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Found 2 structures.
Displayed structures from 1 to 2
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Structure type: structural motif or average structure
Trivial name: β-1,3-glucan
Compound class: EPS, O-polysaccharide, cell wall polysaccharide, glucan
Contained glycoepitopes: IEDB_1397514,IEDB_142488,IEDB_146664,IEDB_153543,IEDB_158555,IEDB_161166,IEDB_2278476,IEDB_2278477,IEDB_558869,IEDB_857743,IEDB_983931,SB_192
The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. Importance: We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain β-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, β-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of β-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.
mass spectrometry, parasites, β-1, 3-glucan, Toxoplasma, Toxoplasma gondii, Cyst wall, Eimeria
NCBI PubMed ID: 23015739The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a β-(1→3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. β-(1→3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The β-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of β-(1→3)- and β-(1→6)-D-glucosidic linkages. The specific activity of the β-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.
cell wall, MALDIMS, β-glucan, Candida spp., limulus test
Publication DOI: 10.1016/S0008-6215(99)00049-XPolysaccharides of foliose thalli of Pseudocyphellaria clathrata (De Not.) Malme, a fungus lichenized with a green photobiont, were extracted and chemically characterized. The thalli were successively extracted with water and 2% aqueous KOH, at 100 degrees C. After neutralization, the solution was subjected to freeze-thawing and Fehling treatment of the supernatant precipitated a structurally complex galactoglucomannan (GGM) that was identical to a similar fraction obtained from Pseudocyphellaria aurata (Ach.) Vainio, a fungus lichenized by the green alga Dictyochloropsis symbiotica. Isolated from the supernatant of the Fehling precipitation, was an arabinogalactomannoglucan (AGMG) that contained arabinose, galactose, mannose, and glucose in a 12:34:40:14 molar ratio. Methylation analysis showed 18 different structures, the principal being nonreducing galactofuranosyl end-units (25%) with the main chain composed by (1→3)-linked β-glucopyranosyl units, as seen by controlled Smith degradation.
polysaccharides, lichen, Pseudocyphellaria clathrata
Publication DOI: 10.1639/0007-2745(2005)108[118:PPBTCP]2.0.CO;2This report describes a method for the solubilization of a micro-particulate β-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8m) and partially phiosphorylated at 100°. The resulting water-soluble product is called glucan phosphate. The conversion rate is 70%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan phosphate is composed of 34.66% C, 6.29% H, 42.83% O, and 2.23% P and has a repeating-unit empirical formula of (C6H10O5)7·PO3H2, indicating a phosphate group substitution on every seventh glucose subunit. Molecular-weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (s.e.c.) with on-line, multi-angle laser light scattering (m.a.1.1.s) photometry and differential viscometry (d.v.). Two polymer peaks were resolved. Peak 1 (Mw = 3570000 daltons), represents ~2% of the total polymers, while peak 2 (Mw = 110000 daltons) comprises ~98% of polymers. 13C- and 31P-n.m.r. spectroscopy confirmed the β-1,3 interchain linkage and the presence of a phosphate group. In solution, glucan phosphate polymers self-associate in a triple-helical arrangement. The ability to prepare a immunologically active, non-toxic, water-soluble β-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.
phosphate, glucan, Saccharomyces cerevisiae, solubilization
NCBI PubMed ID: 1804535The cell wall polysaccharides of brewers spent yeast Saccharomyces pastorianus (BSY) and the inoculum yeast (IY) were studied in order to understand the changes induced by the brewing process. The hot water and alkali extractions performed solubilized mainly mannoproteins, more branched for BSY than those of IY. Also, (31)P solid state NMR showed that the BSY mannoproteins were 3 times more phosphorylated. By electron microscopy it was observed that the final residues of alkali sequential extraction until 4M KOH preserved the yeast three-dimensional structure. The final residues, composed mainly by glucans (92%), showed that the BSY, when compared with IY, contained higher amount of (1→4)-linked Glc (43% for BSY and 16% for IY) and lower (1→3)-linked Glc (17% for BSY and 42% for IY). The enzymatic treatment of final residue showed that both BSY and IY had (α1→4)-linked Glc and (β1→4)-linked Glc, in a 2:1 ratio, showing that S. pastorianus increases their cellulose-like linkages with the brewing process.
glucan, mannoprotein, alcoholic fermentation stress, brewers spent yeast, sequential extraction
NCBI PubMed ID: 25839826Changes in cell wall structure of four strains of Sacccharomyces cerevisiae species (brewer's, baker's and probiotic yeast) after culturing on deproteinated potato juice water (DPJW) with diverse addition of glycerol and different pH were investigated. It allowed to select conditions intensifying biosynthesis of β(1,3)/(1,6)-glucan and mannoproteins of cell walls of tested strains. Yeast cell wall structural polysaccharides show biological activity and technological usability in food industry but also decide about therapeutic properties of yeast biomass. The highest increase in the thickness of walls (by about 100%) and β-glucan layer (by about 120%) was stated after cultivation of S. cerevisiae R9 brewer's yeast in DPJW supplemented with 5 and 10% (w/v) of glycerol and pH 7.0 while S. cerevisiae var. boulardi PAN yeast synthesized by ab. 70% thicker β-glucan layer when the pH of growth medium was equal to 5.0. The cells of brewer's yeast (S. cerevisiae R9), probiotic (S. cerevisiae CNCM 1-745) and baker's (S. cerevisiae 102) intensified the ratio of mannoproteins in the structure of cell walls cultivated in mediums supplemented with above 15% of glycerol what point out the protective action of glycoprotein's under osmotic stress conditions. The study confirms at the first time the possibility of using agro-industrial waste in biosynthesis of functional polysaccharides of S. cerevisiae cell wall. It could be an new advantage in production of yeast biomass with therapeutic properties or β-glucan preparation as a novel food ingredient.
glycerol, Saccharomyces cerevisiae, mannoproteins, β(1, waste potato juice water, 3)/(1, 6)-glucan
NCBI PubMed ID: 29885339Zymoseptoria tritici is a hemibiotrophic pathogen which causes Septoria leaf blotch in wheat. The pathogenesis of the disease consists of a biotrophic phase and a necrotrophic phase. The pathogen infects the host plant by suppressing its immune response in the first stage of infection. Hemibiotrophic pathogens of the genus Fusarium cause Fusarium head blight, and the necrotrophic Parastagonosporanodorum is responsible for Septoria nodorum blotch in wheat. Cell wall-degrading enzymes in plants promote infections by necrotrophic and hemibiotrophic pathogens, and trichothecenes, secondary fungal metabolites, facilitate infections caused by fungi of the genus Fusarium. There are no sources of complete resistance to the above pathogens in wheat. Defense mechanisms in wheat are controlled by many genes encoding resistance traits. In the wheat genome, the characteristic features of loci responsible for resistance to pathogenic infections indicate that at least several dozen genes encode resistance to pathogens. The molecular interactions between wheat and Z. tritici, P. nodorum and Fusarium spp. pathogens have been insufficiently investigated. Most studies focus on the mechanisms by which the hemibiotrophic Z. tritici suppresses immune responses in plants and the role of mycotoxins and effector proteins in infections caused by P. nodorum and Fusarium spp. fungi. Trichothecene glycosylation and effector proteins, which are involved in defense responses in wheat, have been described at the molecular level. Recent advances in molecular biology have produced interesting findings which should be further elucidated in studies of molecular interactions between wheat and fungal pathogens. The Clustered Regularly-Interspaced Short Palindromic Repeats/ CRISPR associated (CRISPR/Cas) system can be used to introduce targeted mutations into the wheat genome and confer resistance to selected fungal diseases. Host-induced gene silencing and spray-induced gene silencing are also useful tools for analyzing wheat-pathogens interactions which can be used to develop new strategies for controlling fungal diseases.
Fusarium, Parastagonospora nodorum, Zymoseptoria tritici, anatomical barriers, pattern-triggered immunity
NCBI PubMed ID: 29642627Fungal β-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cation-π and face-to-face π-π interactions. The lead compound is capable of inhibiting AfGel4 with an IC50 value of 42 μm.
carbohydrates, Oligosaccharides, Glycomimetics, Aspergillus fumigatus, transglycosylases
NCBI PubMed ID: 29164827Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non-albicansCandida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan-Candida species biofilm therapy.IMPORTANCECandida species, the most common fungal pathogens, frequently grow as a biofilm. These adherent communities tolerate extremely high concentrations of antifungal agents, due in large part, to a protective extracellular matrix. The present studies define the structural, functional, and genetic similarities and differences in the biofilm matrix from the four most common Candida species. Each species synthesizes an extracellular mannan-glucan complex (MGCx) which contributes to sequestration of antifungal drug, shielding the fungus from this external assault. Synthesis of a common polysaccharide backbone appears conserved. However, subtle structural differences in the branching side chains likely rely upon unique modification enzymes, which are species specific. Our findings identify MGCx backbone synthesis as a potential pan-Candida biofilm therapeutic target.
Biofilm, Extracellular matrix, Candida, antifungal resistance, non-albicans
NCBI PubMed ID: 29615504There are about 40 species in the Pleurotus genus, including those with high economic significance, i.e. P. ostreatus and P. pulmonarius. The fruiting bodies of oyster mushrooms are of high nutritional and health-promoting value. In addition, many species belonging to the Pleurotus genus have been used as sources of substances with documented medicinal properties, such as high-molecular weight bioactive compounds (polysaccharides, peptides and proteins) and low-molecular weight compounds (terpenoids, fatty acid esters and polyphenols). The bioactive substances contained in the mycelium and fruiting bodies of Pleurotus species exhibit immunostimulatory, anti-neoplastic, anti-diabetic, anti-atherosclerotic, anti-inflammatory, antibacterial and anti-oxidative properties. Their multidirectional positive influence on the human organism is the result of interaction of bioactive substances. Extracts from individual Pleurotus species can be used for the production of dietary supplements increasing the organism’s immunity. They are also used for the production of cosmetics. They can be added to functional foods as probiotics, or used as natural preservatives or ingredients of special foodstuffs for patients with specific diseases.
Functional food, fruiting bodies, health-promoting activities, oyster mushrooms
Publication DOI: 10.2478/fhort-2018-0012Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of β-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.
cell wall, Microscopy, antifungal activity, azoles
NCBI PubMed ID: 30082817Deadly infections from opportunistic fungi have risen in frequency, largely because of the at-risk immunocompromised population created by advances in modern medicine and the HIV/AIDS pandemic. This review focuses on dynamics of the fungal polysaccharide cell wall, which plays an outsized role in fungal pathogenesis and therapy because it acts as both an environmental barrier and as the major interface with the host immune system. Human fungal pathogens use architectural strategies to mask epitopes from the host and prevent immune surveillance, and recent work elucidates how biotic and abiotic stresses present during infection can either block or enhance masking. The signaling components implicated in regulating fungal immune recognition can teach us how cell wall dynamics are controlled, and represent potential targets for interventions designed to boost or dampen immunity.
cell wall, glucan, innate immunity, fungi, evasion
NCBI PubMed ID: 29452950Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and β-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential β-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of β-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast β-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.
Genes, sequencing, Proteomics, Sprassis crispa
NCBI PubMed ID: 30375506The high mortality of invasive fungal infections, and the limited number and inefficacy of antifungals necessitate the development of new agents with novel mechanisms and targets. The fungal cell wall is a promising target as it contains polysaccharides absent in humans, however, its molecular structure remains elusive. Here we report the architecture of the cell walls in the pathogenic fungus Aspergillus fumigatus. Solid-state NMR spectroscopy, assisted by dynamic nuclear polarization and glycosyl linkage analysis, reveals that chitin and α-1,3-glucan build a hydrophobic scaffold that is surrounded by a hydrated matrix of diversely linked β-glucans and capped by a dynamic layer of glycoproteins and α-1,3-glucan. The two-domain distribution of α-1,3-glucans signifies the dual functions of this molecule: contributing to cell wall rigidity and fungal virulence. This study provides a high-resolution model of fungal cell walls and serves as the basis for assessing drug response to promote the development of wall-targeted antifungals
cell wall, cell wall polymers, fungi, solid-state NMR
NCBI PubMed ID: 30013106Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.
β-1, filamentous fungi, Beauveria bassiana, cell wall integrity, alkaline tolerance, high pH, 3-glycanosyltransferase
NCBI PubMed ID: 29802184Glycoengineered yeast cells, which express human-compatible glycan structures, are particularly attractive host cells to produce therapeutic glycoproteins. Disruption of OCH1 gene, which encodes an α-1,6-mannosyltransferase required for mannan-type N-glycan formation, is essential for the elimination of yeast-specific N-glycan structures. However, the gene disruption causes cell wall defects leading to growth defects. Here, we tried to identify factors to rescue the growth defects of och1Δ cells by in vivo mutagenesis using piggyBac (PB)-based transposon. We isolated a mutant strain, named 121, which could grow faster than parental och1Δ cells. The PB element was introduced into the promoter region of BEM4 gene and upregulated the BEM4 expression. Overexpression of BEM4 suppressed growth defects in och1Δ cells. The slow grow phenotypes were partially rescued by expression of Rho1p, whose function is regulated by Bem4p. Our results indicate that BEM4 would be useful to produce therapeutic proteins in glycoengineered yeast without the growth defects.
glycosylation, glycoengineering, budding yeast, piggyBac transposon
NCBI PubMed ID: 29882469| -4)-b-D-GlcpNAc-(1- | Show graphically |
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Structure type: structural motif or average structure
Trivial name: chitin
Compound class: cell wall polysaccharide, glucan
Contained glycoepitopes: IEDB_135813,IEDB_137340,IEDB_141807,IEDB_151531,IEDB_153212,IEDB_241099,IEDB_423114,IEDB_423150,SB_74,SB_85
Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).
biosynthesis, cell wall, glycosyltransferases, polysaccharides, glycosylation, Metabolic engineering
NCBI PubMed ID: 26342143The composition and organization of the cell walls from Saccharomyces cerevisiae, Candida albicans, Aspergillus fumigatus, Schizosaccharomyces pombe, Neurospora crassa, and Cryptococcus neoformans are compared and contrasted. These cell walls contain chitin, chitosan, β-1,3-glucan, β-1,6-glucan, mixed β-1,3-/β-1,4-glucan, α-1,3-glucan, melanin, and glycoproteins as major constituents. A comparison of these cell walls shows that there is a great deal of variability in fungal cell wall composition and organization. However, in all cases, the cell wall components are cross-linked together to generate a cell wall matrix. The biosynthesis and properties of each of the major cell wall components are discussed. The chitin and glucans are synthesized and extruded into the cell wall space by plasma membrane-associated chitin synthases and glucan synthases. The glycoproteins are synthesized by ER-associated ribosomes and pass through the canonical secretory pathway. Over half of the major cell wall proteins are modified by the addition of a glycosylphosphatidylinositol anchor. The cell wall glycoproteins are also modified by the addition of O-linked oligosaccharides, and their N-linked oligosaccharides are extensively modified during their passage through the secretory pathway. These cell wall glycoprotein posttranslational modifications are essential for cross-linking the proteins into the cell wall matrix. Cross-linking the cell wall components together is essential for cell wall integrity. The activities of four groups of cross-linking enzymes are discussed. Cell wall proteins function as cross-linking enzymes, structural elements, adhesins, and environmental stress sensors and protect the cell from environmental changes.
Candida albicans, Aspergillus fumigatus, Saccharomyces cerevisiae, fungal cell wall, Schizosaccharomyces pombe, Neurospora crassa, cell wall biogenesis, glucan; chitin, Cryptococcus neoformas
NCBI PubMed ID: 23419716The structure of cell wall of yeasts (genus Saccharomyces) is one of the factors that determine their health-promoting properties connected to the presence of b-glucans and mannoprotein. The aim of the study was to determine the influence of glycerol as a carbon source on structural polymers of cell wall (b-glucan and mannoprotein) of probiotic yeasts Saccharomyces cerevisiae var. boulardii and brewer’s yeasts S. cerevisiae R9. Significant increase of the percentage of polysaccharide content in the cell wall dry weight of S. cerevisiae R9 brewer’s yeasts was noted (in the range of 10–20 %) after cultivation in medium containing glycerol at a concentration of 2–5 % and pH 4.0. The highest content of carbohydrates in probiotic yeasts’ cell wall (58 %) was observed after cultivation in medium containing 3 % of glycerol and pH 5.0. The cell wall of probiotic yeasts was characterized by higher content of mannoprotein comparing with cell wall preparation of brewer’s yeasts S. cerevisiae R9 composed mainly of b-glucans. After cultivation in mediums with 2 and 3 % of glycerol, the cell of brewer’s yeasts contained the highest amount of b(1,3/1,6)-glucan in dry weight of the cell wall (about 36 %). Glycerol at a concentration of 3 and 5 % also intensified mannoprotein biosynthesis in cell wall of S. cerevisiae R9, approximating their content to those noted in the cells of probiotic yeasts (about 29 % (w/w) of dry weight of the cell wall) after cultivation in a medium of pH 5.0 containing 3 % of glycerol.
cell wall, β-glucan, Saccharomyces cerevisiae, mannoprotein, probiotic yeast
Publication DOI: 10.1007/s00217-013-2016-8Chitin, a small organic molecule commonly found in fungal cell walls and insect exoskeletons, has the ability to elicit an immune response in plants by binding to specific membrane-bound receptors. Understanding how plants detect and fend off deleterious fungi and insects will enable improved defense strategies against these pathogens. A wide array of techniques, including affinity binding studies, isothermal calorimetry, structural analysis, and molecular genomic methods have been used to identify and characterize chitin-binding receptors as well as the kinetic parameters of chitin-receptor interactions. There are a number of newly developed analytical technologies in mechanical, electrochemical, and optical biosensing that have great potential to further elucidate the interactions between chitin and its binding partners. In this review, we provide a detailed examination of the methods currently used to characterize chitin-binding interactions, along with emerging analytical techniques that have the potential to transform this area of study.
biosensors, N-acetyl-chitooligosaccharides, pathogen-associated molecular patterns
Publication DOI: 10.1515/revac-2012-0030Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.
NCBI PubMed ID: 23615555Chitinases, the enzymes responsible for the biological degradation of chitin, are found in a wide range of organisms from bacteria to higher plants and animals. They participate in numerous physiological processes such as nutrition, parasitism, morphogenesis and immunity. Many organisms, in addition to chitinases, produce inactive chitinase-like lectins that despite lacking enzymatic activity are involved in several regulatory functions. Most known chitinases belong to families 18 and 19 of glycosyl hydrolases, however a fewchitinases that belong to families 23 and 48 have also been identified in recent years. In this review, different aspects of chitinases and chi-lectins from bacteria, fungi, insects, plants and mammals are discussed.
Biomarker, chitinase, chi-lectin, gene evolution, biocontrol
NCBI PubMed ID: 24095741Understanding the surface properties of the human opportunistic pathogen Aspergillus fumigatus conidia is essential given the important role they play during the fungal interactions with the human host. Although chitin synthases with myosin motor-like domain (CSM) play a major role in cell wall biosynthesis, the extent to which deletion of the CSM genes alter the surface structural and biophysical-biological properties of conidia is not fully characterized. We used three complementary atomic force microscopy techniques—i.e., structural imaging, chemical force microscopy with hydrophobic tips, and singlemolecule force spectroscopy with lectin tips—to gain detailed insights into the nanoscale surface properties (ultrastructure, hydrophobicity) and polysaccharide composition of the wild-type and the chitin synthase mutant (DcsmA, DcsmB, and ΔcsmA/csmB) conidia of A. fumigatus. Wild-type conidia were covered with a highly hydrophobic layer of rodlet nanostructures. By contrast, the surface of the ΔcsmA mutant was almost completely devoid of rodlets, leading to loss of hydrophobicity and exposure of mannan and chitin polysaccharides. The ΔcsmB and ΔcsmA/csmB mutants showed a different behavior, i.e., the surfaces featured poorly organized rodlet layers, yet with a low hydrophobicity and substantial amounts of exposed mannan and chitin at the surface. As the rodlet layer is important for masking recognition of immunogenic fungal cell wall components by innate immune cells, disappearance of rodlet layers in all three chitin synthase mutant conidia was associated with an activation of human dendritic cells. These nanoscale analyses emphasize the important and distinct roles that the CSMA and CSMB genes play in modulating the surface properties and immune interactions of A. fumigatus and demonstrate the power of atomic force microscopy in fungal genetic studies for assessing the phenotypic characteristics of mutants altered in cell surface organization.
NCBI PubMed ID: 23870253The Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation. We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model.
virulence, cell wall, glycosylation, Candida albicans, mannoproteins, MNN1
NCBI PubMed ID: 23886038In budding yeast, the neck that connects the mother and daughter cell is the site of essential functions such as organelle trafficking, septum formation and cytokinesis. Therefore, the morphology of this region, which depends on the surrounding cell wall, must be maintained throughout the cell cycle. Growth at the neck is prevented, redundantly, by a septin ring inside the cell membrane and a chitin ring in the cell wall. Here, we describe recent work supporting the hypothesis that attachment of the chitin ring, which forms at the mother–bud neck during budding, to β-1,3-glucan in the cell wall is necessary to stop growth at the neck. Thus, in this scenario, chemistry controls morphogenesis.
NCBI PubMed ID: 23949603Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.
NCBI PubMed ID: 23397570The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.
NCBI PubMed ID: 23750903Fungal cell walls contain several types of glycans, which play important roles in the pathogenesis of fungal infection and host immune response. Among them, glycosphingolipids have attracted much attention lately since they contribute actively to the fungi development and fungal-induced pathogenesis. Although glycosphingolipids are present in pathogenic and non-pathogenic fungi, pathogenic strains exhibit distinct glycan structures on their sphingolipids, which contribute to the regulatory processes engaged in inflammatory response. In Candida albicans, phospholipomannan (PLM) represents a prototype of these sphingolipids. Through its glycan and lipid moieties, PLM induces activation of host signaling pathways involved in the initial recognition of fungi, causing immune system disorder and persistent fungal disease. In this review, first we describe the general aspects of C. albicans sphingolipids synthesis with a special emphasize on PLM synthesis and its insertion into the cell wall. Then, we discuss the role of PLM glycosylation in regulating immune system activation and its contribution to the chronic persistent inflammation found in Candida infections and chronic inflammatory diseases.
regulation, glycosphingolipids, yeasts, inflammatory response
NCBI PubMed ID: 25394861Antagonistic actinomycete strains isolated from the environment are valuable tools for an eco-friendly, healthy, and safe control of phytopathogenic fungi. We have evaluated the culture filtrate of Streptomyces griseorubens E44G, an actinomycete strain isolated from soil, on the growth and ultrastructure of hyphal cells of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici, the causal agent of Fusarium wilt disease of tomato. The effect of the Streptomyces culture filtrate on some of the carbohydrate fractions in the hyphal cell of the pathogen using gold-labeled lectin complexes was also elucidated. Of the concentrations of S. griseorubens E44G culture filtrate tested, the highest (400 mu L) had the most potent antifungal effect on the mycelial growth of the fungus. At this concentration, some changes in the morphology of the fungal hyphae were observed by scanning electron microscopy, and a number of dramatic changes in the ultrastructure of the hyphal cells of the fungus were observed by transmission electron microscopy. Ultracytochemical localization of carbohydrate fractions of the hyphal cell of the fungus revealed the presence of a very high quantity of chitin in the cell wall which was digested following exposure to the culture filtrate of S. griseorubens E44G, indicating the presence of a chitinase enzyme in that filtrate. The ultracytochemical investigations also indicated the presence of mannose, glucose, and galactose in the fungal cell wall, as well as the absence of glucosides. Moreover, the fungal cell cytoplasm contained glucosides and galactose but not chitin. These results confirm that the chitinase enzyme was produced by S. griseorubens E44G and that this enzyme may play a role in the potential of this strain as an antifungal agent against F. oxysporum f. sp. lycopersici.
antagonist, TEM, SEM, chitinase, sugar localization, ultracytochemical
Publication DOI: 10.1007/s13213-014-1019-4The complex carbohydrates accessible to the distal gut microbiota (DGM) are key drivers in determining the structure of this ecosystem. Typically, plant cell wall polysaccharides and recalcitrant starch (i.e. dietary fiber), in addition to host glycans are considered the primary nutrients for the DGM; however, we recently demonstrated that α-mannans, highly branched polysaccharides that decorate the surface of yeast, are also nutrients for several members of Bacteroides spp. This relationship suggests that the advent of yeast in contemporary food technologies and the colonization of the intestine by endogenous fungi have roles in microbiome structure and function. Here we discuss the process of yeast mannan metabolism, and the interparagraph between various sources of intestinal fungi and their roles in recognition by the host innate immune system.
polysaccharide, symbiosis, evolution, mannooligosaccharide, fungal cell wall, carbohydrate active enzyme, catabolism, distal gut microbiota, yeast mannan
NCBI PubMed ID: 26440374A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.
cell wall, mannan, N-glycans, endo-mannanases
NCBI PubMed ID: 26048011Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-β-D-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β-1,3-glucan, β-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.
cell wall, glucan, glycoside hydrolase, chitin, transglycosylation, computer modelling, carbohydrate binding module
NCBI PubMed ID: 25495733Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus that activates, suppresses or modulates the immune response by changing its cell wall structure and by secreting proteases. In this study, we show that chitin acts as an adjuvant in a murine model of A. fumigatus protease induced allergy. The mice were immunised intraperitoneally with A. fumigatus culture filtrate antigen either with or without chitin and were subsequently challenged with the culture filtrate antigen intranasally. Alum was used as an adjuvant control. Compared to alum, chitin induced a weaker inflammatory response in the lungs, measured as the total cell efflux in BAL, EPO and chitinase production. However, chitin enhanced the total IgE, specific IgE and specific IgG1 production as efficiently as alum. Pre-treatment with chitin but not with alum depressed the concentration of the Th2 cytokines IL-4 and IL-13 in BAL fluid. These results shows that chitin, in spite of a reduction of the Th2 cytokine levels in the lungs, enhanced the total and specific IgE production in A. fumigatus culture filtrate induced allergy.
innate immunity, IgE, Aspergillus fumigatus, chitin, Fungal cell wall polysaccharides, lung inflammation
NCBI PubMed ID: 25631683The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock.
cell wall, glycosyltransferases, Microscopy
NCBI PubMed ID: 26220968Brewers spent yeast insoluble residue (BSYIR) is the insoluble material that remains after the exhaustive extraction with strong alkali solutions of brewers spent yeast (BSY). BSYIR contains 57% polysaccharides, composed mainly of glucose residues (98%) and minor amount of mannose residues (1%). The glucans were mainly (1→4)-linked (56.6%); with lower amounts of (1→3)-, (1→4,6)-, terminal-, (1→6)-, and (1→3,6)-linked glucose residues, together with terminal- and (1→6)-linked mannose residues. Scanning electron microscopy (SEM) showed that in BSYIR the spherical shape of BSY was preserved. However, when the BSYIR was treated with chlorite solutions, either under alkali or acidic conditions, followed by an extraction with aqueous KOH (0.1 M) solution, it promoted the destruction of the cell wall three-dimensional structure. The treatments with chlorite solutions allowed the solubilisation of yeast non carbohydrate material in BSYIR and the recovered residues were enriched in carbohydrates. The residue left after treatment with chlorite under alkaline solutions was composed of 92% of carbohydrates and the residue left with chlorite/acetic acid treatment contained 89% carbohydrate. Both residues were composed of only glucose. Comparing the BSYIR with the residue obtained after treatment with chlorite under alkaline solutions, it was observed an enrichment in linear glucans, (1→4)-Glc (61%) and (1→3)-Glc (from 17% to 20%), and the decrease in branched glucans, namely (1→4,6)-Glc, from 10% to 4%. The results obtained show that the three-dimensional spherical structure of the yeast cell wall is preserved by a network of glucans and mannoproteins. The oxidation of this network promotes its disruption by formation of an unbranched compact structure of glucans.
glycoproteins, Glucans, Saccharomyces pastorianus, scanning electronmicroscopy(SEM)
Journal NLM ID: 101634698Purified chitin-glucan complex (CGCpure) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a β-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGCpure had an average molecular weight of 4.9 × 10(5)Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGCpure has α-chitin in its structure. CGCpure presented an endothermic decomposition peak at 315°C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products.
structural analysis, molecular weight, chitin–glucan complex (CGC), Komagataella pastoris, thermal properties
NCBI PubMed ID: 26076647Biological activities of medicinal mushrooms have been attributed to β-(1→3),(1→6)-glucans that are present in the cell wall of fungi and some plants. Antitumor, immunomodulatory, antimicrobial, antinociception, antiinflammatory, prebiotic, antioxidant, and antidiabetic are some of different properties already described for β-(1→3),(1→6)-glucans. Immune activation systems, including specific β-glucan receptors like Dectin-1, complement (CR3), and Toll (TLR), have been identified to clarify these biological effects. The β-(1→3)-glucans are synthesized by β-(1→3)-glucan synthase (GLS), an enzyme belonging to the glucosyltransferase group, which has a catalytic unit (FKS) and another regulatory (RHO). The mechanisms for adding β-(1→6) branches to the non-reducing ends of the β-(1→3)-glucan chains are unclear until now. Due to the biological importance of β-(1→3),(1→6)-glucan, it is necessary to understand the biochemical and molecular mechanisms of its synthesis, both to optimize the production of bioactive compounds and to develop antifungal drugs that interrupt this process. Therefore, the aim of this review is to gather information about the potential of β-(1→3),(1→6)-glucans, their methods of isolation, purification, and chemical characterization, as well as how these biomolecules are synthesized by fungi and what studies involving biotechnology or molecular biology have contributed to this subject.
characterization, biotechnology, molecular biology, β-(1→3), (1→6)-glucans, β-(1→3)-glucan synthase, medicinal activities
NCBI PubMed ID: 26252967Zymoseptoria tritici is a hemibiotrophic pathogen which causes Septoria leaf blotch in wheat. The pathogenesis of the disease consists of a biotrophic phase and a necrotrophic phase. The pathogen infects the host plant by suppressing its immune response in the first stage of infection. Hemibiotrophic pathogens of the genus Fusarium cause Fusarium head blight, and the necrotrophic Parastagonosporanodorum is responsible for Septoria nodorum blotch in wheat. Cell wall-degrading enzymes in plants promote infections by necrotrophic and hemibiotrophic pathogens, and trichothecenes, secondary fungal metabolites, facilitate infections caused by fungi of the genus Fusarium. There are no sources of complete resistance to the above pathogens in wheat. Defense mechanisms in wheat are controlled by many genes encoding resistance traits. In the wheat genome, the characteristic features of loci responsible for resistance to pathogenic infections indicate that at least several dozen genes encode resistance to pathogens. The molecular interactions between wheat and Z. tritici, P. nodorum and Fusarium spp. pathogens have been insufficiently investigated. Most studies focus on the mechanisms by which the hemibiotrophic Z. tritici suppresses immune responses in plants and the role of mycotoxins and effector proteins in infections caused by P. nodorum and Fusarium spp. fungi. Trichothecene glycosylation and effector proteins, which are involved in defense responses in wheat, have been described at the molecular level. Recent advances in molecular biology have produced interesting findings which should be further elucidated in studies of molecular interactions between wheat and fungal pathogens. The Clustered Regularly-Interspaced Short Palindromic Repeats/ CRISPR associated (CRISPR/Cas) system can be used to introduce targeted mutations into the wheat genome and confer resistance to selected fungal diseases. Host-induced gene silencing and spray-induced gene silencing are also useful tools for analyzing wheat-pathogens interactions which can be used to develop new strategies for controlling fungal diseases.
Fusarium, Parastagonospora nodorum, Zymoseptoria tritici, anatomical barriers, pattern-triggered immunity
NCBI PubMed ID: 29642627Plants and microbes utilize glycoconjugates as structural entities, energy reserves for cellular processes, and components of cellular recognition or binding events. The structural heterogeneity of carbohydrates in such systems is a result of the ability of the carbohydrate biosynthetic enzymes to reorient sugar monomers in a variety of forms, generating highly complex, linear, branched, or hierarchical structures. During the interaction between plants and their microbial pathogens, the microbial cell surface glycans, cell wall derived glycans, and glycoproteins stimulate the signaling cascades of plant immune responses, through a series of specific or broad spectrum recognition events. The microbial glycan-induced plant immune responses and the downstream modifications observed in host-plant glycan structures that combat the microbial attack have garnered immense interest among scientists in recent times. This has been enabled by technological advancements in the field of glycobiology, making it possible to study the ongoing co-evolution of the microbial and the corresponding host glycan structures, in greater detail. The new glycan analogs emerging in this evolutionary arms race brings about a fresh perspective to our understanding of plant-pathogen interactions. This review discusses the role of diverse classes of glycans and their derivatives including simple sugars, oligosaccharides, glycoproteins, and glycolipids in relation to the activation of classical Pattern-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) defense responses in plants. While primarily encompassing the biological roles of glycans in modulating plant defense responses, this review categorizes glycans based on their structure, thereby enabling parallels to be drawn to other areas of glycobiology. Further, we examine how these molecules are currently being used to develop new bio-active molecules, potent as priming agents to stimulate plant defense response and as templates for designing environmentally friendly foliar sprays for plant protection.
carbohydrates, glycans, plant defense, fungi, elicitors, oomycete, priming
NCBI PubMed ID: 30022987Wild mushrooms are gathered and consumed in many regions of the world. Edible mushrooms are a good source of valuable nutritious ingredients but they also are able to accumulate heavy metals from the environment. Participation of polysaccharide components of mushroom cell walls in biosorption is poorly understood, therefore, our investigations focussed on an alkali-soluble polysaccharide derived from the cell wall of Boletus edulis. Its chemical structure is described based on chemical analysis, GPC, NMR and FT-IR and other instrumental techniques. The polysaccharide contained mainly C-3 substituted α-D-glucose and α-D-mannose. The ratio between these components was 10:4. We postulate the polymer is a (1→3)-linked α-D-glucan decorated by α-(1→3)-D-mannose chains. The molecular mass of the polysaccharide was estimated at about 850 kDa. Biosorption of heavy metal ions (Cd, Cu, Zn and Pb) by the tested polymer was measured using AAS techniques. The polysaccharide has a high ability to accumulate lead and cadmium.
NMR spectroscopy, alkali-soluble polysaccharide, Boletus edulis, biosorption, α-(1→3)-D-Mannoglucans
NCBI PubMed ID: 30381194During the past decades, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Although most of the works concerned bacteria, AFM also permitted major breakthroughs in the understanding of physiology and pathogenic mechanisms of some fungal species associated with cystic fibrosis. Complementary to electron microscopies, AFM offers unprecedented insights to visualize the cell wall architecture and components through three-dimensional imaging with nanometer resolution and to follow their dynamic changes during cell growth and division or following the exposure to drugs and chemicals. Besides imaging, force spectroscopy with piconewton sensitivity provides a direct means to decipher the forces governing cell-cell and cell-substrate interactions, but also to quantify specific and non-specific interactions between cell surface components at the single-molecule level. This nanotool explores new ways for a better understanding of the structures and functions of the cell surface components and therefore may be useful to elucidate the role of these components in the host-pathogen interactions as well as in the complex interplay between bacteria and fungi in the lung microbiome.
molecular recognition, Force spectroscopy, AFM imaging, fungi, lectin–carbohydrate interactions, pathogen–host adhesion
NCBI PubMed ID: 29128932Aspergillus fumigatus mitogen-activated protein kinases (MAPKs) are involved in maintaining the normal morphology of the cell wall and providing resistance against cell wall-damaging agents. Upon cell wall stress, cell wall-related sugars need to be synthesized from carbohydrate storage compounds. Here we show that this process is dependent on cAMP-dependent protein kinase A (PKA) activity and regulated by the high-osmolarity glycerol response (HOG) MAPKs SakA and MpkC. These protein kinases are necessary for normal accumulation/degradation of trehalose and glycogen, and the lack of these genes reduces glucose uptake and glycogen synthesis. Alterations in glycogen synthesis were observed for the sakA and mpkC deletion mutants, which also displayed alterations in carbohydrate exposure on the cell wall. Carbohydrate mobilization is controlled by SakA interaction with PkaC1 and PkaR, suggesting a putative mechanism where the PkaR regulatory subunit leaves the complex and releases the SakA-PkaC1 complex for activation of enzymes involved in carbohydrate mobilization. This work reveals the communication between the HOG and PKA pathways for carbohydrate mobilization for cell wall construction.IMPORTANCE Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections such as invasive pulmonary aspergillosis, especially in immunocompromised patients. The fungal cell wall is the main component responsible for recognition by the immune system, due to the specific composition of polysaccharide carbohydrates exposed on the surface of the fungal cell wall called pathogen-associated molecular patterns (PAMPs). Key enzymes in the fungal cell wall biosynthesis are a good target for fungal drug development. This report elucidates the cooperation between the HOG and PKA pathways in the mobilization of carbohydrates for fungal cell wall biosynthesis. We suggest that the reduced mobilization of simple sugars causes defects in the structure of the fungal cell wall. In summary, we propose that SakA is important for PKA activity, therefore regulating the availability and mobilization of monosaccharides for fungal cell wall biosynthesis during cell wall damage and the osmotic stress response.
cell wall, glycogen, Trehalose, Aspergillus fumigatus, MpkC, SakA, high-osmotic glycerol pathway, protein kinase A
NCBI PubMed ID: 30538182Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of β-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.
cell wall, Microscopy, antifungal activity, azoles
NCBI PubMed ID: 30082817Chitinases are hydrolases that catalyze the cleavage of the β-1,4-O-glycosidic linkages in chitin, a polysaccharide abundantly found in nature. Chitin is an important structural component of the cell wall of most fungi and the exoskeleton of arthropods, including insects and crustaceans. These enzymes are widespread in the living world, being found in organisms from all three domains of life. Due to their hydrolytic activity on chitin, chitinases have great biotechnological potential in different areas, such as human health, agriculture and food technology. The antifungal, insecticidal and nematicidal effects of many chitinases have been intensively investigated in the scientific literature, aiming to exploit these properties to protect crops against phytopathogenic fungi and insect pests and parasitic nematodes. On the other hand, the effects of chitinases on bacteria have been underexploited, possibly because chitin is not present in bacterial cell walls. The aim of this study was to search the scientific literature for works describing chitinases with antibacterial activity. Three bibliographic databases were searched using the keywords “chitinase” and “antibacterial” as descriptors and the chosen articles were selected according to specific inclusion and exclusion criteria. As a result, we identified only 5 reports wherein 6 purified chitinases have been shown experimentally to have antibacterial activity. Three out of these 6 antibacterial chitinases were shown to be bifunctional enzymes, which have chitinase and lysozyme activity. The possible mechanism of action of these antibacterial chitinases is discussed, highlighting their potential as antibacterial agents.
peptidoglycan, antimicrobial, lysozyme, chitin, Hydrolases
Publication DOI: 10.5530/jyp.2018.10.33Chitin is a polysaccharide that provides structure and rigidity to the cell walls of fungi and insects. Mammals possess multiple chitinases, which function to degrade chitin, thereby supporting a role for chitinases in immune defense. However, chitin degradation has been implicated in the pathogenesis of asthma. Here, we determined the impact of acidic mammalian chitinase (AMCase) (Chia) deficiency on host defense during acute exposure to the fungal pathogen Aspergillus fumigatus as well as its contribution to A. fumigatus-associated allergic asthma. We demonstrate that chitin in the fungal cell wall was detected at low levels in A. fumigatus conidia, which emerged at the highest level during hyphal transition. In response to acute A. fumigatus challenge, Chia-/- mice unexpectedly demonstrated lower A. fumigatus lung burdens at 2 days postchallenge. The lower fungal burden correlated with decreased lung interleukin-33 (IL-33) levels yet increased IL-1β and prostaglandin E2 (PGE2) production, a phenotype that we reported previously to promote the induction of IL-17A and IL-22. During chronic A. fumigatus exposure, AMCase deficiency resulted in lower dynamic and airway lung resistance than in wild-type mice. Improved lung physiology correlated with attenuated levels of the proallergic chemokines CCL17 and CCL22. Surprisingly, examination of inflammatory responses during chronic exposure revealed attenuated IL-17A and IL-22 responses, but not type 2 responses, in the absence of AMCase. Collectively, these data suggest that AMCase functions as a negative regulator of immune responses during acute fungal exposure and is a contributor to fungal asthma severity, putatively via the induction of proinflammatory responses.
Aspergillus fumigatus, asthma/allergy, lung defense
NCBI PubMed ID: 29712728Deadly infections from opportunistic fungi have risen in frequency, largely because of the at-risk immunocompromised population created by advances in modern medicine and the HIV/AIDS pandemic. This review focuses on dynamics of the fungal polysaccharide cell wall, which plays an outsized role in fungal pathogenesis and therapy because it acts as both an environmental barrier and as the major interface with the host immune system. Human fungal pathogens use architectural strategies to mask epitopes from the host and prevent immune surveillance, and recent work elucidates how biotic and abiotic stresses present during infection can either block or enhance masking. The signaling components implicated in regulating fungal immune recognition can teach us how cell wall dynamics are controlled, and represent potential targets for interventions designed to boost or dampen immunity.
cell wall, glucan, innate immunity, fungi, evasion
NCBI PubMed ID: 29452950Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present ubiquitously at the cell surface in all eukaryotes. They play a crucial role in the interaction of the cell with its external environment, allowing the cell to receive signals, respond to challenges, and mediate adhesion. In yeast and fungi, they also participate in the structural integrity of the cell wall and are often essential for survival. Roughly four decades after the discovery of the first GPI-APs, this review provides an overview of the insights gained from studies of the GPI biosynthetic pathway and the future challenges in the field. In particular, we focus on the biosynthetic pathway in Saccharomyces cerevisiae, which has for long been studied as a model organism. Where available, we also provide information about the GPI biosynthetic steps in other yeast/ fungi. Although the core structure of the GPI anchor is conserved across organisms, several variations are built into the biosynthetic pathway. The present Review specifically highlights these variations and their implications. There is growing evidence to suggest that several phenotypes are common to GPI deficiency and should be expected in GPI biosynthetic mutants. However, it appears that several phenotypes are unique to a specific step in the pathway and may even be species-specific. These could suggest the points at which the GPI biosynthetic pathway intersects with other important cellular pathways and could be points of regulation. They could be of particular significance in the study of pathogenic fungi and in identification of new and specific antifungal drugs/ drug targets.
cell wall, Candida albicans, Saccharomyces cerevisiae, GPI anchor biosynthesis, antifungal drug target
NCBI PubMed ID: 29679465The cell wall is one of the major keys to fungal identity. Fungi use their cell wall to sense the environment, and localize nutrients and competing microorganism. Pathogenic species additionally modify their cell walls to hide from a host's immune system. With the growing number of fungal infections and alarming shortage of available drugs, we are in need of new approaches to fight pathogens. The cell wall seems to be a natural target, since animal host cells are devoid of it. The current knowledge about fungal cell wall components is often limited, and there is huge diversity both in structure and composition between species. In order to compare the distribution of diverse proteins involved in cell wall biosynthesis and maintenance, we performed sequence homology searches against 24 fungal proteomes from distinct taxonomic groups, all reported as human pathogens. This approach led to identification of 4014 cell wall proteins (CWPs), and enabled us to speculate about cell wall composition in recently sequenced pathogenic fungi with limited experimental information. We found large expansions of several CWP families, in particular taxa, and a number of new CWPs possibly involved in evading host immune recognition. Here, we present a comprehensive evolutionary history of fungal CWP families in the context of the fungal tree of life.
glycosyltransferase, fungal cell wall, fungal pathogen, cell wall proteins, cross-linking enzymes, glycohydrolase
NCBI PubMed ID: 29371499Chitin, a potential allergy-promoting pathogen-associated molecular pattern (PAMP), is a linear polymer composed of N-acetylglucosamine residues which are linked by β-(1,4)-glycosidic bonds. Mammalians are potential hosts for chitin-containing protozoa, fungi, arthropods, and nematodes; however, mammalians themselves do not synthetize chitin and thus it is considered as a potential target for recognition by mammalian immune system. Chitin is sensed primarily in the lungs or gut where it activates a variety of innate (eosinophils, macrophages) and adaptive immune cells (IL-4/IL-13 expressing T helper type-2 lymphocytes). Chitin induces cytokine production, leukocyte recruitment, and alternative macrophage activation. Intranasal or intraperitoneal administration of chitin (varying in size, degree of acetylation and purity) to mice has been applied as a routine approach to investigate chitin's priming effects on innate and adaptive immunity. Structural chitin present in microorganisms is actively degraded by host true chitinases, including acidic mammalian chitinases and chitotriosidase into smaller fragments that can be sensed by mammalian receptors such as FIBCD1, NKR-P1, and RegIIIc. Immune recognition of chitin also involves pattern recognition receptors, mainly via TLR-2 and Dectin-1, to activate immune cells to induce cytokine production and creation of an immune network that results in inflammatory and allergic responses. In this review, we will focus on various immunological aspects of the interaction between chitin and host immune system such as sensing, interactions with immune cells, chitinases as chitin degrading enzymes, and immunologic applications of chitin.
immune system, innate immunity, chitin, adaptive immunity, chitinase
NCBI PubMed ID: 28251581The high mortality of invasive fungal infections, and the limited number and inefficacy of antifungals necessitate the development of new agents with novel mechanisms and targets. The fungal cell wall is a promising target as it contains polysaccharides absent in humans, however, its molecular structure remains elusive. Here we report the architecture of the cell walls in the pathogenic fungus Aspergillus fumigatus. Solid-state NMR spectroscopy, assisted by dynamic nuclear polarization and glycosyl linkage analysis, reveals that chitin and α-1,3-glucan build a hydrophobic scaffold that is surrounded by a hydrated matrix of diversely linked β-glucans and capped by a dynamic layer of glycoproteins and α-1,3-glucan. The two-domain distribution of α-1,3-glucans signifies the dual functions of this molecule: contributing to cell wall rigidity and fungal virulence. This study provides a high-resolution model of fungal cell walls and serves as the basis for assessing drug response to promote the development of wall-targeted antifungals
cell wall, cell wall polymers, fungi, solid-state NMR
NCBI PubMed ID: 30013106Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.
virulence, cell wall, Microscopy, Sporothrix
NCBI PubMed ID: 29522522The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.
virulence, glycosylation, sporotrichosis, cell wall structure, Sporothrix
NCBI PubMed ID: 29538731Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.
β-1, filamentous fungi, Beauveria bassiana, cell wall integrity, alkaline tolerance, high pH, 3-glycanosyltransferase
NCBI PubMed ID: 29802184Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.
adherence, Biofilm, Aspergillus nidulans, GDP-mannose transporters, spore hydrophobicity
NCBI PubMed ID: 29420778Cryptococcus neoformans is an opportunistic fungal pathogen that is ubiquitous in the environment. It causes a deadly meningitis that is responsible for over 180,000 deaths worldwide each year, including 15% of all AIDS-related deaths. The high mortality rates for this infection, even with treatment, suggest a need for improved therapy. Unique characteristics of C. neoformans may suggest directions for drug discovery. These include features of three structures that surround the cell: the plasma membrane, the cell wall around it, and the outermost polysaccharide capsule. We review current knowledge of the fundamental biology of these fascinating structures and highlight open questions in the field, with the goal of stimulating further investigation that will advance basic knowledge and human health.
cell wall, polysaccharide capsule, Cryptococcus neoformans, plasma membrane
NCBI PubMed ID: 29742198Carbohydrate recognition is fundamental to a wide variety of interkingdom interactions. For example, bacterial peptidoglycan, an N-acetyl-D-glucosamine (GlcNAc)±N-acetylmuramic acid (MurNAc) polymer [1], and fungal chitin, a GlcNAc polymer [2, 3], are both immunostimulatory to vertebrates. In addition, bacterially produced Nodulation (Nod) factors, which consist of a modified GlcNAc, play a key role in symbiotic interactions with plants [4]. The structural similarity of these GlcNAc-containing molecules (Fig 1) suggests possible overlap in their physiological action, although many of the mechanisms underlying the detection of these molecules in different organisms appear unrelated. However, a single phylogenetically conserved domain, called Lysin (LysM), is found in specific receptors in signaling pathways responsive to one or more of these three related carbohydrates in bacteria, plants, and fungi and possibly mammalian systems. Thus, promiscuous activation could occur when a structurally similar but physiologically inappropriate ligand binds and thereby aberrantly activates an incorrect LysM domain-containing receptor. Here, I will discuss this possibility and its implications for immune pathologies such as asthma in which chitin is relevant.
nod factors, GlcNAc, carbohydrate recognition, chitin, LysM domain
NCBI PubMed ID: 30308080Fungal cell walls, which are essential for environmental adaptation and host colonization by the fungus, have been evolutionarily selected by plants and animals as a source of microbe-associated molecular patterns (MAMPs) that, upon recognition by host pattern recognition receptors (PRRs), trigger immune responses conferring disease resistance. Chito-oligosaccharides [β-1,4-N-acetylglucosamine oligomers, (GlcNAc)n ] are the only glycosidic structures from fungal walls that have been well-demonstrated to function as MAMPs in plants. Perception of (GlcNAc)4-8 by Arabidopsis involves CERK1, LYK4 and LYK5, three of the eight members of the LysM PRR family. We found that a glucan-enriched wall fraction from the pathogenic fungus Plectosphaerella cucumerina which was devoid of GlcNAc activated immune responses in Arabidopsis wild-type plants but not in the cerk1 mutant. Using this differential response, we identified the non-branched 1,3-β-D-(Glc) hexasaccharide as a major fungal MAMP. Recognition of 1,3-β-D-(Glc)6 was impaired in cerk1 but not in mutants defective in either each of the LysM PRR family members or in the PRR-co-receptor BAK1. Transcriptomic analyses of Arabidopsis plants treated with 1,3-β-D-(Glc)6 further demonstrated that this fungal MAMP triggers the expression of immunity-associated genes. In silico docking analyses with molecular mechanics and solvation energy calculations corroborated that CERK1 can bind 1,3-β-D-(Glc)6 at effective concentrations similar to those of (GlcNAc)4 . These data support that plants, like animals, have selected as MAMPs the linear 1,3-β-D-glucans present in the walls of fungi and oomycetes. Our data also suggest that CERK1 functions as an immune co-receptor for linear 1,3-β-D-glucans in a similar way to its proposed function in the recognition of fungal chito-oligosaccharides and bacterial peptidoglycan MAMPs.
cell wall, glucan, Plant immunity, chitin, CERK, BAK1, necrotrophic fungi
NCBI PubMed ID: 29083116Asthma is a heterogeneous disease that affects approximately 300 million people worldwide, largely in developed countries. The etiology of the disease is poorly understood, but is likely to involve specific innate and adaptive responses to inhaled microbial components that are found in allergens. Fungal-derived allergens represent a major contributing factor in the initiation, persistence, exacerbation, and severity of allergic asthma. C-type lectin like receptors, such as dectin-1, dectin-2, DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, and mannose receptor, recognize many fungal-derived allergens and other structurally similar allergens derived from house dust mites (HDM). In some cases, the fungal derived allergens have been structurally and functionally identified alongside their respective receptors in both humans and mice. In this review, we discuss recent understanding on how selected fungal and HDM derived allergens as well as their known or unknown receptors shape allergic airway diseases.
fungi, Dectin-1, C-type lectin receptors, allergens, sensitization
NCBI PubMed ID: 29696023The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased β-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.
polysaccharide, Biofilm, Extracellular matrix, Aspergillus fumigatus, mitogen activated kinases, phosphatases
Publication DOI: 10.1016/j.tcsw.2018.03.002Scedosporium/Lomentospora species are opportunistic mould pathogens, presenting notable antifungal resistance. We analysed the conidia and germinated conidia of S. apiospermum (Sap), S. aurantiacum (Sau), S. minutisporum (Smi) and L. prolificans (Lpr) by scanning electron microscopy and exposition of surface molecules by fluorescence microscopy. FINDINGS Conidia of Sap, Smi and Sau had oval, ellipsoidal and cylindrical shape, respectively, with several irregularities surrounding all surface areas, whereas Lpr conidia were rounded with a smooth surface. The germination of Sap occurred at the conidial bottom, while Smi and Sau germination primarily occurred at the centre of the conidial cell, and Lpr germination initiated at any part of the conidial surface. The staining of N-acetylglucosamine-containing molecules by fluorescein-labelled WGA primarily occurred during the germination of all studied fungi and in the conidial scars, which is the primary location of germination. Calcofluor white, which recognises the polysaccharide chitin, strongly stained the conidial cells and, to a lesser extent, the germination. Both mannose-rich glycoconjugates (evidenced by fluoresceinated-ConA) and cell wall externally located polypeptides presented distinct surface locations and expression according to both morphotypes and fungal species. In contrast, sialic acid and galactose-containing structures were not detected at fungal surfaces. MAIN CONCLUSIONS The present study demonstrated the differential production/exposition of surface molecules on distinct morphotypes of Scedosporium/Lomentospora species.
glycoconjugates, polysaccharides, cell surface, Scedosporium/Lomentospora, germination, polypeptides
NCBI PubMed ID: 29924142Cryptococcus neoformans is a human fungal pathogen that often causes infections in immunocompromised individuals. Upon inhalation into the lungs C. neoformans differentiates into cells with altered size and morphology, including production of large titan cells. Titan cells possess thickened cell wall and dense, cross-linked capsule when compared to in vitro grown cells. In addition, titan cells have increased cell wall chitin that is associated with a detrimental anti-inflammatory immune response. Here we examined the cell wall and capsule composition of in vitro, in vivo typical-sized and in vivo titan cells using High Performance Liquid Chromatography (HPLC). The monomer composition of cell wall polysaccharides showed that in vivo C. neoformans cells contained more glucosamine and less glucose than in vitro cells, suggesting alteration in abundance of both chitin and glucans, respectively. Low levels of galactosamine were also detected in carbohydrates from both in vivo and vitro cells. Within the in vivo cell population, differences in the proportions of cell wall and capsule monomers between typical and titan cells were also observed. Taken together, these results demonstrate that C. neoformans reshapes its cell wall and capsule composition during infection. These cell wall and capsule alterations likely help C. neoformans escape recognition by, and allow modulation of, the host immune system.
cell wall, capsule, Cryptococcus, C. neoformans, titan cells
NCBI PubMed ID: 30123851Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and β-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and β-1,3-glucan concentrations were detected at 36 A degrees C than those incubated at 28 A degrees C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 A degrees C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.
structure, cell wall, Trichoderma, Pleurotus ostreatus, integrity
NCBI PubMed ID: 29846777Lactase1 (Lcc1 ) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of lcc1 on the hyphal morphology and cell wall structure of L edodes. A thick cell wall and fibrous layer were clearly observed in the Icc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lccl secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.
cell wall polysaccharides, 6-glucan, 3-1, chitin, beta-1, white rot fungi, Basidiomycota
NCBI PubMed ID: 30449357Brewer's yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of β-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.
cell wall polysaccharides, β-Glucans, autolysis, brewer’s yeast
NCBI PubMed ID: 30128783Chitin is the second abundant polysaccharide in the world after cellulose. It is a vital structural component of the fungal cell wall but not for plants. In plants, fungi are recognised through the perception of conserved microbe-associated molecular patterns (MAMPs) to induce MAMP-triggered immunity (MTI). Chitin polymers and their modified form, chitosan, induce host defence responses in both monocotyledons and dicotyledons. The plants' response to chitin, chitosan, and derived oligosaccharides depends on the acetylation degree of these compounds which indicates possible biocontrol regulation of plant immune system. There has also been a considerable amount of recent research aimed at elucidating the roles of chitin hydrolases in fungi and plants as chitinase production in plants is not considered solely as an antifungal resistance mechanism. We discuss the importance of chitin forms and chitinases in the plant-fungal interactions and their role in persistent and possible biocontrol.
chitosan, chitin, chitinase, PR protein, plant–fungal interaction
NCBI PubMed ID: 30181925Cryptococcus neoformans mutants lacking each of the eight putative chitin synthase genes (CHS) have been previously generated. However, it is still unclear how deletion of chitin synthase genes affects the cryptococcal capsule. Since the connections between chitin metabolism and capsular polysaccharides in C. neoformans are numerous, we analyzed the effects of deletion of CHS genes on capsular and capsule-related structures of C. neoformans. CHS deletion affected capsular morphology in multiple ways, as determined by scanning electron microscopy and immunofluorescence analysis. Molecular diameter, serological reactivity and export of capsular polysaccharide were also affected in most of the chsΔ mutants, but the most prominent alterations were observed in the chs3Δ strain. C. neoformans cells lacking CHS genes also had altered formation of extracellular vesicles and variable chitinase activity under stress conditions. These results reveal previously unknown functions of CHS genes that greatly impact the physiology of C. neoformans.
structure, capsule, chitin synthase, Cryptococcus meoformans
NCBI PubMed ID: 32743128Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the β-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect β-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata.
chitin, Candida glabrata, cell wall integrity, calnexin, ER stress, cne1
NCBI PubMed ID: 29248112Chitin is an essential component of the cell wall of Cryptococcus neoformans conferring structural rigidity and integrity under diverse environmental conditions. Chitin deacetylase genes encode the enyzmes (chitin deacetylases [Cdas]) that deacetylate chitin, converting it to chitosan. The functional role of chitosan in the fungal cell wall is not well defined, but it is an important virulence determinant of C. neoformans Mutant strains deficient in chitosan are completely avirulent in a mouse pulmonary infection model. C. neoformans carries genes that encode three Cdas (Cda1, Cda2, and Cda3) that appear to be functionally redundant in cells grown under vegetative conditions. Here we report that C. neoformans Cda1 is the principal Cda responsible for fungal pathogenesis. Point mutations were introduced in the active site of Cda1 to generate strains in which the enzyme activity of Cda1 was abolished without perturbing either its stability or localization. When used to infect CBA/J mice, Cda1 mutant strains produced less chitosan and were attenuated for virulence. We further demonstrate that C. neoformans Cda genes are transcribed differently during a murine infection from what has been measured in vitro IMPORTANCE Cryptococcus neoformans is unique among fungal pathogens that cause disease in a mammalian host, as it secretes a polysaccharide capsule that hinders recognition by the host to facilitate its survival and proliferation. Even though it causes serious infections in immunocompromised hosts, reports of infection in hosts that are immunocompetent are on the rise. The cell wall of a fungal pathogen, its synthesis, composition, and pathways of remodelling are attractive therapeutic targets for the development of fungicides. Chitosan, a polysaccharide in the cell wall of C. neoformans is one such target, as it is critical for pathogenesis and absent in the host. The results we present shed light on the importance of one of the chitin deacetylases that synthesize chitosan during infection and further implicates chitosan as being a critical factor for the pathogenesis of C. neoformans.
chitosan, fungal virulence, catalytic site, cell wall localization, chitin deacetylase, metal binding site
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