Galactosaminogalactan Research Articles

Characterization of the Neurospora crassa Galactosaminogalactan Biosynthetic Pathway.

The Neurospora crassa genome has a gene cluster for the synthesis of galactosaminogalactan (GAG). The gene cluster includes the following: (1) UDP-glucose-4-epimerase to convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine (NCU05133), (2) GAG synthase for the synthesis of an acetylated GAG (NCU05132), (3) GAG deacetylase (/NCW-1/NCU05137), (4) GH135-1, a GAG hydrolase with specificity for N-acetylgalactosamine-containing GAG (NCU05135), and (5) GH114-1, a galactosaminidase with specificity for galactosamine-containing GAG (NCU05136). The deacetylase was previously shown to be a major cell wall glycoprotein and given the name of NCW-1 (non-GPI anchored cell wall protein-1). Characterization of the polysaccharides found in the growth medium from the wild type and the GAG synthase mutant demonstrates that there is a major reduction in the levels of polysaccharides containing galactosamine and N-acetylgalactosamine in the mutant growth medium, providing evidence that the synthase is responsible for the production of a GAG. The analysis also indicates that there are other galactose-containing polysaccharides produced by the fungus. Phenotypic characterization of wild-type and mutant isolates showed that deacetylated GAG from the wild type can function as an adhesin to a glass surface and provides the fungal mat with tensile strength, demonstrating that the deacetylated GAG functions as an intercellular adhesive. The acetylated GAG produced by the deacetylase mutant was found to function as an adhesive for chitin, alumina, celite (diatomaceous earth), activated charcoal, and wheat leaf particulates.

CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan.

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

Amino sugars influence Aspergillus fumigatus cell wall polysaccharide biosynthesis, and biofilm formation through interfering galactosaminogalactan deacetylation

Aspergillus fumigatus is a ubiquitous fungal pathogen responsible for a significant number of deaths annually due to invasive aspergillosis infection. While the utilization of diverse carbon sources, including amino sugars, has been explored in other fungi, its impact on A. fumigatus remains uncharted territory. In this study, we investigated A. fumigatus responses to glucose (Glc), glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) as carbon sources. GlcN inhibited growth, reduced sporulation and delayed germination, while GlcNAc had no such effects. Both amino sugars induced alterations in cell wall composition, leading to a reduction in glucan and galactomannan levels while increasing chitin and mannan content, rendering A. fumigatus susceptible to cell wall stress and osmotic stress. GlcN repressed biofilm formation via downregulation of galactosaminogalactan (GAG) cluster genes, notably agd3, which encodes a GAG-specific deacetylase. Moreover, GlcN increased biofilm susceptibility to echinocandins, suggesting its potential for enhancing the effectiveness of antifungal treatments. This study sheds light on the multifaceted effects of amino sugars on A. fumigatus, encompassing growth, cell wall biosynthesis, and biofilm formation, offering promising avenues for innovative aspergillosis treatment strategies.

Transcription factor CreA is involved in the inverse regulation of biofilm formation and asexual development through distinct pathways in Aspergillus fumigatus.

The exopolysaccharide galactosaminogalactan (GAG) contributes to biofilm formation and virulence in the pathogenic fungus Aspergillus fumigatus. Increasing evidence indicates that GAG production is inversely linked with asexual development. However, the mechanisms underlying this regulatory relationship are unclear. In this study, we found that the dysfunction of CreA, a conserved transcription factor involved in carbon catabolite repression in many fungal species, causes abnormal asexual development (conidiation) under liquid-submerged culture conditions specifically in the presence of glucose. The loss of creA decreased GAG production independent of carbon sources. Furthermore, CreA contributed to asexual development and GAG production via distinct pathways. CreA promoted A. fumigatus GAG production by positively regulating GAG biosynthetic genes (uge3 and agd3). CreA suppressed asexual development in glucose liquid-submerged culture conditions via central conidiation genes (brlA, abaA, and wetA) and their upstream activators (flbC and flbD). Restoration of brlA expression to the wild-type level by flbC or flbD deletion abolished the abnormal submerged conidiation in the creA null mutant but did not restore GAG production. The C-terminal region of CreA was crucial for the suppression of asexual development, and the repressive domain contributed to GAG production. Overall, CreA is involved in GAG production and asexual development in an inverse manner.

Regulation of Surfactant Protein Gene Expression by Aspergillus fumigatus in NCl-H441 Cells.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

Identification of Compounds Preventing A. fumigatus Biofilm Formation by Inhibition of the Galactosaminogalactan Deacetylase Agd3.

The opportunistic fungus Aspergillus fumigatus causes a set of diseases ranging from allergy to lethal invasive mycosis. Within the human airways, A. fumigatus is embedded in a biofilm that forms not only a barrier against the host immune defense system, but also creates a physical barrier protecting the fungi from chemicals such as antifungal drugs. Novel therapeutic strategies aim at combining drugs that inhibit biofilm synthesis or disrupt existing biofilm with classical antimicrobials. One of the major constituents of A. fumigatus biofilm is the polysaccharide galactosaminogalactan (GAG) composed of α1,4-linked N-acetylgalactosamine, galactosamine, and galactose residues. GAG is synthesized on the cytosolic face of the plasma membrane and is extruded in the extracellular space, where it is partially deacetylated. The deacetylase Agd3 that mediates this last step is essential for the biofilm formation and full virulence of the fungus. In this work, a previously described enzyme-linked lectin assay, based on the adhesion of deacetylated GAG to negatively charged plates and quantification with biotinylated soybean agglutinin was adapted to screen microbial natural compounds, as well as compounds identified in in silico screening of drug libraries. Actinomycin X2, actinomycin D, rifaximin, and imatinib were shown to inhibit Agd3 activity in vitro. At a concentration of 100 µM, actinomycin D and imatinib showed a clear reduction in the biofilm biomass without affecting the fungal growth. Finally, imatinib reduced the virulence of A. fumigatus in a Galleria mellonella infection model in an Agd3-dependent manner.

In Silico Structural Analysis, Classification, and Functional Annotation of an Uncharacterized Protein from an Aquatic Fungus Lindgomyces ingoldianus

An uncharacterized protein from Lindgomyces ingoldianus was initially annotated to contain various domains with promising biotechnological applications. Thus, this study was conducted to determine the structural characteristics, classification, and potential function of this protein through in silico methods. Results revealed that this protein has a neutral charge and is unstable and non-polar. It is predicted to have a signal peptide, glycoside hydrolase family 114 (GH114) domain, low complexity region, and fungal type cellulose-binding domain (fCBD) or type 1 carbohydrate-binding module (CBM1) region. Structural characterization and phylogenetic analysis revealed that this protein is an endo-α-1,4-polygalactosaminidase enzyme. This protein was also predicted to contain 36 active sites and is extracellularly secreted. Molecular docking analysis showed that it could bind galactosaminogalactan (GAG), a key virulence factor for Aspergillus fumigatus chronic infections. The binding of this protein to GAG was much better than Ega3, which could be attributed to the presence of the fCBD region that is unique to this protein. It is hypothesized that the fCBD domain helps in carbohydrate recognition and holds them in place for maximum catalysis in the GH114 domain. Finally, this protein is found to be related to its orthologue from the plant pathogenic fungus Zopfia rhizophila.

Aspergillus fumigatus Elongator complex subunit 3 affects hyphal growth, adhesion and virulence through wobble uridine tRNA modification.

The eukaryotic multisubunit Elongator complex has been shown to perform multiple functions in transcriptional elongation, histone acetylation and tRNA modification. However, the Elongator complex plays different roles in different organisms, and the underlying mechanisms remain unexplored. Moreover, the biological functions of the Elongator complex in human fungal pathogens remain unknown. In this study, we verified that the Elongator complex of the opportunistic fungal pathogen Aspergillus fumigatus consists of six subunits (Elp1-6), and the loss of any subunit results in similarly defective colony phenotypes with impaired hyphal growth and reduced conidiation. The catalytic subunit-Elp3 of the Elongator complex includes a S-adenosyl methionine binding (rSAM) domain and a lysine acetyltransferase (KAT) domain, and it plays key roles in the hyphal growth, biofilm-associated exopolysaccharide galactosaminogalactan (GAG) production, adhesion and virulence of A. fumigatus; however, Elp3 does not affect H3K14 acetylation levels in vivo. LC-MS/MS chromatograms revealed that loss of Elp3 abolished the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNA wobble uridine (U34), and the overexpression of tRNAGlnUUG and tRNAGluUUC, which normally harbor mcm5s2U modifications, mainly rescues the defects of the Δelp3 mutant, suggesting that tRNA modification rather than lysine acetyltransferase is responsible for the primary function of Elp3 in A. fumigatus. Strikingly, global proteomic comparison analyses showed significantly upregulated expression of genes related to amino acid metabolism in the Δelp3 mutant strain compared to the wild-type strain. Western blotting showed that deletion of elp3 resulted in overexpression of the amino acid starvation-responsive transcription factor CpcA, and deletion of CpcA markedly reversed the defective phenotypes of the Δelp3 mutant, including attenuated virulence. Therefore, the findings of this study demonstrate that A. fumigatus Elp3 functions as a tRNA-modifying enzyme in the regulation of growth, GAG production, adhesion and virulence by maintaining intracellular amino acid homeostasis. More broadly, our study highlights the importance of U34 tRNA modification in regulating cellular metabolic states and virulence traits of fungal pathogens.

S2.3d Mixed biofilm of Aspergillus fumigatus and Stenotrophomonas maltophilia: Microscopic visualization of galactosaminogalactan and galactomannan polysaccharides in the extracellular matrix

S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PM Introduction Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are commonly co-isolated from the airways of cystic fibrosis patients, especially in mixed biofilms. A mixed Af-Sm biofilm model, developed by our lab, demonstrated that Sm exhibits an antibiosis effect on Af by (1) inhibiting fungal growth, (2) rendering the fungal mycelia highly branched, and (3) increasing the fungal cell wall thickness.1 The presence of three Af cell wall polysaccharides, galactomannan (GM), galactosaminogalactan (GAG), and α-1,3-glucan, have been described in the extracellular matrix (ECM) of Af biofilms.2 GM and GAG are known to be released out from the cell wall of Af.3 GAG contains N-acetylgalactosamines (GalNAc) which undergo partial deacetylation extracellularly, to be converted to GalN, and thus transform this polysaccharide into a polycation with strong surface binding properties.4Aim of the studyAnalyze the structure of fungal cell wall polysaccharides in the formation of Af biofilm alone or mixed with Sm.MethodsThe Af-Sm co-culture was prepared on an 8-chambers Lab-Tek slide as described previously1. Anti-GAG and anti-GM monoclonal antibodies produced at the Aspergillus Unit at Pasteur Institute of Paris, were used to focus on the 3D structure of GAG and GM in the ECM of the biofilm. These polysaccharides were analyzed by fluorescence confocal microscopy at 24 h.Results and ConclusionGM was found, as expected, in the hyphal Af cell wall but was very little in the ECM. GAG was also found in the cell wall but mainly formed a beautiful fibrillary network between the hyphae, showing the importance of this polysaccharide in cell-cell interaction and in the structuration of Af biofilm. GAG could be the surface receptor for Sm, which would promote strong adhesion between Sm and Af in the biofilm.Sources1. Melloul E et al. Front Microbiol. 2018;2. Beauvais A et al. Microbiol Spectr. 2015;3. Fontaine T et al. J Fungi. 2020;4. Briard B et al. Mycologia 2016.

P364 Quantitative monitoring of Aspergillus fumigatus mycelial growth by optical density

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesFilamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes (Δags1Δgtb3) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density.MethodsTo measure the optical density of conidia and mycelia in shake-flask culture, conidia (final concentration, 1.0 × 10⁷/ml) of AfS35 or Δags1Δgtb3 strain were inoculated into 50 ml of Aspergillus minimal medium (AMM) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture (2 ml) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD₆₀₀ was measured in a microplate reader. To evaluate the susceptibility of the Δags1Δgtb3 strain to antifungal agents, conidia (final concentration, 5.0 × 10⁶/ml) were inoculated into 500 μL of RPMI medium containing an antifungal agent (voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD₆₀₀ was measured in triplicate.ResultsIn AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the Δags1Δgtb3 hyphae were continuously dispersed (Fig. 1a). The OD₆₀₀ measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD₆₀₀ (Fig. 1b). In the Δags1Δgtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h (Fig. 1b), suggesting that the measurement of OD₆₀₀ is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the Δags1Δgtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate (Fig. 2). Growth was repressed in the presence of antifungal agents tested, except for micafungin (Fig. 2). Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine (Fig. 2), which was in agreement with MICs determined by CLSI M38-A2.ConclusionWe established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.

Co-Operative Biofilm Interactions between Aspergillus fumigatus and Pseudomonas aeruginosa through Secreted Galactosaminogalactan Exopolysaccharide.

The mold Aspergillus fumigatus and bacterium Pseudomonas aeruginosa form biofilms in the airways of individuals with cystic fibrosis. Biofilm formation by A. fumigatus depends on the self-produced cationic exopolysaccharide galactosaminogalactan (GAG), while P. aeruginosa biofilms can contain the cationic exopolysaccharide Pel. GAG and Pel are rendered cationic by deacetylation mediated by either the secreted deacetylase Agd3 (A. fumigatus) or the periplasmic deacetylase PelA (P. aeruginosa). Given the similarities between these polymers, the potential for biofilm interactions between these organisms were investigated. P. aeruginosa were observed to adhere to A. fumigatus hyphae in a GAG-dependent manner and to GAG-coated coverslips of A. fumigatus biofilms. In biofilm adherence assays, incubation of P. aeruginosa with A. fumigatus culture supernatants containing de-N-acetylated GAG augmented the formation of adherent P. aeruginosa biofilms, increasing protection against killing by the antibiotic colistin. Fluorescence microscopy demonstrated incorporation of GAG within P. aeruginosa biofilms, suggesting that GAG can serve as an alternate biofilm exopolysaccharide for this bacterium. In contrast, Pel-containing bacterial culture supernatants only augmented the formation of adherent A. fumigatus biofilms when antifungal inhibitory molecules were removed. This study demonstrates biofilm interaction via exopolysaccharides as a potential mechanism of co-operation between these organisms in chronic lung disease.

An Alanine Aminotransferase Is Required for Biofilm-Specific Resistance of Aspergillus fumigatus to Echinocandin Treatment.

ABSTRACTAlanine metabolism has been suggested as an adaptation strategy to oxygen limitation in organisms ranging from plants to mammals. Within the pulmonary infection microenvironment, Aspergillus fumigatus forms biofilms with steep oxygen gradients defined by regions of oxygen limitation. An alanine aminotransferase, AlaA, was observed to function in alanine catabolism and is required for several aspects of A. fumigatus biofilm physiology. Loss of alaA, or its catalytic activity, results in decreased adherence of biofilms through a defect in the maturation of the extracellular matrix polysaccharide galactosaminogalactan (GAG). Additionally, exposure of cell wall polysaccharides is also impacted by loss of alaA, and loss of AlaA catalytic activity confers increased biofilm susceptibility to echinocandin treatment, which is correlated with enhanced fungicidal activity. The increase in echinocandin susceptibility is specific to biofilms, and chemical inhibition of alaA by the alanine aminotransferase inhibitor β-chloro-l-alanine is sufficient to sensitize A. fumigatus biofilms to echinocandin treatment. Finally, loss of alaA increases susceptibility of A. fumigatus to in vivo echinocandin treatment in a murine model of invasive pulmonary aspergillosis. Our results provide insight into the interplay of metabolism, biofilm formation, and antifungal drug resistance in A. fumigatus and describe a mechanism of increasing susceptibility of A. fumigatus biofilms to the echinocandin class of antifungal drugs.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density.

ABSTRACTFilamentous 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.

Improved recombinant protein production in Aspergillus oryzae lacking both α-1,3-glucan and galactosaminogalactan in batch culture with a lab-scale bioreactor

Filamentous fungi are used as production hosts for various commercially valuable enzymes and chemicals including organic acids and secondary metabolites. We previously revealed that α-1,3-glucan and galactosaminogalactan (GAG) contribute to hyphal aggregation in the industrial fungus Aspergillus oryzae, and that production of recombinant protein in shake-flask culture is higher in a mutant lacking both α-1,3-glucan and GAG (AGΔ-GAGΔ) than in the parental strain. Here, we compared the productivity of the wild type, AGΔ-GAGΔ, and mutants lacking α-1,3-glucan (AGΔ) or GAG (GAGΔ) in batch culture with intermittent addition of glucose in a 5-L lab-scale bioreactor. The hyphae of the wild type and all mutants were dispersed by agitation, although the wild type and AGΔ formed small amounts of aggregates. Although mycelial weight was similar among the strains, the concentration of a secreted recombinant protein (CutL1) was the highest in AGΔ-GAGΔ. Evaluation of fluid properties revealed that the apparent viscosities of mycelial cultures of the wild type and AGΔ-GAGΔ decreased as the agitation speed was increased. The apparent viscosity of the AGΔ-GAGΔ culture tended to be lower than that of the wild-type strain at each agitation speed, and was significantly lower at 600rpm. Overall, the lack of α-1,3-glucan and GAG in the hyphae improved culture rheology, resulting in an increase in recombinant protein production in AGΔ-GAGΔ. This is the first report of flow behavior improvement by a cell-surface component defect in a filamentous fungus.

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts.

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.

Fungal Polysaccharides Promote Protective Immunity.

Fungal pathogens represent a rising threat against immunocompromised patients. By using Aspergillus fumigatus, Briard et al. showed that the cell wall galactosaminogalactan (GAG) triggers macrophage inflammasome activation, promoting protective immunity. This provides new insights into the role of GAG in host-pathogen interactions and also perspectives for developing GAG-based anti-inflammatory therapeutics.

Galactosaminogalactan activates the inflammasome to provide host protection.

SummaryInflammasomes are important sentinels of innate immune defense activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defense against aspergillosis2,3, a major health concern for immunocompromised patients; however, the Aspergillus fumigatus PAMPs responsible for inflammasome activation are not known. Here we discovered that A. fumigatus galactosaminogalactan (GAG) is a novel PAMP that activates the NLRP3 inflammasome. Binding of GAG to ribosomal proteins inhibited cellular translation machinery, thereby activating the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, triggering NLRP3 inflammasome activation. In mice, a GAG-deficient Aspergillus mutant Δgt4c failed to elicit protective inflammasome activation and exhibited enhanced virulence. Moreover, administration of GAG protected mice from DSS-induced colitis in an inflammasome-dependent manner. Thus, ribosomes connect sensing of this fungal PAMP to activation of an innate immune response.

The Transcription Factor SomA Synchronously Regulates Biofilm Formation and Cell Wall Homeostasis in Aspergillus fumigatus.

Polysaccharides are key components of both the fungal cell wall and biofilm matrix. Despite having distinct assembly and regulation pathways, matrix exopolysaccharide and cell wall polysaccharides share common substrates and intermediates in their biosynthetic pathways. It is not clear, however, if the biosynthetic pathways governing the production of these polysaccharides are cooperatively regulated. Here, we demonstrate that cell wall stress promotes production of the exopolysaccharide galactosaminogalactan (GAG)-depend biofilm formation in the major fungal pathogen of humans Aspergillus fumigatus and that the transcription factor SomA plays a crucial role in mediating this process. A core set of SomA target genes were identified by transcriptome sequencing and chromatin immunoprecipitation coupled to sequencing (ChIP-Seq). We identified a novel SomA-binding site in the promoter regions of GAG biosynthetic genes agd3 and ega3, as well as its regulators medA and stuA Strikingly, this SomA-binding site was also found in the upstream regions of genes encoding the cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Thus, SomA plays a direct regulation of both GAG and cell wall polysaccharide biosynthesis. Consistent with these findings, SomA is required for the maintenance of normal cell wall architecture and compositions in addition to its function in biofilm development. Moreover, SomA was found to globally regulate glucose uptake and utilization, as well as amino sugar and nucleotide sugar metabolism, which provides precursors for polysaccharide synthesis. Collectively, our work provides insight into fungal adaptive mechanisms in response to cell wall stress where biofilm formation and cell wall homeostasis were synchronously regulated.IMPORTANCE The cell wall is essential for fungal viability and is absent from human hosts; thus, drugs disrupting cell wall biosynthesis have gained more attention. Caspofungin is a member of a new class of clinically approved echinocandin drugs to treat invasive aspergillosis by blocking β-1,3-glucan synthase, thus damaging the fungal cell wall. Here, we demonstrate that caspofungin and other cell wall stressors can induce galactosaminogalactan (GAG)-dependent biofilm formation in the human pathogen Aspergillus fumigatus We further identified SomA as a master transcription factor playing a dual role in both biofilm formation and cell wall homeostasis. SomA plays this dual role by direct binding to a conserved motif upstream of GAG biosynthetic genes and genes involved in cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Collectively, these findings reveal a transcriptional control pathway that integrates biofilm formation and cell wall homeostasis and suggest SomA as an attractive target for antifungal drug development.

Synthesis and Structural Analysis of Aspergillus fumigatus Galactosaminogalactans Featuring α-Galactose, α-Galactosamine and α-N-Acetyl Galactosamine Linkages.

Galactosaminogalactan (GAG) is a prominent cell wall component of the opportunistic fungal pathogen Aspergillus fumigatus. GAG is a heteropolysaccharide composed of α-1,4-linked galactose, galactosamine and N-acetylgalactosamine residues. To enable biochemical studies, a library of GAG-fragments was constructed featuring specimens containing α-galactose-, α-galactosamine and α-N-acetyl galactosamine linkages. Key features of the synthetic strategy include the use of di-tert-butylsilylidene directed α-galactosylation methodology and regioselective benzoylation reactions using benzoyl-hydroxybenzotriazole (Bz-OBt). Structural analysis of the Gal, GalN and GalNAc oligomers by a combination of NMR and MD approaches revealed that the oligomers adopt an elongated, almost straight, structure, stabilized by inter-residue H-bonds, one of which is a non-conventional C-H⋅⋅⋅O hydrogen bond between H5 of the residue (i+1) and O3 of the residue (i). The structures position the C-2 substituents almost perpendicular to the oligosaccharide main chain axis, pointing to the bulk solvent and available for interactions with antibodies or other binding partners.

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.