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Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C‐type lectin receptor‐like kinase 1 (CLRLK1) in the aspect of PD callose‐modulated symplasmic continuity. Here, we found that loss‐of‐function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine‐tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis. Summary statement: In the current research article, we have identified a novel C‐TYPE LECTIN RECEPTOR‐LIKE KINASE 1 (CLRLK1) in Arabidopsis, which modulates symplasmic connectivity and gravitropic response via plasmodesmata callose regulation. And this modulation might recruit an actin‐depolymerizing factor 3 (ADF3) function. [ABSTRACT FROM AUTHOR]

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti- Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δ ags1 Δ gtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δ ags1 Δ gtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δ ags1 Δ ags2 Δ ags3 Δ gtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti- Aspergillus agents. [ABSTRACT FROM AUTHOR]

Iron affects the diversity of the oral microbial landscape. Laboratory-strain CAI4 of Candida albicans that causes oropharyngeal candidiasis (OPC) exhibits iron-induced changes to the cell wall, impacting phagocytosis (by macrophages) and susceptibility of fungal cells to cell wall-perturbing antifungals, in vitro. To understand the effect of iron on the CAI4-strain, wild type (WT) SC5314-strain, and oral isolates of C. albicans. An immunosuppressed murine model of OPC was used to assess the effect of iron on oral-to-gut infection and antifungal susceptibility of the CAI4-strain. In vitro antifungal susceptibility, cell wall analysis, and phagocytic assays were performed under low and high iron, for the SC5314-strain and oral isolates. High iron enhanced oral and gut fungal levels for the CAI4-strain in mice; CAI4 cells from low iron mice were more susceptible to antifungals. The SC5314-strain and oral isolates showed enhanced antifungal-resistance towards most antifungals tested, under high iron. Iron-mediated cell wall changes and phagocytic response in the SC5315-strain were similar to CAI4; oral isolates showed a variable response. Host iron can potentially alter infection severity and dissemination, efficacy of antifungal treatment, and host immune response during OPC. Clinical isolates showed most of these effects of iron, despite exhibiting a varied cell wall composition-change response to iron. [ABSTRACT FROM AUTHOR]

Natural products serve as a valuable resource in drug discovery and the identification of bioactive molecules in the field of epimedicine, which targets epigenetic regulator enzymes through epidrugs. In this study, β-1,3-glucan (BG), a natural storage polysaccharide in Euglena gracilis, a well-known immunostimulatory agent, is propounded as a promising epidrug. To elucidate the therapeutic efficacy of BG against ovarian cancer, the molecular interactions between BG and epigenetic regulators, Protein Arginine Methyltransferases (PRMTs) and Sirtuins (SIRTs) were investigated using computational methods followed by in vitro gene expression studies in SKOV-3 ovarian cancer cell line. The binding energies of PRMT5 and SIRT5 against BG were observed as -65.5 and -68.2 kcal/mol, respectively. The in vitro cytotoxic effects of BG against human ovarian cancer cell line, SKOV-3 showed an IC 50 of 150 µg/mL at 48 h. Significant epigenetic modifications were observed to be influenced by BG which increased the gene expression of PRMT5, SIRT5 and Nrf2 to 0.3, 0.5, and 0.7 fold-change respectively, while the Nrf1/2 plasmid showed reduced reporter activity by 29%. Collectively, both in silico and in vitro studies provided valuable insights into the epigenetic regulation of PRMT5 and SIRT5 by BG via Nrf1/2. Nonetheless, further preclinical and clinical investigations are essential to validate the therapeutic properties of BG as an epidrug against ovarian cancer.

GH16 chitin transferases, GH17 β-1,3-glucan transferases, and GH72 β-1,3-glucan/lichenin transferases are important fungal cell wall crosslinking enzymes. The Neurospora crassa genome encodes three genes from the GH17 gene family and five members in the GH16 subfamily 18 and 19 fungal chitin transferases. We created deletion mutants lacking all three GH17 genes and determined that they had wild type morphology and are more sensitive to cell wall perturbation reagents than the wild type. We also created deletion mutants lacking all five GH16 subfamily 18 and 19 genes and found that they had wild type morphology and are more sensitive to cell wall perturbation reagents than the wild type. We conclude that GH16 and GH17 enzymes play roles in cell wall biogenesis. In N. crassa, GH72 enzymes have been reported to be lichenin transferases, while in other fungi they have been shown to be the β-1,3-glucan transferases. Neurospora triple GH72 deletions give rise to a tight colonial morphology, sensitivity to cell wall perturbation reagents, and release of cell wall proteins into the medium. To ask if GH72 and GH17 enzymes might be redundant in N. crassa, we created sextuple mutants lacking the three GH72 genes and the three GH17 genes and found that they were indistinguishable from the GH72 triple mutant. We also found that a recombinant GH72 enzyme is able to form a lichenin-enzyme intermediate demonstrating that GH72 enzymes are lichenin transferases. The N. crassa GH72 enzymes are lichenin transferases and are not redundant with the GH17 β-1,3-glucan transferases.

Patulin (PAT) contaminant in apple juice has attracted extensive attention. As a biosorbent, β -1,3-D-glucan shows great advantages in removing toxic contaminants. The aim of this study was to reveal the adsorption mechanisms of PAT by β -1,3-D-glucan. Four β -1,3-D-glucans with different number of side chains: curdlan, a hypothetical glucan, schizophyllan (SPG) and lentinan were simulated. Performance of molecular docking was observed to predict the binding mode and affinity. The most potential compounds were performed molecular dynamics (MD) simulation. The results show that the interaction between β -1,3-D-glucan and PAT is driven by steric complementarity, intermolecular H-bond and Pi–Sigma interaction. SPG has a large internal helical space to facilitate the insertion of PAT into the helix, and has relatively strong interaction with PAT. The side and main chains form a cavity to wrap PAT inside the helix. Furthermore, the binding free energy calculation was carried out using Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) approach. SPG showed high negative Δ G bind value. Structural and energetic analyses demonstrated the adsorption characteristics of the four β -1,3-D-glucans. SPG is the most promising adsorbent for PAT. The side chain plays a major role in the adsorption process of the β -1,3-D-glucans. It provides a basis for the development of β -1,3-D-glucan as PAT adsorbent. The binding modes between the four β-1,3-D-glucans and patulin were investigated using molecular docking and MD simulation. The results show that the interaction between β-1,3-D-glucan and PAT is driven by steric complementarity, intermolecular H-bond and Pi-Sigma interaction. SPG has a large internal helical space to facilitate the insertion of PAT into the helix, and shows high negative ΔGbind value. [ABSTRACT FROM AUTHOR]

Copyright of China Brewing is the property of China Brewing Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

ABSTRACT Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

Complement system and dendritic cells (DCs) form – beside neutrophils and macrophages – the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with Aspergillus fumigatus lacking ß-1,3-glucans (fks1tetOnrep under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking β-glucan synthase, making the A. fumigatus fks1tetOn mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the fks1-deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the A. fumigatus fks1tetOnrep mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that fks1tetOnrep induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DC-SIGN significantly reduced the fks1tetOnrep-mediated induction of an inflammatory cytokine profile. Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of Aspergillus fumigatus by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms. Abbreviations: C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal–regulated kinases; JNK : c-Jun N-terminal kinases; MAPK: mitogen-activated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th :T helper; TLR :Toll-like receptor; WT: wild type.

Invasive fungal infections (IFI) cause considerable morbidity and mortality in pediatric patients. Serum biomarkers such as 1,3-beta-D glucan (BDG) and galactomannan (GM) have been evaluated for the IFI diagnosis. However, most evidence regarding their utility is derived from studies in adult oncology patients. This systematic review aimed to compare the diagnostic accuracy of BDG and GM individually or in combination for diagnosing IFI in pediatric patients. PubMed, CINAHL, Embase, and Cochrane Library were searched until March 2019 for diagnostic studies evaluating both serum GM and BDG for diagnosing pediatric IFI. The pooled diagnostic odds ratio (DOR), specificity and sensitivity were computed. Receiver operating characteristics (ROC) curve and area under the curve (AUC) were used for summarizing overall assay performance. Six studies were included in the meta-analysis. The summary estimates of sensitivity, specificity, pooled DOR, AUC of the GM assay for proven or probable IFI were 0.74, 0.76, 13.25, and 0.845. The summary estimates of sensitivity, specificity, pooled DOR, AUC of the BDG assay were 0.70, 0.69, 4.3, and 0.722. The combined predictive ability of both tests was reported in two studies (sensitivity: 0.67, specificity: 0.877). Four studies were performed in hematology-oncology patients, while two were retrospective studies from pediatric intensive care units (ICUs). In the subgroup of hematology-oncology patients, DOR of BDG remained similar at 4.25 but increased to 40.28 for GM. We conclude that GM and BDG have a modest performance for identifying IFI in pediatric patients. GM has a better accuracy over BDG. Combining both improves the specificity at the cost of sensitivity. [ABSTRACT FROM AUTHOR]

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of β-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii, which had naturally more β-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and β-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells.

Alpha-1,3-glucan, in addition to β-1,3-glucan, is an important polysaccharide component of fungal cell walls. It is reported for many fungal species, including human pathogenic genera: Aspergillus , Blastomyces , Coccidioides , Cryptococcus , Histoplasma and Pneumocystis , plant pathogens, e.g. Magnaporthe oryzae and entomopathogens, e.g. Metarhizium acridum. In human and plant pathogenic fungi, α-1,3-glucan is considered as a shield for the β-1,3-glucan layer preventing recognition of the pathogen by the host. However, its role in induction of immune response is not clear. In the present study, the cellular immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan was investigated for the first time. The changes detected in the total hemocyte count (THC) and differential hemocyte count (DHC), formation of hemocyte aggregates and changes in apolipophorin III localization indicated activation of G. mellonella cellular mechanisms in response to immunization with A. niger α-1,3-glucan. Our results, which have clearly demonstrated the response of the insect immune system to this fungal cell wall component, will help in understanding the α-1,3-glucan role in immune response against fungal pathogens not only in insects but also in mammals, including humans. [ABSTRACT FROM AUTHOR]

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of β-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii , which had naturally more β-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and β-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells. [ABSTRACT FROM AUTHOR]

Complement system and dendritic cells (DCs) form - beside neutrophils and macrophages - the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with Aspergillus fumigatus lacking ß-1,3-glucans (fks1tetOnrep under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking β-glucan synthase, making the A. fumigatus fks1tetOn mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the fks1-deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the A. fumigatus fks1tetOnrep mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that fks1tetOnrep induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DCSIGN significantly reduced the fks1tetOnrep-mediated induction of an inflammatory cytokine profile. Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of Aspergillus fumigatus by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms. Abbreviations: C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal-regulated kinases; JNK: c-Jun N-terminal kinases; MAPK: mitogenactivated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th:T helper; TLR: Toll-like receptor; WT: wild type. [ABSTRACT FROM AUTHOR]

Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan paramylon, is suggested to function as a dietary fiber and enhance immunity. Here, we aimed to investigate the effects of E. gracilis EOD-1 biomass (EOD1BM) ingestion on immunoglobulin A (IgA) antibody titers in saliva, its reactivity, and the health-related quality of life (QOL) in humans. Reacting human immunoglobulin preparations and saliva with paramylon granules revealed the presence of anti-paramylon antibodies in the blood and saliva. We conducted a placebo-controlled, double-blind, crossover study involving 13 healthy subjects who ingested the placebo or EOD1BM for 4 weeks. Saliva was collected from each subject before and after ingestion, and IgA titers and E. gracilis EOD-1 paramylon (EOD1PM) reactivity were compared. In the EOD1BM Ingestion group, the anti-EOD1PM IgA content and titer increased after EOD1BM ingestion. No such change was observed in the Placebo group. Furthermore, the health-related QOL, especially mental health, increased in the EOD1BM Ingestion group. Thus, EOD1BM ingestion led to the production of paramylon (PM)-specific IgA antibody and increased salivary IgA antibody titers. We demonstrate that EOD1BM ingestion enhanced the immunity in the mucosal surface, evoked an antigen-specific response, and increased the health-related QOL, thereby contributing to health improvement. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Southern Agriculture is the property of Journal of Southern Agriculture and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE) or agsB (agsBOE), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE, 41.6 ± 5.8 kDa; agsBOE, 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain–fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans.

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10 g L−1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0 g L−1 hr−1 and 600 rpm, respectively, and at the w-glucan production stage, they were 0.5 g L−1 hr−1 and 400 rpm, respectively. W-glucan production reached 17.31 g L−1, with a degree of polymerization of 19-25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan. [ABSTRACT FROM AUTHOR]

The aim of this study was to discuss the functions of FKS family genes which encode β-1, 3-glucan synthase regarding the viability and autolysis of yeast strain. Loss of FKS1 gene severely influences the viability and anti-autolytic ability of yeast. Mutation of FKS1 and FKS2 genes led to cell reconstruction, resulting in a sharp shrinkage of cell volume and decreased stress resistance, viability, and anti-autolytic ability. Deletion of FKS3 gene did not clearly influence the synthesis of β-1, 3-glucan of yeast but increased the strain’s stress resistance, viability, and anti-autolytic ability. It is suggested that FKS3 would be the potential target for improving the stress resistance of yeast. The results revealed the relationship among FKS family genes and demonstrated their functions on yeast cell wall construction and anti-autolytic ability. [ABSTRACT FROM AUTHOR]

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization. [ABSTRACT FROM AUTHOR]

α-1,3-Glucan, a component of the fungal cell wall, is a refractory polysaccharide for most plants. Previously, we showed that various fungal plant pathogens masked their cell wall surfaces with α-1,3-glucan to evade plant immunity. This surface accumulation of α-1,3-glucan was infection specific, suggesting that plant factors might induce its production in fungi. Through immunofluorescence observations of fungal cell walls, we found that carrot (Daucus carota) extract induced the accumulation of α-1,3-glucan on germlings in Colletotrichum fioriniae, a polyphagous fungal pathogen that causes anthracnose disease in various dicot plants. Bioassay-guided fractionation of carrot leaf extract successfully identified two active substances that caused α-1,3-glucan accumulation in this fungus: lutein, a carotenoid widely distributed in plants, and stigmasterol, a plant-specific membrane component. Lutein, which had a greater effect on C. fioriniae, also induced α-1,3-glucan accumulation in other Colletotrichum species and in the phylogenetically distant rice pathogen Cochliobolus miyabeanus, but not in the rice pathogen Magnaporthe oryzae belonging to the same phylogenetic subclass as Colletotrichum. Our results suggested that fungal plant pathogens reorganize their cell wall components in response to specific plant-derived compounds, which these pathogens may encounter during infection. [ABSTRACT FROM AUTHOR]

Mast cells are primary mediators of allergic inflammation. Beta-1,3- glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 μg/ml BG, 100 mg/ml peptidoglycan (PGN), or 10 μM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation. [ABSTRACT FROM AUTHOR]

1,3-ß-D-glucan ( BDG) is increasingly used to diagnose invasive fungal infections ( IFI), although false positive results are a concern. To evaluate the potential interaction of blood products with the BDG assay, human albumin ( HA), fresh frozen plasma ( FFP), undiluted platelet transfusion ( UPT) and packed red blood cells ( PRBC) were tested for their BDG content using two different b-D-glucan tests. UPTs tested negative, FFP, PBRC and HA tested positive for BDG. In serial dilution, BDG concentration correlated with blood product concentration. To investigate the clinical impact of blood product transfusions, we measured BDG levels before and after the transfusion in three patients (2 PRBC, 1 HA). In the patients receiving PRBC transfusions, BDG values increased from 13 and 17 pg ml−1 to 183 and 361 pg ml−1, the HA transfusion increased the serum level from 42 to 58 pg ml−1. BDG concentrations measured in blood products can be used to predict false positive BDG results. [ABSTRACT FROM AUTHOR]

Purpose: To determine the impact of β-l,3-glucan isolated from Euglena gracilis on corneal epithelial cell migration and on wound healing in a rat alkali burn model. Methods: Immortalized human corneal epithelial cells (HCECs) were cultured in media with 50,100 and 200 μg/ mL laminarin (β-1,3- and β-l,6-glucans), β-l,3-glucan and hyaluronic acid (HA)-conjugated β-l,3-glucan; Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 (negative control) and serum containing DMEM/nutrient mixture F-12 (positive control). Migration assays were conducted via the manual scraping of HCECs. Next, alkali injuries were induced using 1 N NaOH in 40 eyes of 40 Sprague-Dawley male rats. The injury-only group (n = 10) received topical phosphate-buffered saline four times a day for 2d. The study groups received 200 pg/mL topical laminarin (n = 10), β-l,3-glucan (n = 10) and β-l,3-glucan HA (n = 10). Using slit-lamp biomicroscopy, injured corneas were evaluated in terms of re-epithelialization and opacity, and tissue sections were histologically analyzed. Results: Migration assay rates were enhanced as laminarin and β-l,3-glucan increased, compared to negative control cells (all p<0.05). In the comparison between β-l,3-glucan and its HA conjugate form, β-l,3-glucan-HA showed more enhanced migration rate than β-l,3-glucan (p < 0.05). In rat alkali burn model, wound-healing ratio was greatest in β-l,3-glucan-HA groups (96.0±4.1%), followed by β-l,3-glucan (86.0±6.5%), laminarin (67.0 ± 7.5%) and injury-only group (54.0 ±6.5%) (p< 0.0001; ANOVA). The opacity score was also lowest in β-1,3-glucan-HA groups (3.0 ±0.75), followed by β-l,3-glucan (3.4 ±0.5), laminarin (3.7±0.8) and injury-only group (4.7 ±0.46) (p< 0.0001; ANOVA) Histologically, relatively fewer polymorphonuclear leukocytes infiltrated the corneal stroma in the β-1,3-glucan and β-l,3-glucan-HA groups, compared to the injury-only group. Conclusions: β-l,3-Glucan, particularly when conjugated with HA, promoted epithelial wound healing in vitro and suppressed the acute inflammatory reaction in corneal alkali burns. [ABSTRACT FROM AUTHOR]

Expression of immune-related genes in muscle was evaluated in a 84-day feeding trial in white shrimp, Litopenaeus vannamei, fed diets containing 0, 250, 500, or 1000 mg/kg β-1,3-glucan from Laminaria digitata. The transcript abundance of hemocyanin, lipopolysaccharide/b-glucan binding protein (LGBP), penaeidin-3, prophenoloxidase (proPO), serine proteinase, and cytosolic manganese superoxide dismutase (cMnSOD) was quantified by realtime PCR. The dietary inclusion of β-1,3-glucan significantly affected the mRNA expression of all six genes. LGBP, cMnSOD, and hemocyanin were significantly up-regulated by day 14, serine proteinase peaked on day 28, and penaeidin-3 and ProPO peaked on day 56. The dosage of β-1,3-glucan also affected mRNA expression. cMnSOD was significantly higher in shrimp fed the diets containing 250 or 500 mg/kg β-1,3-glucan than in those fed the control. ProPO and penaeidin-3 were significantly higher in shrimp fed the diet containing 500 mg/kg β-1,3-glucan than in those fed the control. LGBP, serine proteinase, and hemocyanin were significantly higher in shrimp fed the 500 or 1000 mg/kg than in those fed the control. Thus, feeding diets with 250, 500, or 1000 mg β-1,3-glucan/kg dry diet can help improve the expression of immune-related genes in L. vannamei. [ABSTRACT FROM AUTHOR]

β-1,3-Glucan with 1.0 g kg−1 was supplemented to a basal diet [control (C)] with different feeding schedules: permanently β-1,3-glucan diet (BG) and 14 days BG-14 days control diet (C + BG). Growth and immunological responses [respiratory burst (RB), superoxide dismutase (SOD), catalase (CAT), lysozyme and total protein] in haemolymph, hepatopancreas and muscle of Litopenaeus vannamei were recorded after 84-day feeding and exposure to nitrite-N (20 mg L−1) for 120 h. β-1,3-Glucan administration did not affect growth performance. However, as compared with control, elevated CAT and lysozyme activities were seen in haemolymph of both BG groups, while significantly higher activities of SOD, lysozyme and RB in hepatopancreas, and higher activities of CAT and lysozyme in muscle were only seen in C + BG group. After nitrite-N stress, significantly higher haemolymph protein, and hepatopancreas activities of lysozyme and RB were observed in both BG groups, but significantly higher activities of haemolymph SOD was only seen in C + BG group. The mortality in groups BG and C + BG was significantly lower than that in group C, but C + BG group showed a trend of higher nitrite-N resistance compared with BG group. Considering dietary cost and immunostimulatory effects, the feeding schedule with 14 days BG-14 days control diet is more recommended. [ABSTRACT FROM AUTHOR]

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC values from 2.33 × 10 M to 36.0 × 10 M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections. [ABSTRACT FROM AUTHOR]

Laetiporus sulphureus is a source of α-1,3-glucan that can substitute for the commercially-unavailable streptococcal mutan used to induce microbial mutanases. The water-insoluble fraction of its fruiting bodies from 0.15 to 0.2% (w/v) induced mutanase activity in Paenibacillus sp. MP-1 at 0.35 μ ml. The mutanase extensively hydrolyzed streptococcal mutan, giving 23% of saccharification, and 83% of solubilization of glucan after 6 h. It also degraded α-1,3-polymers of biofilms, formed in vitro by Streptococcus mutans, even after only 3 min of contact. [ABSTRACT FROM AUTHOR]

∙ Brown algae exhibit a unique carbon (C) storage metabolism. The photoassimilate D-fructose 6-phosphate is not used to produce sucrose but is converted into D-mannitol. These seaweeds also store C as β-1,3-glucan (laminarin), thus markedly departing from most living organisms, which use α-1,4-glucans (glycogen or starch). ∙ Using a combination of bioinformatic and phylogenetic approaches, we identified the candidate genes for the enzymes involved in C storage in the genome of the brown alga Ectocarpus siliculosus and traced their evolutionary origins. ∙ Ectocarpus possesses a complete set of enzymes for synthesis of mannitol, laminarin and trehalose. By contrast, the pathways for sucrose, starch and glycogen are completely absent. ∙ The synthesis of β-1,3-glucans appears to be a very ancient eukaryotic pathway. Brown algae inherited the trehalose pathway from the red algal progenitor of phaeoplasts, while the mannitol pathway was acquired by lateral gene transfer from Actinobacteria. The starch metabolism of the red algal endosymbiont was entirely lost in the ancestor of Stramenopiles. In light of these novel findings we question the validity of the 'Chromalveolate hypothesis'. [ABSTRACT FROM AUTHOR]

Abstract: Time-series changes in transcript abundance of nine genes encoding important immune proteins in haemocytes or hepatopancreas of Pacific white shrimp Litopenaeus vannamei fed daily in a 1-week feeding trial diets containing three levels (0%, 0.2% or 1%) of β-1,3-glucan from Schizophyllum commune were quantified by real-time PCR. As a whole, the immune modulation elicited by β-glucan is bimodal, one swift reaction of up- or down-regulation occurred within 24h and a delayed regulation was commenced as late as 3–7days. Haemocyanin, crustin, prophenoloxidase (proPO) and transglutaminase (TGase) did not respond to the glucan treatment. While penaeidin 3 (Litvan PEN3) was swiftly down-regulated (0–24h), lysozyme and cytosolic manganese superoxide dismutase (cMnSOD) were swiftly up-regulated (0–24h). In contrast, the two pattern recognition proteins (PRPs), β-glucan binding protein–high density lipoprotein (BGBP-HDL) and lipopolysaccharide/β-glucan binding protein (LGBP), showed a delayed up-regulation. Their expressions were not maximized until as late as 72h or 7days, respectively, which coincide with the initiation of reported immune enhancement (6–24days) of PO and SOD activity, phagocytosis and superoxide anion production in penaeid shrimp receiving glucan-containing diet. These immune responses could be the downstream effects of the two PRP gene up-regulation that predispose the shrimp to a state of high immune responsiveness. Increased dosage of β-glucan from 2 to 10gkg−1 diet did not affect the expressions of the genes, indicating the sufficiency of β-glucan supplementation at 2gkg−1 diet. [Copyright &y& Elsevier]

Abstract: A cDNA clone encoding possible prophenoloxidase-activating serine protease (PAP) was isolated by screening the cDNA library from immunized larval fat body of the wild silkmoth, Samia cynthia ricini. The cDNA encodes a 438 amino acid open reading frame with a predicted 20 residue signal peptide. Samia PAP has high sequence similarity to Bombyx mori and Manduca sexta PAPs, which contain two amino terminal clip domains followed by a carboxyl-terminal catalytic domain. The expression of the gene was barely detectable in the fat body of naive larvae, but induced after injection of the larvae with β-1,3-glucans or bacterial cells. [Copyright &y& Elsevier]

Background: The incidence of allergic symptoms to cedar pollen has reached epidemic proportions in Japan. Intravenous injection of β-1,3-glucan in human subjects is known to induce a TH1 response, whereas oral uptake does not. Objective: It was examined whether orally ingested, superfine dispersed β-1,3-glucan (SDG), easily absorbed by intestinal mucosa, would alleviate allergic symptoms. Methods: Allergic patients were orally administrated either SDG (n = 30) or nondispersed β-1,3-glucan (n = 30), and allergic symptoms were assessed clinically in a double-blind, placebo-controlled randomized study. Results: SDG alleviated ongoing symptoms of Japanese cedar pollen–induced rhinorrhea, sneezing, nasal congestion, and itchy watery eyes, and its oral uptake before symptom onset exhibited preventive effects. Alleviation of allergic symptoms was evident not only for seasonal allergy to cedar pollen but also for perennial allergy. Oral ingestion of β-1,3-glucan in individuals with allergic tropism could reduce the spontaneous increase in both allergen-specific and total IgE titers. The clinical responses to treatment were well correlated with the capacity of monocytes to bind to β-1,3-glucan. Although SDG reduced allergic symptoms, the oral uptake of nondispersed β-1,3-glucan produced no clinical effects, despite the identical amount of β-1,3-glucan in both preparations. Conclusion: We postulate that orally taken β-1,3-glucan prepared in a form easily absorbed by intestinal mucosa is able to alleviate cedar pollen–induced allergic symptoms. Clinical implications: Orally effective SDG might greatly contribute to the resolution of epidemic medical problems of seasonal cedar pollen–induced allergy. [Copyright &y& Elsevier]

Abstract: All glucan elicitors from fungal cell walls have been described as ß-glucans. In a previous work, we have isolated and characterized an α-1, 3-glucan from a non-pathogenic Rhizoctonia isolate which induced glucanase activity in potato sprouts. In this work we showed that, in addition to eliciting a wide array of defense reactions, the α-1, 3-glucan induces protection against Rhizoctonia canker and dry rot in plants and tubers, respectively. The α-1, 3-glucan strongly induced ß-1, 3-glucanase and chitinase activities. Immunological analysis showed that the level of the pathogenesis-related proteins increased. Histological studies showed an increase in cell wall deposition of callose and lignin, and ultrastructural analysis showed changes in the cytoplasm and the accumulation of electron-dense bodies in vacuoles. Protection assays showed that pre-treatments of potato sprouts and potato tubers with the α-1, 3-glucan results in a 40% of protection against Rhizoctonia canker and 60% protection against dry rot, respectively. [Copyright &y& Elsevier]

Abstract: The optimal culture condition of basidiomycete Sparassis crispa mycelia was determined for producing a large amount of antineoplastic constituent, i.e. β-1,3-glucan. Furthermore, a new extraction method with steam explosion as a pretreatment was attempted to increase the amount of antineoplastic constituent extracted from S. crispa mycelia. The shake-flask liquid culture of fungal mycelia could shorten a culture time up to about 1/3 compared with the conventional stationary liquid culture. The productivity of S. crispa mycelia reached its maximum at an initial glucose concentration of 30g/L, a culture temperature of 25–30°C, and pH 5. Steam explosion at a steam temperature of 225°C for 5min not only enhanced the amount of antineoplastic constituent extracted from the mycelia but also shortened the extraction time significantly. [Copyright &y& Elsevier]

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 μm, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for β-1,4-glucan and β-1,3-glucan. [ABSTRACT FROM AUTHOR]

The mode of action of an extracellular ∐-1,3-glucanase from Bacillus clausii NM-1 on β-1,3-3glucooligosaccharides and their alditols was studied. The enzyme could not hydrolyze laminaribiose or laminaritriose. β-1,3-Glucooligosaccharides higher than laminarihexaose were rapidly hydrolyzed, while laminaritetraose was slowly hydrolyzed. The kcat/Km ratios for a series of β-1,3-glucooligosaccharides from laminaritetraose to laminariheptaose showed that the substrate binding site of the enzyme covered a wide range of β-1,3-glucooligosaccharides having six glucose residues. The action pattern of the enzyme on the alditols corresponding to each laminarioligosaccharide suggested that the catalytic site of the enzyme existed between the third and fourth glucose residue from the non-reducing terminal. The value of kcat/Km also suggested that the sixth binding position contributed to the catalytic efficiency and stability. [Copyright &y& Elsevier]

A novel plant gene CFL1 was cloned from cotton (Gossypium hirsutum L.) fibers by expressed sequence tag (EST) database searching and 5′-RACE (rapid amplification of cDNA ends). This gene shows sequence homology with FKS1 which has been identified as the putative catalytic subunit of the yeast β-1,3-glucan synthase. It encodes a protein (CFL1p) of 219 kDa with 13 deduced transmembrane helices and 2 large hydrophilic domains, one of which is at the N-terminus and the other in the internal region of the polypeptide. CFL1 displays 21% identity and 41% similarity to FKS1 at the amino acid level over its entire length, with 31% identity and 52% similarity for the hydrophilic central domain. Using RNA and protein blot analysis, CFL1 was found to be expressed at higher levels in cotton fibers during primary wall development. CFL1 also had a strong expression in young roots. Using a calmodulin (CaM)-gel overlay assay, the hydrophilic N-terminal domain of CFL1p was shown to bind to CaM, while the hydrophilic central domain did not. A putative CaM-binding domain, 16 amino acids long, was predicted in the hydrophilic N-terminal domain. Moreover, a product-entrapment assay demonstrated that a protein associated with an in vitro-synthesized callose pellet could be labeled by anti-CFL1 antibodies. Our finding suggests that CFL1 is a putative plant homolog of the yeast β-1,3-glucan synthase subunit FKS1 and could be involved in callose synthesis. [ABSTRACT FROM AUTHOR]

The effects of growth conditions on paramylon (a β-1,3-glucanreserve carbohydrate) content were examined in the photosyntheticwild-type and a spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. This carbohydrate is known toshow interesting applications in human and veterinary medicine, asimmunostimulant and immunopotentiator. For both strains, the synthesisof reserve depends mainly on the growth conditions, i.e. light or dark. Thehighest amount of paramylon is accumulated by the mutant (90% dryweight) under dark conditions and with glucose as the carbon source. Thesefindings are discussed in terms of feasibility of exploitation of both strainsas an alternative source of β-glucan, and of the importance of thechloroplast compartment in the synthesis of this compound. [ABSTRACT FROM AUTHOR]

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

α-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans . We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA -overexpressing ( agsA OE ) strain was larger than that in an agsB -overexpressing ( agsB , which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in OE ) strain. The mechanism that regulates its MM is poorly understood. Although the gene amyD , which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in A . nidulans , how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD (Δ amyD ) or overexpressed amyD ( amyD strain, and characterized the chemical structure of α-1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of α-1,3-glucan from the OE ) in the genetic background of the ABPU1 (wild-type), agsA strain. In addition, the MM of α-1,3-glucan from the OE , or agsB OE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.agsB OE amyD OE strain was smaller than that in the parental agsB OE strain. In addition, the MM of α-1,3-glucan from the agsA OE Δ amyD strain was greater than that in the agsA OE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Miyazawa, Yamashita, Takeuchi, Kamachi, Yoshimi, Tashiro, Koizumi, Ogata, Yano, Kasahara, Sano, Yamagata, Nakajima and Abe.)