Sterol-binding polysaccharides of Rhizopus arrhizus, Penicillium roquefortii and Saccharomyces carlsbergensis

Polysaccharides that bind with sterols and render them water-soluble were isolated from two mycelial fungi, Rhizopus arrhizus and Penicillium roquefortii and a yeast Saccharomyces carlsbergensis. The polysaccharides from R. arrhizus and S. carlsbergensis were accompanied by small quantities of phosphorus, protein and lipid, none of which significantly influenced the binding of sterol to polysaccharide. The chemical composition and sterol-binding properties of the polysaccharides from the filamentous species were almost identical, but differed significantly from those of the yeast polysaccharide. The principal sterol-binding polysaccharide of S. carlsbergensis was identified as a mannan and that of the filamentous fungi as a glucan(s). The binding capacity of the purified yeast polysaccharide was almost two-fold greater than that of R. arrhizus and P. roquefortii.     

Metabolism of 2-deoxy-d-glucose by baker's yeast. VI. A study on cell wall mannan

The incorporation of 2-deoxy-d-glucose into cell wall mannan of growing Saccharomyces cerevisiae proceeded continuously during culture growth and followed the cell multiplication. About 10% of mannan labelled with deoxyglucose was concurrently released into the medium. The distribution of deoxyglucose between the side-chains and the main chain of mannan has been established. Approximately 90% of deoxyglucose present in the polysaccharide was bound in the side-chains and only 10% was located in the (1 å 6)-linked main chain. This result suggested that deoxyglucose metabolites serving as glycosyl donors in mannan biosynthesis were much worse substrates for the enzyme(s) responsible for the formation of the main chain of the polysaccharide than for the mannosyl transferases involved in the formation of the mannan side-chains. Degradation of deoxyglucose-containing mannan by α-mannosidase of Arthrobacter GJM-1 stopped at the deoxyglucosyl residues.     

Preparation of Anti-protein and Anti-mannan Antisera against Fungal Cell Wall by Affinity Chromatography

Iranzo, M., Marcilla, A., Elorza, M. V., Mormeneo, S., and Sentandreu, R. 1994. Preparation of anti-protein and anti-mannan antisera against fungal cell wall by affinity chromatography. Experimental Mycology 18, 159-167. A novel and easy chromatographic method has been developed for the isolation of anti-protein and anti-mannan antisera from a population of polyclonal antibodies obtained against Candida albicans and Yarrowia lipolytica cell wall mannoproteins. The technique is based on the immobilization of mannan (to be used as immunoadsorbent) by Affi-Prep H_z resin after the oxidation of neighboring hydroxyl groups of the polysaccharide with sodium periodate. For Y. lipolytica polyclonal antiserum, a single chromatographic step using the homologous mannan was sufficient to obtain an antiprotein antibody preparation free of antimannan antibodies. For C. albicans, three chromatographic processes using homologous and heterologous mannan were needed to obtain a satisfactory antiprotein antiserum. The potential application of the anti-protein antiserum obtained has been demonstrated by indirect immunofluorescence assays of whole cells and electrophoretic analysis of wall proteins in C. albicans and Saccharomyces cerevisiae .     

Production of an exocellular acid phosphatase by the yeast Hansenula holstii NCYC 560

1. The yeast Hansenula holstii NCYC 560 produced invertase and an inducible acid phosphatase located betweent the cytoplasmic membrane and the yeast cell wall. 2. These enzymes were also found in the culture medium outside the cell boundaries. 3. The amount of cell wall mannan in cells grown in phosphate-limited medium decreased in comparison with that of cells grown in phospahte-rich medium. 4. It is proposed that the mannan in this yeast is a loose and highly permeable structure, allowing external enzymes to leave the cell boundaries.     

Candidacidal activity of myeloperoxidase: characterization of myeloperoxidase-yeast complex formation

We have previously demonstrated the ability of human neutrophil myeloperoxidase to bind to cell wall mannan polysaccharide isolated from Candida albicans. This binding capacity provides for association of the enzyme with target yeast which is essential for efficient candidacidal activity. In this report, we further consider the role of the mannan-binding property of myeloperoxidase in the candidacidal activity of the enzyme. Solubilized mannan antagonizes binding of the enzyme to yeast, suggesting that mannan may be a primary component of the fungal cell wall which serves as a target for binding of myeloperoxidase. Myeloperoxidase is shown to form complexes with both solubilized mannan and Candida yeast, with Kds of 0.97 x 10(-5) M and 1.2 x 10(-5) M, respectively. The interaction between myeloperoxidase and mannan does not allow the enzyme to readily dissociate from the surface of target yeast. As a result, the enzyme may be unable to dissociate from dead yeast to become available for binding to additional fungal targets.     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Isolation and structural determination of a unique polysaccharide containing mannofuranose from the cell wall of the fungus <Emphasis Type="Italic">Acrospermum compressum</Emphasis>

The alkali-extractable and water-soluble fungal polysaccharide F1SS isolated from the cell wall of Acrospermum compressum has been studied by methylation analyses, reductive cleavage and 1D- and 2D-NMR spectroscopy. The polysaccharide consists of a regular disaccharide repeating unit with the structure: The mannan core was obtained by mild hydrolysis of the polysaccharide F1SS and its structure was deduced to be composed of a skeleton of α-(1→6)-mannopyranan, with around 1 out of 11 residues substituted at position 2 by short chains (one to six units) of 2-substituted mannopyranoses. DOSY experiments provided molecular sizes of 60 kDa and 2.5 kDa for the polysaccharide F1SS and the mannan core, respectively. This is the first report of a fungal mannofuranose-containing cell wall polysaccharide.     

Chemical structure of a polysaccharide isolated from the cell wall of Arachniotus verruculosus and A. ruber.

The structure of a cell wall alkali-extractable and water-soluble polysaccharide isolated from two species of Arachniotus has been established by reductive cleavage and NMR spectroscopy. The linear polysaccharide consists of a regular disaccharide-repeating unit with the structure: [-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-(1-->](n)-->mannan core.     

Yeast cell-wall synthesis

1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Structure of polysaccharides from the Polyporus tumulosus cell wall

Cell walls of the Basidiomycete fungus Polyporus tumulosus (Cooke) were fractionated, and the polysaccharide content of the fractions investigated. The major constituents of the cell wall include four polysaccharides, chitin, a β-1, 3-glucan and the alkali soluble α-glucan and xylomannan.The glucan is highly dextrotatory with an [α]_D²¹ of + 221° and gave on partial acid hydrolysis and acetolysis an homologous series of oligosaccharides. The disaccharide was shown to be nigerose 3-0-α-D-glucopyranosyl-D-glucose. Periodate oxidation and methylation studies provided supporting evidence that the polysaccharide is an essentially unbranched polymer of 1,3-linked glucose residues.The other alkali-soluble polysaccharide, a xylomannan, is a polymer of mannose and xylose in the approximate molar proportions of 1.2:1. It has an [α]_D = + 56° and on partial acid hydrolysis and acetolysis gave an homologous series of 1,3-linked mannodextrins but no oligosaccharides containing xylose were obtained. An α-1,3-linked mannan was prepared from the xylomannan by degradation with mild acid or by degradation of the periodate-oxidased and reduced xylomannan. The structure therefore is visualised as having a backbone of 1,3-linked mannan, to which xylose residues are attached. Methylation studies showed that branching occurs at C-4 of the mannopyranose units; the presence of 2,3-di-o-methyl-d-xylose in the hydrolysate of the methylated polysaccharide indicated that some of the xylose residues are 1,4-linked. The possible structure of the fungal cell wall is discussed in the light of the results obtained.     

Yeast mannan increases Bacteroides thetaiotaomicron abundance and suppresses putrefactive compound production in in vitro fecal microbiota fermentation

ABSTRACT

Yeast mannan is a part of yeast cell wall and can potentially affect gut microflora as a soluble dietary fiber. We demonstrated that yeast mannan suppressed putrefactive production and increased the relative abundance of Bacteroides thetaiotaomicron in in vitro fecal fermentation. These results suggest that yeast mannan can be used as a novel prebiotic food ingredient.     

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

Fungal cell walls frequently contain a polymer of mannose and galactose called galactomannan. In the pathogenic filamentous fungus Aspergillus fumigatus, this polysaccharide is made of a linear mannan backbone with side chains of galactofuran and is anchored to the plasma membrane via a glycosylphosphatidylinositol or is covalently linked to the cell wall. To date, the biosynthesis and significance of this polysaccharide are unknown. The present data demonstrate that deletion of the Golgi UDP-galactofuranose transporter GlfB or the GDP-mannose transporter GmtA leads to the absence of galactofuran or galactomannan, respectively. This indicates that the biosynthesis of galactomannan probably occurs in the lumen of the Golgi apparatus and thus contrasts with the biosynthesis of other fungal cell wall polysaccharides studied to date that takes place at the plasma membrane. Transglycosylation of galactomannan from the membrane to the cell wall is hypothesized because both the cell wall-bound and membrane-bound polysaccharide forms are affected in the generated mutants. Considering the severe growth defect of the A. fumigatus GmtA-deficient mutant, proving this paradigm might provide new targets for antifungal therapy.     

A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation

AIM: The polysaccharide composition of the Saccharomyces cerevisiae cell wall was measured under various growth conditions and was compared with the cell wall structure. METHODS AND RESULTS: Chemical and enzymatic methods were used to determine levels of beta-1,3-glucan and 1,6-glucan, mannan and chitin of the yeast cell wall, whereas the structure/resistance of the wall was qualitatively assessed by the sensibility to the lytic action by zymolyase. It was found that the dry mass and polysaccharides content of the cell wall could vary by more than 50% with the nature of the carbon source, nitrogen limitation, pH, temperature and aeration, and with the mode of cell cultivation (shake flasks vs controlled fermentors). While no obvious correlation could be found between beta-glucan or mannan levels and the susceptibility of whole yeast cells to zymolyase, increase of beta-1,6-glucan levels, albeit modest with respect to the growth conditions investigated, and to a lesser extent that of chitin, was associated with decreased sensitivity of yeast cells to the lytic action by zymolyase. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that the cell wall structure is merely determined by cross-linking between cell wall polymers, pointed out the role of beta-1,6-glucan in this process. Hence, this study reinforces the idea that enzymes involved in these cross-linking reactions are potential targets for antifungal drugs.     

Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus

The galactomannan is a major cell wall molecule of Aspergillus fumigatus. This molecule is composed of a linear mannan with a repeating unit composed of four α1,6 and α1,2 linked mannose with side chains of galactofuran. To obtain a better understanding of the mannan biosynthesis in A. fumigatus, it was decided to undertake the successive deletion of the 11 genes which are putative orthologs of the mannosyltransferases responsible for establishing α1,6 and α1,2 mannose linkages in yeast. These deletions did not lead to a reduction of the mannan content of the cell wall of the mycelium of A. fumigatus. In contrast, the mannan content of the conidial cell wall was reduced and this reduction was associated with a partial disorganization of the cell wall leading to defects in conidial survival both in vitro and in vivo.     

C. albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5% serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6% increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3%) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8-100%) and a decrease in cell wall mannan (5.7-17.3%). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.     

The structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

LeMAN4 endo-β-mannanase from ripe tomato fruit can act as a mannan transglycosylase or hydrolase

Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590–600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-β-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529–538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-β-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6–11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-β-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-β-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes.     

Studies on the Discrimination of SCP-related Yeast by Proton Magnetic Resonance Spectroscopy: Structural Changes in Cell Wall Mannan of Candida subtropicalis Grown in Different Media

Applicability of PMR spectroscopy to the discrimination of the SCP-related yeast was investigated because the yeast was inactivated by heat treatment. When Candida subtropicalis was cultured in a medium containing glucose as a sole source of carbon, its cell wall mannan (mannan A) showed a PMR spectral pattern characterized by three intense peaks at δ: 4.97, 5.10, 5.29 and two small peaks at δ: 4.90, 5.19. Mannan (mannan B) from the yeast cultured in a medium containing n-pentadecane and triton X–100 showed a different spectral pattern in which the signals were observed at δ: 4.97, 5,10, 5.29, the intensity ratios of the signals were also different from those of mannan A. Acetolysis mannan was analyzed to compare the difference between two specified structures by using a gel elution method, methylation analysis and PMR spectra. Mannan B contained a less amount of the a (1→3) linkage than mannan A did, and differed from mannan A in its distribution pattern of side chain units. Our previous results together with the present ones proved the PMR method to be effective for the discrimination of the yeast.     

Purification of Phosphomannanase and Its Action on the Yeast Cell Wall

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.