Biochemical characterization and antifungal activity of a recombinant ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659.

ß-1,3-glucanases can degrade ß-1,3-glucoside bonds in ß-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the ß-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-ß-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Effects of ß-1,3-glucan on growth, immune responses, and intestinal microflora of the river prawn (Macrobrachium nipponense) and its resistance against Vibrio parahaemolyticus.

In this study, we investigated the impact of ß-1,3-glucan on the immune responses and gut microbiota of the river prawn (Macrobrachium nipponense) in the presence of Vibrio parahaemolyticus stress. Shrimps were fed one of the following diets: control (G1), 0.2% curdlan (G2), 0.1% ß-1,3-glucan (G3), 0.2% ß-1,3-glucan (G4), or 1.0% ß-1,3-glucan (G5) for 6 weeks and then challenged with V. parahaemolyticus for 96 h. Under Vibrio stress, shrimps in G4 exhibited the highest length gain rate, weight gain rate, and survival rate. They also showed increased intestinal muscle thickness and villus thickness compared to the control and 0.2% curdlan groups. The apoptosis rate was lower in G4 than in the control group, and the digestive enzyme activities (pepsin, trypsin, amylase, and lipase), immune enzyme activities (acid phosphatase, alkaline phosphatase, lysozyme, and phenoxidase), and energy metabolism (triglyceride, cholesterol, glycogen, and lactate dehydrogenase) were enhanced. Expression levels of growth-related genes (ecdysone receptor, calmodulin-dependent protein kinase I, chitin synthase, and retinoid X receptor) and immune-related genes (toll-like receptor 3, myeloid differentiation primary response 88, mitogen-activated protein kinase 7, and mitogen-activated protein kinase 14) were higher in G4 than in the control. Microbiota analysis indicated higher bacterial abundance in shrimps fed ß-1,3-glucan, as evidenced by Sob, Chao1, and ACE indices. Moreover, 0.2% ß-1,3-glucan increased the relative abundances of Bacteroidota and Firmicutes while reducing those of Corynebacteriales and Lactobacillales. In summary, ß-1,3-glucan enhances immune enzyme activities, alters immune-related gene expression, and impacts gut microbial diversity in shrimp. These findings provide valuable insights into the mechanisms underlying ß-1,3 glucan's immune-enhancing effects.

Effects of dietary soluble ß-1,3-glucan on the growth performance, antioxidant status, and immune response of the river prawn (Macrobrachium nipponense).

The effects of dietary ß-1,3-glucan on the growth performance, body composition, hepatopancreas tissue structure, antioxidant activities, and immune response of the river prawn (Macrobrachium nipponense) were investigated. In total, 900 juvenile prawns were fed one of five diets with different contents of ß-1,3-glucan (0%, 0.1%, 0.2%, and 1.0%) or 0.2% curdlan for 6 weeks. The growth rate, weight gain rate, specific growth rate, specific weight gain rate, condition factor, and hepatosomatic index of juvenile prawns fed 0.2% ß-1,3-glucan were significantly higher than those fed 0% ß-1,3-glucan and 0.2% curdlan (p < 0.05). The whole-body crude lipid content of prawns supplemented with curdlan and ß-1,3-glucan was significantly higher than that of the control group (p < 0.05). The antioxidant and immune enzyme activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), catalase (CAT), lysozyme (LZM), phenoloxidase (PO), acid phosphatase (ACP), and alkaline phosphatase (AKP) in the hepatopancreas of juvenile prawns fed 0.2% ß-1,3-glucan were significantly higher than those of the control and 0.2% curdlan groups (p < 0.05), and tended to increase and then decrease with increasing dietary ß-1,3-glucan. The highest malondialdehyde (MDA) content was observed in juvenile prawns without ß-1,3-glucan supplementation. The results of real-time quantitative PCR indicated that dietary ß-1,3-glucan promoted expression of antioxidant and immune-related genes. Binomial fit analysis of weight gain rate and specific weight gain rate showed that the optimum ß-1,3-glucan requirement of juvenile prawns was 0.550%-0.553%. We found that suitable dietary ß-1,3-glucan improved juvenile prawns growth performance, antioxidant capacity, and non-specific immunity, which provide reference for shrimp healthy culture.

ß-1,3-glucan improved the health and immunity of juvenile African catfish (Clarias gariepinus) and neutralized the histological changes caused by lead and fipronil pollutants.

BACKGROUND: Water pollutants cause adverse effects in aquatic ecosystems. The immunomodulatory and mitigating effects of dietary 1,3-glucan on fipronil and lead-induced intoxication in African catfish (Clarias gariepinus) were investigated. Two hundred forty catfish were randomly divided into four equal groups: those in the first group were fed basic diet and served as controls; those in the second group were supplemented with ß-1,3-glucan (0.1%); those in the third group were exposed to combination of lead nitrate at 0.041 mg/L (1/10 96 h LC50) and fipronil at 2.8 mg/l (1/10 96 h LC50); and those in the fourth group were exposed to combination of fipronil, lead, and ß-1,3-glucan. The health status, haematological, immunological, and histological changes were all evaluated. RESULT: Swelling on the dorsolateral side, spinal column deviation, sluggish movement, skin bleaching, excessive mucus secretion, significant variations in blood indices-related measures, and a 45% death rate were observed in the third group. There was a significant reduction in interleukin-1 (IL-1) and interleukin-6 (IL-6) and immunoglobulin M (IgM) concentrations, as well as decrease in their corresponding gene expression, indicating that fipronil and lead had immunosuppressive activity. Severe catarrhal enteritis and mucinous degeneration of the lining epithelium, and notable depletion of white pulp, congested red pulp and hemosiderosis were common pathological findings in the spleen. ß-1,3-glucan alone or in combination with fipronil and lead provoked physical activity, blood indices, with elevations in IL-1ß, IL-2, IL-6, and IgM concentrations, as well as up-regulation in their genes' expression in splenic tissues, when compared to the third group. The spleen and intestine had normal histological architecture with 5% mortalities. There were no fish deaths in the ß-1,3-glucan-alone or control groups. CONCLUSION: The use of ß-1,3-glucan (0.1%) as dietary supplement could be implemented to protect against the toxic effects of fipronil and lead toxicity by improving the health and immunological parameters of intoxicated catfish.

Poacic acid, a ß-1,3-glucan-binding antifungal agent, inhibits cell-wall remodeling and activates transcriptional responses regulated by the cell-wall integrity and high-osmolarity glycerol pathways in yeast.

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits ß-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to ß-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on ß-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting ß-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.

Molecular properties and ligand specificity of zebrafish intelectin-2.

Intelectins are immune lectins expressed in chordates, including several fish species, in which intelectins are known to be upregulated upon infection. However, the basic biochemical properties and bacteria binding specificities of several fish intelectins are not well studied. We focus our investigation on zebrafish intelectin-2 (DrIntL-2) that is predominantly expressed in the gastrointestinal tract. The disulfide-linked oligomeric state and the cysteine responsible for intermolecular disulfide bonds are identified. DrIntL-2 is a globular particle of around 30 nm. In addition to the typical exocyclic 1,2-diol ligands, DrIntL-2 binds ß-1,3-glucan and recognizes Salmonella typhimurium and Pseudomonas aeruginosa. This investigation not only shed light on the fish innate immunity that will be essential for the aquaculture industry, but will also provide a foundation for further application of DrIntL-2 in bacteria detection and identification.

ß-1,3-Glucan/chitin unmasking in the Saccharomyces cerevisiae mutant, Δmnn9, promotes immune response and resistance of the Pacific oyster (Crassostrea gigas) to Vibrio coralliilyticus infection.

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component ß-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, ß-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of ß-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for ß-glucan-binding protein (CgßGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of ß-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to ß-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgßGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked ß-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding.

Ibrexafungerp: An orally active ß-1,3-glucan synthesis inhibitor.

We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious ß-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.

Reduced vacuolar ß-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.

The ß-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative ß-1,3-glucan synthase (PtBGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that PtBGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of PtBGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in PtBGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.

Effect of Vitamin C/Hydrocortisone Immobilization within Curdlan-Based Wound Dressings on In Vitro Cellular Response in Context of the Management of Chronic and Burn Wounds.

Bioactive dressings are usually produced using natural or synthetic polymers. Recently, special attention has been paid to ß-glucans that act as immunomodulators and have pro-healing properties. The aim of this research was to use ß-1,3-glucan (curdlan) as a base for the production of bioactive dressing materials (curdlan/agarose and curdlan/chitosan) that were additionally enriched with vitamin C and/or hydrocortisone to improve healing of chronic and burn wounds. The secondary goal of the study was to compressively evaluate biological properties of the biomaterials. In this work, it was shown that vitamin C/hydrocortisone-enriched biomaterials exhibited faster vitamin C release profile than hydrocortisone. Consecutive release of the drugs is a desired phenomenon since it protects wounds against accumulation of high and toxic concentrations of the bioactive molecules. Moreover, biomaterials showed gradual release of low doses of the hydrocortisone, which is beneficial during management of burn wounds with hypergranulation tissue. Among all tested variants of biomaterials, dressing materials enriched with hydrocortisone and a mixture of vitamin C/hydrocortisone showed the best therapeutic potential since they had the ability to significantly reduce MMP-2 synthesis by macrophages and increase TGF-ß1 release by skin cells. Moreover, materials containing hydrocortisone and its blend with vitamin C stimulated type I collagen deposition by fibroblasts and positively affected their migration and proliferation. Results of the experiments clearly showed that the developed biomaterials enriched with bioactive agents may be promising dressings for the management of non-healing chronic and burn wounds.