Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal β-glucan from the immune system.
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ABSTRACT:
SUBMITTER: Gravelat FN
PROVIDER: S-EPMC3749958 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that α-1,3-glucan functions in hyphal aggregation. However, Aspergillus oryzae α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.
| S-EPMC6753227 | biostudies-literature
Project description:A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of ?1-4 linked galactose and ?1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.
| S-EPMC3213105 | biostudies-literature
Project description:Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18-/- and Dectin-1-/- murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4-/- mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4-/- neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18-/- neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4-/- corneas; however, we found no difference between C57BL/6 and PAD4-/- mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.
| S-EPMC5986955 | biostudies-literature
Project description: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.
| S-EPMC8647855 | biostudies-literature
Project description:1. The components of an enzyme preparation from Aspergillus niger, which hydrolysed substrates containing beta-(1-->3)- and beta-(1-->4)-glucosidic linkages, were separated by calcium phosphate and Dowex 1 column chromatography. 2. The hydrolytic activity of each fraction from both types of column towards laminaribiose, laminarin, carboxymethylpachyman, pachydextrins, salicin, cellobiose, cellopentaose and swollen cellulose was tested. 3. The activity towards the beta-(1-->3)-glucosidic substrates was found in three well-separated groups of fractions. The differences in action pattern of these groups is discussed. 4. Preparative-scale chromatography that enabled the separation of a beta-(1-->4)-glucan-glucanohydrolase component substantially free of activity towards beta-(1-->3)-glucosidic substrates is described. Residual beta-(1-->3)-glucan-hydrolase activity was removed by adsorption on to insoluble laminarin at pH3.5.
| S-EPMC1207219 | biostudies-other
Project description:Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3-glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1alpha (MIP-1alpha), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte-CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan-initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense.
| S-EPMC1311140 | biostudies-literature
Project description:Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.
| S-EPMC2670538 | biostudies-literature
Project description:Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.
| S-EPMC8086055 | biostudies-literature
Project description:Invasive fungal infections caused by Aspergillus (A.) and Mucorales species still represent life-threatening diseases in immunocompromised individuals, and deeper knowledge about fungal interactions with elements of innate immunity, such as complement and platelets, appears essential for optimized therapy. Previous studies showed that galactosaminogalactan secreted by A. fumigatus and A. flavus is deposited on platelets, thereby inducing their activation. Since the altered platelet surface is a putative trigger for complement activation, we aimed to study the interplay of platelets with complement in the presence of fungal GAG. Culture supernatants (SN) of A. fumigatus and A. flavus both induced not only GAG deposition but also subsequent deposition of complement C3 fragments on the platelet surface. The SN of a Δuge3 mutant of A. fumigatus, which is unable to synthesize GAG, did not induce complement deposition on platelets, nor did the SN of other Aspergillus species and all tested Mucorales. Detailed analysis revealed that GAG deposition itself triggered the complement cascade rather than the GAG-induced phosphatidylserine exposure. The lectin pathway of complement could be shown to be crucially involved in this process. GAG-induced complement activation on the platelet surface was revealed to trigger processes that might contribute to the pathogenesis of invasive aspergillosis by A. fumigatus or A. flavus. Both pro-inflammatory anaphylatoxins C3a and C5a arose when platelets were incubated with SN of these fungal species; these processes might favor excessive inflammation after fungal infection. Furthermore, platelets were stimulated to shed microparticles, which are also known to harbor pro-inflammatory and pro-coagulant properties. Not only did early processes of the complement cascade proceed on platelets, but also the formation of the terminal complement C5b-9 complex was detected on platelets after incubation with fungal SN. Subsequently, reduced viability of the platelets could be shown, which might contribute to the lowered platelet numbers found in infected patients. In summary, fungal GAG initiates an interplay between complement and platelets that can be supposed to contribute to excessive inflammation, thrombocytopenia, and thrombosis, which are important hallmarks of fatal invasive mycoses.
| S-EPMC7573070 | biostudies-literature
Project description:1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.
| S-EPMC1207220 | biostudies-other