste11.     

The Sho1 adaptor protein links oxidative stress to morphogenesis and cell wall biosynthesis in the fungal pathogen Candida albicans

The Sho1 adaptor protein is an important element of one of the two upstream branches of the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. In the present work, we describe its role in the pathogenic yeast Candida albicans by the construction of mutants altered in this gene. We report here that sho1 mutants are sensitive to oxidative stress but that Sho1 has a minor role in the transmission of the phosphorylation signal to the Hog1 MAP kinase in response to oxidative stress, which mainly occurs through a putative Sln1-Ssk1 branch of the HOG pathway. Genetic analysis revealed that double ssk1 sho1 mutants were still able to grow on high-osmolarity media and activate Hog1 in response to this stress, indicating the existence of alternative inputs of the pathway. We also demonstrate that the Cek1 MAP kinase is constitutively active in hog1 and ssk1 mutants, a phenotypic trait that correlates with their resistance to the cell wall inhibitor Congo red, and that Sho1 is essential for the activation of the Cek1 MAP kinase under different conditions that require active cell growth and/or cell wall remodeling, such as the resumption of growth upon exit from the stationary phase. sho1 mutants are also sensitive to certain cell wall interfering compounds (Congo red, calcofluor white), presenting an altered cell wall structure (as shown by the ability to aggregate), and are defective in morphogenesis on different media, such as SLAD and Spider, that stimulate hyphal growth. These results reveal a role for the Sho1 protein in linking oxidative stress, cell wall biogenesis, and morphogenesis in this important human fungal pathogen.     

Differential susceptibility of mitogen-activated protein kinase pathway mutants to oxidative-mediated killing by phagocytes in the fungal pathogen Candida albicans

The role of four mitogen-activated protein (MAP) kinase pathways in the survival of Candida albicans following infection of human phagocytes has been addressed through the analysis of mutants defective in their respective MAP kinase. While the contribution of the cell integrity (Mkc1-mediated) or mating (Cek2-mediated) pathways is relatively minor to survival, clear and opposite effects were observed for cek1 and hog1 mutants, despite the fact that these two MAP kinases are important virulence determinants in the mouse model of experimental infection. The Cek1-mediated pathway is involved in sensitivity to phagocyte-mediated killing, while the HOG pathway contributes to the survival of the fungal cells in this interaction. Furthermore, reporter genes have been developed to quantify oxidative and nitrosative stress. hog1 mutants show an oxidative and nitrosative stress response augmented - albeit non-protective - when challenged with oxidants and NO donors in vitro or phagocytic cells (macrophages, neutrophils and the myelomonocytic cell line HL-60), suggesting this as the cause of their reduced virulence in the murine model of infection. These data have important consequences for the development of novel antifungal therapies to combat against fungal infection.     

Cdc37p is involved in osmoadaptation and controls high osmolarity-induced cross-talk via the MAP kinase Kss1p

Cdc37p, the p50 homolog of Saccharomyces cerevisiae, is an Hsp90 cochaperone involved in the targeting of protein kinases to Hsp90. Here we report a role for Cdc37p in osmoadaptive signalling in this yeast. The osmosensitive phenotype that is displayed by the cdc37-34 mutant strain appears not to be the consequence of deficient signalling through the high osmolarity glycerol (HOG) MAP kinase pathway. Rather, Cdc37p appears to play a role in the filamentous growth (FG) pathway, which mediates adaptation to high osmolarity parallel to the HOG pathway. The osmosensitive phenotype of the cdc37-34 mutant strain is aggravated upon the deletion of the HOG gene. We report that the hyper-osmosensitive phenotype of the cdc37-34, hog1 mutant correlates to a reduced of activity of the FG pathway. We utilized this phenotype to isolate suppressor genes such as KSS1 that encodes a MAP kinase that functions in the FG pathway. We report that Kss1p interacts physically with Cdc37p. Like Kss1p, the second suppressor that we isolated, Dse1p, is involved in cell wall biogenesis or maintenance, suggesting that Cdc37p controls osmoadapation by regulating mitogen-activated protein kinase signalling aimed at adaptive changes in cell wall organization.     

HOG1基因对白念珠菌超微结构的影响

目的探讨HOG1基因对白念珠菌超微结构的影响。方法设置实验组、HOG21组(hog1/hog1双等位基因缺陷株);对照组、WT组(标准株),分别在扫描电镜及透射电镜下观察两组菌株细胞的超微结构。结果扫描电镜下观察两组细胞均呈圆形或椭圆形,呈多边出芽繁殖的生长方式。但HOG1基因缺陷株细胞表面粗糙、凹凸不平,出芽数目比标准株少;标准株细胞表面光滑,出芽数量较多,可见"花瓣样"结构的芽痕;透射电镜下HOG1基因缺陷株细胞壁结构不完整,电子透明层厚薄不一,部分细胞可见棉絮状电子致密外层局灶性缺失、细胞膜外凸、不连续以及细胞膜周围见囊泡聚集等现象。标准株细胞壁各层结构完整。结论HOG1基因对白念珠菌细胞壁结构具有一定影响。     

The high-osmolarity glycerol response pathway in the human fungal pathogen Candida glabrata strain ATCC 2001 lacks a signaling branch that operates in baker's yeast

The high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway mediates adaptation to high-osmolarity stress in the yeast Saccharomyces cerevisiae. Here we investigate the function of HOG in the human opportunistic fungal pathogen Candida glabrata. C. glabrata sho1Delta (Cgsho1Delta) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1Delta mutants. However, deletion of CgSHO1 in other genetic backgrounds fails to cause osmostress hypersensitivity, whereas cells lacking the downstream MAP kinase Pbs2 remain osmosensitive. Notably, ATCC 2001 Cgsho1Delta cells also display methylglyoxal hypersensitivity, implying the inactivity of the Sln1 branch in ATCC 2001. Genomic sequencing of CgSSK2 in different C. glabrata backgrounds demonstrates that ATCC 2001 harbors a truncated and mutated Cgssk2-1 allele, the only orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC 2001 is caused by a point mutation in Cgssk2-1, which debilitates the second HOG pathway branch. Functional complementation experiments unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in osmostress adaptation. In contrast to yeast, however, Cgsho1Delta mutants display hypersensitivity to weak organic acids such as sorbate and benzoate. Hence, CgSho1 is also implicated in modulating weak acid tolerance, suggesting that HOG signaling in C. glabrata mediates the response to multiple stress conditions.     

Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue

Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.     

Osmolarity hypersensitivity of hog1 deleted mutants is suppressed by mutation in KSS1 in budding yeast Saccharomyces cerevisiae

An osmosensing mechanism of Saccharomyces cerevisiae involves a mitogen-activated protein kinase (MAPK) cascade (HOG pathway). This study aimed to investigate the response of the yeast to osmotic stress. A mutant strain, in which the HOG1 gene was disrupted by TRP1, was constructed. A spontaneous mutant, named YJY45, which suppresses the osmosensitive growth phenotype of the hog1 deletion mutant, was selected and showed a secondary phenotype of temperature sensitivity on YPD containing 0.5 M NaCl at 37 degrees C. Our data indicate that the spontaneous mutation in YJY45 mutant was mapped in KSS1, which is one of the MAPK family. The mutation in KSS1 suppresses the osmolarity-hypersensitive phenotype of the hog1 deletion mutation and restores GPD1 induction.     

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.     

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.     

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.