Separation and Partial Purification of 1,3-beta-Glucan and 1,4-beta-Glucan Synthases from Saprolegnia

Enriched 1,3-β-glucan and 1,4-β-glucan synthase fractions from the fungus Saprolegnia were isolated by rate zonal centrifugation on glycerol gradient. Purification was improved by entrapment of the enzymes in their reaction product, i.e. microfibrillar glucans. 1,3-β-Glucan synthases were separated from 1,4-β-glucan synthases following resuspension of entrapped enzymes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated that 1,3-β-glucan and 1,4-β-glucan synthases may have a different polypeptide composition because they were enriched for different protein subunits (34, 48, and 50 kD for the 1,3-β-glucan synthase and 60 kD for the 1,4-β-glucan synthase).     

β(1,3)-glucanosyl-transferase activity is essential for cell wall integrity and viability of Schizosaccharomyces pombe

Background

The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1⁺, gas2⁺, gas4⁺ and gas5⁺- are present in S. pombe, although their function has not been analyzed.

Methodology/Principal Findings

Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast.

Conclusions/Significance

We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth.     

Surface ��-1,3-Glucan Facilitates Fungal Stealth Infection by Interfering with Innate Immunity in Plants

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant''s defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants'' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.     

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae: STRUCTURAL CHARACTERIZATION AND IN SITU SYNTHESIS*S?

Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (¹H and ¹³C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[¹⁴C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts contains two types of β-glucans. In the former yeast, branched β-(1,3)-glucan accounts for ∼50–55%, whereas β-(1,6)-glucan represents 10–15% of the total yeast cell wall polysaccharides, each chain of the latter extending up to 140–350 glucose residues in length. The amount of 3,6-branched glucose residues varies with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida albicans, and Schizosaccharomyces pombe, respectively (1). β-(1,6)-Glucan stabilizes the cell wall, since it plays a central role as a linker for specific cell wall components, including β-(1,3)-glucan, chitin, and mannoproteins (2, 3). However, the exact structure of the β-(1,6)-glucan and the mode of biosynthesis of this polymer are largely unknown. In S. pombe, immunodetection studies suggested that synthesis of this polymer backbone begins in the endoplasmic reticulum, with extension occurring in the Golgi (4) and final processing at the plasma membrane. In S. cerevisiae, Montijn and co-workers (5), by immunogold labeling, detected β-(1,6)-glucan at the plasma membrane, suggesting that the synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and their homologues, SKN1 and KNH1, have been reported to be involved in β-(1,6)-glucan synthesis in S. cerevisiae, C. albicans, and Candida glabrata (6–10). Among all of these genes, the ones that seem to play the major synthetic role are KRE5 and KRE9, since their disruption caused significant reduction (100 and 80%, respectively, relative to wild type) in the cell wall β-(1,6)-glucan content (11–13).To date, the biochemical reaction responsible for the synthesis of β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in most cases, when membrane preparations are incubated with UDP-glucose, only linear β-(1,3)-glucan polymers are produced, although some studies have reported the production of low amounts of β-(1,6)-glucans by membrane preparations (14–17). These data suggest that disruption of the fungal cell prevents or at least has a strong negative effect on β-(1,6)-glucan synthesis. The use of permeabilized cells, which allows substrates, such as nucleotide sugar precursors, to be readily transported across the plasma membrane, is an alternative method to study in situ cell wall enzyme activities (18–22). A number of methods have been developed to permeabilize the yeast cell wall (23), of which osmotic shock was successfully used to demonstrate β-(1,3)-glucan and chitin synthase activities (20, 24). Herein, we describe the biochemical activity responsible for β-(1,6)-glucan synthesis using permeabilized S. cerevisiae cells and UDP-[¹⁴C]glucose as a substrate. We also have analyzed the physicochemical parameters of this activity and chemically characterized the end product and its structural organization within the mature yeast cell wall.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

Factors Influencing beta-Glucan Synthesis by Particulate Enzymes from Suspension-Cultured Lolium multiflorum Endosperm Cells

Particulate enzymes from suspension-cultured ryegrass (Lolium multiflorum Lam.) endosperm cells incorporated glucosyl residues from UDP-glucose and GDP-glucose into β-glucans. Three types of β-glucans were produced from UDP-glucose: 1,3-β-glucan; 1,4-β-glucan; and mixed-linkage 1,3;1,4-β-glucan. As in other systems, relatively more 1,4-β-glucan was produced from a low (10 micromolar) UDP-glucose concentration, and relatively more 1,3-β-glucan was produced from a high (1 millimolar) UDP-glucose concentration. However, in ryegrass, 1,3;1,4-β-glucan represented a major proportion of the products at both low and high UDP-glucose concentrations. The arrangement of linkages in the 1,3;1,4-β-glucan was different at the two concentrations; at the low UDP-glucose concentration, more sequences of three consecutive 1,4-linkages were produced.     

The Fission Yeast SEL1 Domain Protein Cfh3p: A NOVEL REGULATOR OF THE GLUCAN SYNTHASE Bgs1p WHOSE FUNCTION IS MORE RELEVANT UNDER STRESS CONDITIONS*S?

In Schizosaccharomyces pombe, Bgs1/Cps1p is a β(1,3)-glucan synthase required for linear β(1,3)-glucan synthesis and primary septum formation. Here, we have studied the regulation of Bgs1p by Cfh3/Chr4p, a member of a family of conserved adaptor proteins, which resembles the chitin synthase regulator Chs4p from Saccharomyces cerevisiae and Candida albicans. cfh3Δ cells showed a genetic interaction with cps1-191, and Cfh3p co-immunoprecipitated with Bgs1/Cps1p. In the absence of cfh3⁺, cells were more sensitive to digestion by glucanases, and both Calcofluor staining and glucan synthesis were reduced. We found that in a wild-type strain, β(1,3)-glucan synthesis was reduced under stress conditions. In the cfh3Δ, cps1-191, and cfh3Δ cps1-191 strains, β(1,3)-glucan synthesis was further reduced, and growth was impaired under stress conditions, suggesting that Cfh3p and Bgs1p might play a role in ensuring growth in unfavorable environments. In a cfh3Δ mutant, Bgs1p was delocalized when the cells were distressed, but a blockade in endocytosis prevented this delocalization. Finally, we found that the SEL1 repeats are required for Cfh3p function. These results show that Cfh3p is a regulatory protein for Bgs1p and that its function is particularly necessary when the cells are undergoing stress.In Schizosaccharomyces pombe, the primary septum, composed of linear and branchedβ(1,3)-glucan, is surrounded by a secondary septum with a composition similar to that of the lateral cell wall (branched β(1,3)-glucan, β(1,6)-glucan, α(1,3)-glucan, and mannoproteins (1, 2)). Bgs1/Cps1p is the β(1,3)-glucan synthase responsible for the synthesis of linear β(1,3)-glucan and the primary septum structure (3). In the absence of this activity, the cells are able to form remedial septa that do not contain linear β(1,3)-glucan. These septa do not stain with low concentrations of Calcofluor and cannot be degraded by glucanases, so the cells remain chained, forming hyphal structures (3). In S. pombe there are four glucan synthase homologues, bgs1⁺/cps1⁺, bgs2⁺, bgs3⁺, and bgs4⁺ (4–9). In the case of the bgs1Δ cells, apical growth takes place in two opposite directions, producing branched cells with a dichotomic growth. This phenotype is not observed in bgs2Δ, bgs3Δ,or bgs4Δ mutants, thus suggesting that Bgs1p might play a specific role in the control of cell growth (3). Finally, Bgs1p is a component of the cytokinesis checkpoint, which coordinates mitosis with actomyosin ring contraction and septum synthesis (10, 11).The study of Bgs1p regulation should help us to understand the control of cell wall synthesis and cytokinesis in S. pombe. Bgs1p requires an active Septation Initiation Network (SIN), an assembled contractile actomyosin ring, and the type-V myosin Myo52p to localize properly at the division site (9, 12, 13). Regarding the regulation of biochemical activity, the PKC homologues Pck1p and Pck2p activate β(1,3)-glucan synthesis in an unknown way (14) while the Rho1p GTPase is a direct activator of the β(1,3)-glucan synthase catalytic subunit (15).The cell wall is a morphogenetic determinant, but also an essential cellular structure that protects the organism against cell lysis in hypoosmotic environments. In S. pombe, the mitogen-activated protein (MAP)³ kinase Spm1/Pmk1p pathway (also known as the cell integrity pathway) regulates growth and morphogenesis in response to multiple stresses, including hyper- or hypoosmotic conditions, nutrient limitation, and cell wall-damaging compounds (16, 17). Proper growth and morphology under hyperosmotic conditions also requires Skb1p and Skb5p, which are regulators of the Shk1/Orb2/Pak1p kinase (18, 19). However, little is known about the role of the cell wall as a protective element against hyperosmotic conditions, the presence of high concentrations of chloride ions, or nutrient limitation.Here we examine the role of the Cfh3/Chr4 protein in cell wall synthesis and response to stress. Cfh3p shares significant similarity with the Chs4 proteins from Saccharomyces cerevisiae and from Candida albicans, which are chitin synthase regulators (20, 21). In S. cerevisiae, regulation of the chitin synthase Chs3p by Chs4p is complex and still not well understood. Chs4p is required for the correct localization of Cfh3p at the bud neck by mediating its anchorage to septins through the adaptor protein Bni4p, but it also acts as a biochemical activator and is required for the stability of Chs3p at the plasma membrane (20, 22–25). In S. pombe, no chitin synthesis occurs during vegetative growth (26–29), glucan being the main cell wall component, there is no Bni4p homologue, and septins are involved in cell separation but not in septum synthesis (30, 31). We wanted to investigate whether Cfh3p played any role in cell wall synthesis and/or morphogenesis in the fission yeast. We found that in the absence of cfh3⁺, the cells showed reduced β-glucan synthesis and impaired growth under stress conditions. These phenotypes were aggravated in a double cfh3Δ cps1-191 mutant. Our results suggest that Cfh3p is a regulator of the β-glucan synthase Bgs1p whose presence is more critical when the cells are undergoing environmental stress. Cfh3p belongs to a conserved family of scaffold proteins characterized by the presence of tandem repeats of SEL1 domains, which are involved in signal transduction during different cellular processes (32). Here we found that the SEL1 domains in Cfh3p were required for its function.     

Assimilate Unloading from Maize (Zea mays L.) Pedicel Tissues : I. Evidence for Regulation of Unloading by Cell Turgor

β-Glucan synthase activity in plant membranes can be markedly altered by a multiplicity of apparently unrelated factors. In pea epicotyl membranes it is enhanced by low and inhibited by high concentrations of added Ca²⁺, trypsin or soluble pea protease. Ca²⁺ stimulates preexisting synthase activity, particularly in the presence of polycations (spermidine), but protease treatments activate and, with time, inactivate synthase zymogen. Endogenous pea protease activity is also associated with washed pea membrane and appears to be responsible for the decay observed with time in the β-glucan synthase activity. Endogenous pea protease activity is inhibited by thiol inhibitors, e.g. iodoacetamide and Hg²⁺, and by a heat-stable peptide, molecular weight approximately 10,000, that is found in supernatants of pea extracts. These protease inhibitors have the capacity to protect β-glucan synthase activity from denaturation or its zymogen from activation due to endogenous or added protease activity. Evidence is described which supports the proposal that 1,4-β-glucan synthase is destroyed and possibly converted to 1,3-β-glucan synthase activity by protease action, and that the latter may then be greatly enhanced by Ca²⁺ and polycations.     

Infection Structure–Specific Expression of β-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of β-Glucan–Triggered Immunity in Maize

β-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of β-1,3-glucan is unknown. We functionally characterized the β-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of β-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading β-glucan–triggered immunity.     

Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants

A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study β-1,3-glucan synthase activity changes. All tissues contained an active β-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active β-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in β-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that β-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development.     

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln¹⁰⁶ is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.     

Influence of Free Fatty Acids, Lysophosphatidylcholine, Platelet-Activating Factor, Acylcarnitine, and Echinocandin B on 1,3-beta-d-Glucan Synthase and Callose Synthesis

The activity of 1,3-β-d-glucan synthase assayed in the presence of digitonin in a microsomal preparation from suspension-cultured cells of Glycine max can be fully inhibited by unsaturated fatty acids, trienoic acids being most effective. Lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B can also fully inhibit the enzyme. Inhibition is observed both when the enzyme is activated by Ca²⁺ or by trypsinization. At low amounts some of the substances can also cause stimulation. These effects all may result from a displacement of certain endogenous phospholipids necessary for optimal activity of the 1,3-β-d-glucan synthase.     

Differential effects of two fermentable carbohydrates on central appetite regulation and body composition

Background

Obesity is rising at an alarming rate globally. Different fermentable carbohydrates have been shown to reduce obesity. The aim of the present study was to investigate if two different fermentable carbohydrates (inulin and β-glucan) exert similar effects on body composition and central appetite regulation in high fat fed mice.

Methodology/Principal Findings

Thirty six C57BL/6 male mice were randomized and maintained for 8 weeks on a high fat diet containing 0% (w/w) fermentable carbohydrate, 10% (w/w) inulin or 10% (w/w) β-glucan individually. Fecal and cecal microbial changes were measured using fluorescent in situ hybridization, fecal metabolic profiling was obtained by proton nuclear magnetic resonance (¹H NMR), colonic short chain fatty acids were measured by gas chromatography, body composition and hypothalamic neuronal activation were measured using magnetic resonance imaging (MRI) and manganese enhanced MRI (MEMRI), respectively, PYY (peptide YY) concentration was determined by radioimmunoassay, adipocyte cell size and number were also measured. Both inulin and β-glucan fed groups revealed significantly lower cumulative body weight gain compared with high fat controls. Energy intake was significantly lower in β-glucan than inulin fed mice, with the latter having the greatest effect on total adipose tissue content. Both groups also showed an increase in the numbers of Bifidobacterium and Lactobacillus-Enterococcus in cecal contents as well as feces. β- glucan appeared to have marked effects on suppressing MEMRI associated neuronal signals in the arcuate nucleus, ventromedial hypothalamus, paraventricular nucleus, periventricular nucleus and the nucleus of the tractus solitarius, suggesting a satiated state.

Conclusions/Significance

Although both fermentable carbohydrates are protective against increased body weight gain, the lower body fat content induced by inulin may be metabolically advantageous. β-glucan appears to suppress neuronal activity in the hypothalamic appetite centers. Differential effects of fermentable carbohydrates open new possibilities for nutritionally targeting appetite regulation and body composition.     

Intervening with urinary tract infections using anti-adhesives based on the crystal structure of the FimH-oligomannose-3 complex

Background

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and α3β1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.

Methodology/Principal Findings

We demonstrate that α-d-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl α-d-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl α-d-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl α-d-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Manα1,3Manβ1,4GlcNAcβ1,4GlcNAc in an extended binding site. The interactions along the α1,3 glycosidic bond and the first β1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl α-d-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.

Conclusions/Significance

The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.     

Fission Yeast Rgf2p Is a Rho1p Guanine Nucleotide Exchange Factor Required for Spore Wall Maturation and for the Maintenance of Cell Integrity in the Absence of Rgf1p

Schizosaccharomyces pombe Rho1p is essential, directly activates β-1,3-glucan synthase, and participates in the regulation of morphogenesis. In S. pombe, Rho1p is activated by at least three guanine nucleotide exchange factors (GEFs): Rgf1p, Rgf2p, and Rgf3p. In this study we show that Rgf2p is a Rho1p GEF required for sporulation. The rgf2⁺ deletion did not affect forespore membrane formation and the nuclei were encapsulated properly. However, the mutant ascospores appeared dark and immature. The rgf2Δ zygotes were not able to release the ascospores spontaneously, and the germination efficiency was greatly reduced compared to wild-type (wt) spores. This phenotype resembles that of the mutants in bgs2⁺, which encodes a sporulation-specific glucan synthase subunit. In fact, glucan synthase activity was diminished in sporulating rgf2Δ diploids. Rgf2p also plays a role in β-glucan biosynthesis during vegetative growth. Overexpression of rgf2⁺ specifically increased GTP-bound Rho1p, caused changes in cell morphology, and elicited an increase in β-1,3-glucan synthase activity. Moreover, the simultaneous disruption of rgf1⁺ and rgf2⁺ was lethal and both Rgf1p and Rgf2p were able to partially substitute for each other. Our results suggest that Rgf1p and Rgf2p are alternative GEFs with an essential overlapping function in Rho1p activation during vegetative growth.     

The Effects of Orally Administered Beta-Glucan on Innate Immune Responses in Humans,a Randomized Open-Label Intervention Pilot-Study

Rationale

To prevent or combat infection, increasing the effectiveness of the immune response is highly desirable, especially in case of compromised immune system function. However, immunostimulatory therapies are scarce, expensive, and often have unwanted side-effects. β-glucans have been shown to exert immunostimulatory effects in vitro and in vivo in experimental animal models. Oral β-glucan is inexpensive and well-tolerated, and therefore may represent a promising immunostimulatory compound for human use.

Methods

We performed a randomized open-label intervention pilot-study in 15 healthy male volunteers. Subjects were randomized to either the β -glucan (n = 10) or the control group (n = 5). Subjects in the β-glucan group ingested β-glucan 1000 mg once daily for 7 days. Blood was sampled at various time-points to determine β-glucan serum levels, perform ex vivo stimulation of leukocytes, and analyze microbicidal activity.

Results

β-glucan was barely detectable in serum of volunteers at all time-points. Furthermore, neither cytokine production nor microbicidal activity of leukocytes were affected by orally administered β-glucan.

Conclusion

The present study does not support the use of oral β-glucan to enhance innate immune responses in humans.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01727895","term_id":"NCT01727895"}}NCT01727895     

Loss of the Plasma Membrane-Bound Protein Gas1p in Saccharomyces cerevisiae Results in the Release of β1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity

Deletion of GAS1/GGP1/CWH52 results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that gas1Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.     

Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide.