Sphingolipids regulate the yeast high-osmolarity glycerol response pathway

The yeast high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase pathway is activated in response to hyperosmotic stress via two independent osmosensing branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the osmosensing machinery activates the downstream MAP kinase cascade has been well studied, the mechanism by which the machinery senses and responds to hyperosmotic stress remains to be clarified. Here we report that inhibition of the de novo sphingolipid synthesis pathway results in activation of the HOG pathway via both branches. Inhibition of ergosterol biosynthesis also induces activation of the HOG pathway. Sphingolipids and sterols are known to be tightly packed together in cell membranes to form partitioned domains called rafts. Raft-enriched detergent-resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion and hyperosmotic stress have similar effects on the osmosensing machinery of the HOG pathway: dissociation of an Sln1-containing protein complex and elevated association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated regulation of the osmosensing machinery of the HOG pathway.     

Proteomic analysis of the pH response in the fungal pathogen Candida glabrata

Micro-organisms must adapt to environmental change to survive, and this is particularly true for fungal pathogens such as Candida glabrata. C. glabrata is found both in the environment and in diverse niches in its human host. The ambient pH of these niches varies considerably, and therefore we have examined the response of C. glabrata to changes in ambient pH using a proteomic approach. Proteins expressed in C. glabrata cells growing at pH 4.0, 7.4 or 8.0 were compared by 2-DE, and 174 spots displaying reproducible and statistically significant changes in expression level were identified by peptide mass fingerprinting, thereby extending our 2-DE map of the C. glabrata proteome to a total of 272 identified spots. Proteins involved in glucose metabolism, the TCA cycle, respiration and protein synthesis were expressed at lower levels during growth at pH 7.4 and/or 8.0, whereas proteins involved in stress responses and protein catabolism were expressed at higher levels under these alkaline conditions. Our data suggest that C. glabrata perceives low pH as less stressful than higher pH. This contrasts with another opportunistic fungal pathogen of humans, Candida albicans.     

Interaction between the Candida albicans high-osmolarity glycerol (HOG) pathway and the response to human beta-defensins 2 and 3

Human β-defensins 2 and 3 are small cationic peptides with antimicrobial activity against the fungal pathogen Candida albicans. We found that hog1 and pbs2 mutants were hypersensitive to treatment with these peptides, pointing to a role of the high-osmolarity glycerol (HOG) pathway in the response to defensin-induced cell injury.     

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.     

Synthesis of a mannose heptasaccharide of the pathogenic yeast,Candida glabrata IFO 0622 strain

An effective synthesis of the mannose heptasaccharide existing in the pathogenic yeast, Candida glabrata IFO 0622 strain was achieved via TMSOTf-promoted condensation of a tetrasaccharide donor 13 with a trisaccharide acceptor 16, followed by deprotection. The tetrasaccharide 13 was constructed by coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7) with allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (10), followed by deallylation and trichloroacetimadation. The trisaccharide 16 was obtained by coupling of 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate with 10, and subsequent 6-O-deacetylation. The disaccharide 7 was prepared through coupling of perbenzoylated mannosyl trichloroacetimidate with 4,6-O-benzylidene-1,2-O-ethylidene-beta-D-mannopyranose, then simultaneous debenzylidenation and deethylidenation, and subsequent acetylation, selective 1-O-deacetylation, and trichloroacetimidation. The disaccharide 10 was obtained by self-condensation of 3,4,6-tri-O-benzoyl-1,2-O-allyloxyethylidene-beta-D-mannopyranose, followed by selective 2-O-deacetylation.     

Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans

The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.     

Rad6p represses yeast-hypha morphogenesis in the human fungal pathogen Candida albicans

Rad6p plays important roles in post-replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast-hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5'-proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60-83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast-hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast-hypha morphogenesis.     

Structural study of a cell wall mannan-protein complex of the pathogenic yeast Candida glabrata IFO 0622 strain.

We conducted a structural analysis of the cell wall mannan-protein complex (mannan) isolated from a pathogenic yeast, Candida glabrata IFO 0622 strain. The chemical structure of mannobiose released from this mannan by treatment with 10 mM HCl at 100 degrees C for 1 h was identified as Manp beta 1-2Man. The treatment of this mannan with 100 mM NaOH at 25 degrees C for 18 h gave a mixture of alpha-1,2- and alpha-1,3-linked oligosaccharides, from tetraose to biose, and mannose. The acid- and alkali-stable mannan moiety was subjected to mild acetolysis with a 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 36 h. The resultant three novel oligosaccharides, tetraose, hexaose, and heptaose, were identified as Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Man, Manp alpha 1-2Manp alpha 1-2Manp alpha 1-6Manp alpha 1-2Manp alpha 1-2Man, and Manp alpha 1-3Manp alpha 1-2Manp alpha 1-2Manp alpha 1-6Manp alpha 1- 2Manp alpha 1-2Man, respectively, in addition to the three known oligosaccharides, Manp alpha 1-2Man, Manp alpha 1-2Manp alpha 1-2Man, and Manp alpha 1-3Manp alpha 1-2Manp alpha 1-2Man. A sequential analytical procedure involving partial acid hydrolysis with hot 0.3 M H2SO4, methylation, fast atom bombardment mass, and 1H NMR analyses was quite effective in the structural determination of the novel oligosaccharides. The results indicate that this mannan possesses a structure closely resembling that of Saccharomyces cerevisiae X2180-1A wild type strain, with the presence of small amounts of oligomannosyl residue, Manp beta 1-2Manp alpha 1-X, corresponding to one of the epitopes dominating serotype-A specificity of Candida spp., in addition to branches corresponding to hexaose and heptaose each containing one intermediary alpha-1,6 linkage.     

The cell wall of the human pathogen Candida glabrata: differential incorporation of novel adhesin-like wall proteins

The cell wall of the human pathogen Candida glabrata governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of C. glabrata using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that C. glabrata walls incorporate 50% more protein than Saccharomyces cerevisiae walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that C. glabrata walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-β-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-β-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi, C. glabrata contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like EPA family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of C. glabrata contributes to its adaptability and virulence.     

Tec1 and Ste12 transcription factors play a role in adaptation to low pH stress and biofilm formation in the human opportunistic fungal pathogen Candida glabrata

International Microbiology - Eukaryotic cells respond to environmental cues through mitogen activated protein kinase (MAPK) signaling pathways. Each MAPK cascade is specific to particular stimuli...     

Characterization and functional analysis of zinc trafficking in the human fungal pathogen Candida parapsilosis

The zinc restriction and zinc toxicity are part of host defence, called nutritional immunity. The crucial role of zinc homeostasis in microbial survival within a host is established, but little is known about these processes in the opportunistic human fungal pathogen Candida parapsilosis. Our in silico predictions suggested the presence of at least six potential zinc transporters (ZnTs) in C. parapsilosis—orthologues of ZRC1, ZRT3 and ZRT101—but an orthologue of PRA1 zincophore was not found. In addition, we detected a species-specific gene expansion of the novel ZnT ZRT2, as we identified three orthologue genes in the genome of C. parapsilosis. Based on predictions, we created homozygous mutant strains of the potential ZnTs and characterized them. Despite the apparent gene expansion of ZRT2 in C. parapsilosis, only CpZRT21 was essential for growth in a zinc-depleted acidic environment, in addition we found that CpZrc1 is essential for zinc detoxification and also protects the fungi against the elimination of murine macrophages. Significantly, we demonstrated that C. parapsilosis forms zincosomes in a Zrc1-independent manner and zinc detoxification is mediated by the vacuolar importer CpZrc1. Our study defines the functions of C. parapsilosis ZnTs, including a species-specific survival and zinc detoxification system.