M1 macrophages polarized by crude polysaccharides isolated from Auricularia polytricha exhibit anti-tumor effect on human breast cancer cells.

Breast cancer has been reported to correlate with the infiltration of tumor-associated macrophages (TAMs) or M2-like macrophages in tumor microenvironment (TME) that could promote breast cancer progression. In contrast, M1-like macrophages displayed anti-tumor activity toward cancer. This study was focused on Auricularia polytricha (AP), a cloud ear mushroom, which has been reported for anti-tumor activity and immunomodulation. AP extracts were screened on differentiated THP-1 macrophages (M0). Results demonstrated that water extract (APW) and crude polysaccharides (APW-CP) could upregulate M1-related genes and cytokines production (IL-6, IL-1 ß and TNF-α) significantly. Moreover, APW and APW-CP showed a high expression of CD86 (M1 marker) compared to M0. The NF-κB signaling pathway is crucial for pro-inflammatory gene regulation. The APW and APW-CP treatment showed the induction of the NF-κB pathway in a dose-dependent manner, which related to the ß-glucan content in the extracts. Furthermore, APW-CP polarized macrophages were investigated for anti-tumor activity on human breast cancer cells (MCF-7 and MDA-MB-231). Results showed that APW-CP could inhibit the invasion of breast cancer cells and induce apoptosis. Therefore, M1 macrophages polarized by APW-CP showed anti-tumor activity against the breast cancer cells and ß-glucan may be the potential M1-phenotype inducer.

Creating a zero amylose barley with high soluble sugar content by genome editing.

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and ß-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and ß-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to ß-glucans.

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to ß-1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Alpha1-antitrypsin impacts innate host-pathogen interactions with Candida albicans by stimulating fungal filamentation.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic ß-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

Exploring Aegilops caudata: A Comprehensive Study of the CslF6 Gene and ß-Glucan.

In the quest for sustainable and nutritious food sources, exploration of ancient grains and wild relatives of cultivated cereals has gained attention. Aegilops caudata, a wild wheatgrass species, stands out as a promising genetic resource due to its potential for crop enhancement and intriguing nutritional properties. This manuscript investigates the CslF6 gene sequence and protein structure of Aegilops caudata, employing comparative analysis with other grass species to identify potential differences impacting ß-glucan content. The study involves comprehensive isolation and characterization of the CslF6 gene in Ae. caudata, utilizing genomic sequence analysis, protein structure prediction, and comparative genomics. Comparisons with sequences from diverse monocots reveal evolutionary relationships, highlighting high identities with wheat genomes. Specific amino acid motifs in the CslF6 enzyme sequence, particularly those proximal to key catalytic motifs, exhibit variations among monocot species. These differences likely contribute to alterations in ß-glucan composition, notably impacting the DP3:DP4 ratio, which is crucial for understanding and modulating the final ß-glucan content. The study positions Ae. caudata uniquely within the evolutionary landscape of CslF6 among monocots, suggesting potential genetic divergence or unique functional adaptations within this species. Overall, this investigation enriches our understanding of ß-glucan biosynthesis, shedding light on the role of specific amino acid residues in modulating enzymatic activity and polysaccharide composition.

Structure-function analysis of the cyclic ß-1,2-glucan synthase from Agrobacterium tumefaciens.

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic ß-1,2-glucan (CßG) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). CßG is produced by the cyclic glucan synthase (Cgs), a multi-domain membrane protein. So far, its structure as well as the mechanism underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We determine the structure of this complex protein machinery and clarify key aspects of CßG synthesis, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain. Our research opens possibilities for combating pathogens that rely on polysaccharide virulence factors and may lead to synthetic biology approaches for producing complex cyclic sugars.

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii ß-glucans.

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. ß-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. ß-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae ß-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal ß-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

How do the carbon and nitrogen sources affect the synthesis of ß-(1,3/1,6)-glucan, its structure and the susceptibility of Candida utilis yeast cells to immunolabelling with ß-(1,3)-glucan monoclonal antibodies?

BACKGROUND: The need to limit antibiotic therapy due to the spreading resistance of pathogenic microorganisms to these medicinal substances stimulates research on new therapeutic agents, including the treatment and prevention of animal diseases. This is one of the goals of the European Green Deal and the Farm-To-Fork strategy. Yeast biomass with an appropriate composition and exposure of cell wall polysaccharides could constitute a functional feed additive in precision animal nutrition, naturally stimulating the immune system to fight infections. RESULTS: The results of the research carried out in this study showed that the composition of Candida utilis ATCC 9950 yeast biomass differed depending on growth medium, considering especially the content of ß-(1,3/1,6)-glucan, α-glucan, and trehalose. The highest ß-(1,3/1,6)-glucan content was observed after cultivation in deproteinated potato juice water (DPJW) as a nitrogen source and glycerol as a carbon source. Isolation of the polysaccharide from yeast biomass confirmed the highest yield of ß-(1,3/1,6)-glucan after cultivation in indicated medium. The differences in the susceptibility of ß-(1,3)-glucan localized in cells to interaction with specific ß-(1,3)-glucan antibody was noted depending on the culture conditions. The polymer in cells from the DPJW supplemented with glycerol and galactose were labelled with monoclonal antibodies with highest intensity, interestingly being less susceptible to such an interaction after cell multiplication in medium with glycerol as carbon source and yeast extract plus peptone as a nitrogen source. CONCLUSIONS: Obtained results confirmed differences in the structure of the ß-(1,3/1,6)-glucan polymers considering side-chain length and branching frequency, as well as in quantity of ß-(1,3)- and ß-(1,6)-chains, however, no visible relationship was observed between the structural characteristics of the isolated polymers and its susceptibility to immunolabeling in whole cells. Presumably, other outer surface components and molecules can mask, shield, protect, or hide epitopes from antibodies. ß-(1,3)-Glucan was more intensely recognized by monoclonal antibody in cells with lower trehalose and glycogen content. This suggests the need to cultivate yeast biomass under appropriate conditions to fulfil possible therapeutic functions. However, our in vitro findings should be confirmed in further studies using tissue or animal models.

(1 → 3),(1 → 6) and (1 → 3)-ß-D-glucan physico-chemical features drive their fermentation profile by the human gut microbiota.

Mushroom polysaccharides consist of a unique set of polymers that arrive intact in the human large intestine becoming available for fermentation by resident gut bacteria with potential benefits to the host. Here we have obtained four glucans from two mushrooms (Pholiota nameko and Pleurotus pulmonarius) under different extraction conditions and their fermentation profile by human gut bacteria in vitro was evaluated. These glucans were isolated and characterized as (1 â†’ 3),(1 â†’ 6)-ß-D-glucans varying in branching pattern and water-solubility. An aliquot of each (1 â†’ 3),(1 â†’ 6)-ß-D-glucan was subjected to controlled smith degradation process in order to obtain a linear (1 â†’ 3)-ß-D-glucan from each fraction. The four ß-D-glucans demonstrated different water solubilities and molar mass ranging from 2.2 × 105 g.mol-1 to 1.9 × 106 g.mol-1. In vitro fermentation of the glucans by human gut microbiota showed they induced different short chain fatty acid production (52.0-97.0 mM/50 mg carbohydrates), but an overall consistent high propionate amount (28.5-30.3 % of total short chain fatty acids produced). All glucans promoted Bacteroides uniformis, whereas Anaerostipes sp. and Bacteroides ovatus promotion was strongly driven by the ß-D-glucans solubility and/or branching pattern, highlighting the importance of ß-D-glucan discrete structures to their fermentation by the human gut microbiota.

ß-Glucans obtained from fungus for wound healing: A review.

The cell surface of fungus contains a large number of ß-glucans, which exhibit various biological activities such as immunomodulatory, anti-inflammatory, and antioxidation. Fungal ß-glucans with highly branched structure show great potential as wound healing reagents, because they can stimulate the expression of many immune- and inflammatory-related factors beneficial to wound healing. Recently, the wound healing ability of many fungal ß-glucans have been investigated in animals and clinical trials. Studies have proved that fungal ß-glucans can promote fibroblasts proliferation, collagen deposition, angiogenesis, and macrophage infiltration during the wound healing process. However, the development of fungal ß-glucans as wound healing reagents is not systematically reviewed till now. This review discusses the wound healing studies of ß-glucans obtained from different fungal species. The structure characteristics, extraction methods, and biological functions of fungal ß-glucans with wound healing ability are summarized. Researches about fungal ß-glucan-containing biomaterials and structurally modified ß-glucans for wound healing are also involved.

Suppression of Drosophila antifungal immunity by a parasite effector via blocking GNBP3 and GNBP-like 3, the dual receptors for ß-glucans.

The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can bind to ß-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease cascade, Spätzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.

Dectin-1 ligands produce distinct training phenotypes in human monocytes through differential activation of signaling networks.

Cells of the innate immune system retain memory of prior exposures through a process known as innate immune training. ß-glucan, a Dectin-1 ligand purified from the Candida albicans cell wall, has been one of the most widely utilized ligands for inducing innate immune training. However, many Dectin-1 ligands exist, and it is not known whether these all produce the same phenotype. Using a well-established in vitro model of innate immune training, we compared two commercially available Dectin-1 agonists, zymosan and depleted zymosan, with the gold standard ß-glucan in the literature. We found that depleted zymosan, a ß-glucan purified from Saccharomyces cerevisiae cell wall through alkali treatment, produced near identical effects as C. albicans ß-glucan. However, untreated zymosan produced a distinct training effect from ß-glucans at both the transcript and cytokine level. Training with zymosan diminished, rather than potentiated, induction of cytokines such as TNF and IL-6. Zymosan activated NFκB and AP-1 transcription factors more strongly than ß-glucans. The addition of the toll-like receptor (TLR) ligand Pam3CSK4 was sufficient to convert the training effect of ß-glucans to a phenotype resembling zymosan. We conclude that differential activation of TLR signaling pathways determines the phenotype of innate immune training induced by Dectin-1 ligands.

ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Characterization of the Pneumocystis jirovecii and Pneumocystis murina phosphoglucomutases (Pgm2s): a potential target for Pneumocystis therapy.

Pneumocystis cyst life forms contain abundant ß-glucan carbohydrates, synthesized using ß-1,3 and ß-1,6 glucan synthase enzymes and the donor uridine diphosphate (UDP)-glucose. In yeast, phosphoglucomutase (PGM) plays a crucial role in carbohydrate metabolism by interconverting glucose 1-phosphate and glucose 6-phosphate, a vital step in UDP pools for ß-glucan cell wall formation. This pathway has not yet been defined in Pneumocystis. Herein, we surveyed the Pneumocystis jirovecii and Pneumocystis murina genomes, which predicted a homolog of the Saccharomyces cerevisiae major PGM enzyme. Furthermore, we show that PjPgm2p and PmPgm2p function similarly to the yeast counterpart. When both Pneumocystis pgm2 homologs are heterologously expressed in S. cerevisiae pgm2Δ cells, both genes can restore growth and sedimentation rates to wild-type levels. Additionally, we demonstrate that yeast pgm2Δ cell lysates expressing the two Pneumocystis pgm2 transcripts individually can restore PGM activities significantly altered in the yeast pgm2Δ strain. The addition of lithium, a competitive inhibitor of yeast PGM activity, significantly reduces PGM activity. Next, we tested the effects of lithium on P. murina viability ex vivo and found the compound displays significant anti-Pneumocystis activity. Finally, we demonstrate that a para-aryl derivative (ISFP10) with known inhibitory activity against the Aspergillus fumigatus PGM protein and exhibiting 50-fold selectivity over the human PGM enzyme homolog can also significantly reduce Pmpgm2 activity in vitro. Collectively, our data genetically and functionally validate phosphoglucomutases in both P. jirovecii and P. murina and suggest the potential of this protein as a selective therapeutic target for individuals with Pneumocystis pneumonia.

Regulation of baking quality and starch digestibility in whole wheat bread based on ß-glucans and protein addition strategy: Significance of protein-starch-water interaction in dough.

Whole wheat bread has high nutritional value but is characterized by inferior quality and a high glycemic index. Studies have shown that adding ß-glucans and protein can improve bread quality. This study investigated the effects of added oat ß-glucan, barley ß-glucan, or yeast ß-glucan on protein synergy and whole wheat dough and bread quality. The mixing properties, rheological properties, and scanning electron microscopy observations showed that the addition of ß-glucan promoted the formation of gluten networks, while the synergy between the wheat proteins and ß-glucan resulted in a more robust and stable gluten network and a stronger physical starch envelope. Rapid visco-analysis and thermal property evaluations showed that ß-glucan addition inhibited the thermal degradation, gelatinization, and retrogradation of starch. Based on the bread quality results, it was found the ß-glucan could cause some damage to the bread baking quality. For example, the hardness of samples with oats, barley, and yeast increased to 881.69 g, 952.97 g, and 631.75 g, respectively, compared to samples without ß-glucan (317.49 g), whereas the inclusion of yeast ß-glucan proved to be less detrimental. Protein and ß-glucan both reduced starch digestion to some degree, and showed better synergistic effects, with the lowest estimated glycemic index of 70.08 observed in bread containing added yeast ß-glucan and protein. Therefore, yeast ß-glucan and protein mixtures could be selected as viable formulations for enhancing the quality of whole wheat bread.

Anti-inflammatory activity of ß-glucans from different sources before and after fermentation by fecal bacteria in vitro.

BACKGROUND: ß-Glucans are widely sourced and have various physiological effects, including anti-inflammatory effects. However, the strength of the anti-inflammatory activity of ß-glucans from different sources remains unknown due to the lack of rapid and effective biomarkers. This study therefore aimed to screen out the ß-glucans with strong anti-inflammatory activity from five different sources and to further screen out possible biomarkers in metabolites after fermenting the ß-glucans with gut microorganisms. RESULTS: The results showed that all five ß-glucans inhibited the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators and suppressed the mRNA expression level of TLR4/MyD88. Their anti-inflammatory mechanisms involved the inhibition of intracellular reactive oxygen species (ROS) production and suppression of mRNA expression of the NF-κB pathway and JNK pathway. Among them, barley ß-glucan exhibited the strongest anti-inflammatory effect, followed by Ganoderma lucidum ß-glucan. Enhanced anti-inflammatory activity of ß-glucan was found after fermentation and may be related to the increased abundance of metabolites such as vanillin, dihydroxyphenylacetic acid, caffeic acid, acetic acid, butyric acid, and lactic acid. They were strongly positively correlated to the abundance of beneficial bacteria such as Blautia, suggesting that the production of those metabolites may be responsible for the flourishing of the beneficial bacteria. CONCLUSION: In conclusion, barley was a preferred raw material for the preparation of ß-glucans with strong anti-inflammatory activity. Vanillin, dihydroxyphenylacetic acid, caffeic acid, acetic acid, butyric acid, and lactic acid were the possible biomarkers that could be utilized to evaluate the anti-inflammatory effect of ß-glucans. © 2023 Society of Chemical Industry.

Obligatory role of microglia-mobilized hippocampal CREB-BDNF signaling in the prophylactic effect of ß-glucan on chronic stress-induced depression-like behaviors in mice.

Our previous studies have reported that pre-stimulation of microglia before stress stimulation is a possible strategy to prevent depression-like phenotypes; however, the molecular mechanisms underlying this effect are still unclear. Here, we used ß-glucan, a polysaccharide from Saccharomyces cerevisiae with immunomodulatory activities that cannot elicit pro-inflammatory responses in microglia, to address this issue. Our results showed that a single injection of ß-glucan one day before stress exposure dose-dependently prevented the depression-like behaviors triggered by chronic unpredictable stress (CUS), which peaked at 20 mg/kg and prevented the impairment of hippocampal brain-derived neurotrophic factor (BDNF) signaling, a pathological process critical for the progression of depression-like phenotypes. Inhibition of BDNF signaling by infusion of an anti-BDNF antibody into the hippocampus, knock-in of the mutant BDNF Val68Met allele, or blockade of the BDNF receptor in the hippocampus abolished the preventive effect of ß-glucan on CUS-induced depression-like behaviors. Further analysis showed that cAMP-response element binding protein (CREB)-mediated increase of BDNF expression in the hippocampus was essential for the prevention of depression-like phenotypes by ß-glucan. Pretreatment with minocycline or PLX3397 before ß-glucan injection to suppress microglia abolished the preventive effect of ß-glucan on impaired CREB-BDNF signaling in the hippocampus and depression-like behaviors in CUS mice. These results suggest that an increase in hippocampal BDNF following CREB activation triggered by ß-glucan-induced microglia stimulation and subsequent TrkB signaling mediates the preventive effect of ß-glucan on depression. ß-Glucan may be a more suitable immunostimulant for the prevention of depression due to its inability to promote pro-inflammatory responses in microglia.

Characterization of an antifungal ß-1,3-glucanase from Ficus microcarpa latex and comparison of plant and bacterial ß-1,3-glucanases for fungal cell wall ß-glucan degradation.

MAIN CONCLUSION: Each ß-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of ß-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and ß-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal ß-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall ß-glucan, it was compared with ß-1,3-glucanase with different substrate specificities. We obtained recombinant ß-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I ß-1,3-glucanase) and CcGluA (GH64 ß-1,3-glucanase).

Identification of 10 genes on Candida albicans chromosome 5 that control surface exposure of the immunogenic cell wall epitope ß-glucan and cell wall remodeling in caspofungin-adapted mutants.

IMPORTANCE: Candida infections are often fatal in immuno-compromised individuals, resulting in many thousands of deaths per year. Caspofungin has proven to be an excellent anti-Candida drug and is now the frontline treatment for infections. However, as expected, the number of resistant cases is increasing; therefore, new treatment modalities are needed. We are determining metabolic pathways leading to decreased drug susceptibility in order to identify mechanisms facilitating evolution of clinical resistance. This study expands the understanding of genes that modulate drug susceptibility and reveals new targets for the development of novel antifungal drugs.

A GH81-type ß-glucan-binding protein enhances colonization by mutualistic fungi in barley.

Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of ß-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated ß-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a ß-glucan interactor. Mutation of GBP1 and its only paralog, GBP2, in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, GBP mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen Bipolaris sorokiniana and inhibited the penetration success of the obligate biotrophic leaf pathogen Blumeria hordei. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi-a role that has potentially been appropriated by barley-adapted pathogens.