The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on... Show moreThe cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5′ donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal. Show less
Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the... Show moreAmong opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A. niger. We found that both species evoke similar immune cell migratory responses, but A. fumigatus is more efficiently phagocytized than A. niger. Though efficiently phagocytized, A. fumigatus conidia retains the ability to germinate and form hyphae from inside macrophages leading to serious infection even at relatively low infectious burdens. By contrast, A. niger appears to rely on extracellular germination, and rapid hyphal growth to establish infection. Despite these differences in the mechanism of infection between the species, galactofuranose mutant strains of both A. fumigatus and A. niger display attenuated pathogenesis. However, deficiency in this cell wall component has a stronger impact on A. niger, which is dependent on rapid extracellular hyphal growth. In conclusion, we uncover differences in the interaction of the two fungal species with innate immune cells, noticeable from very early stages of infection, which drive a divergence in their route to establishing infections. Show less
Arentshorst, M.; Lange, D. de; Park, J.; Lagendijk, E.L.; Alazi, E.; Hondel, C.A.M.J.J. van den; Ram, A.F.J. 2019
Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans... Show moreGalactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions. Show less
Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward... Show morePlant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown. Show less
Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the... Show moreAmong opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A. niger. We found that both species evoke similar immune cell migratory responses, but A. fumigatus is more efficiently phagocytized than A. niger. Though efficiently phagocytized, A. fumigatus conidia retains the ability to germinate and form hyphae from inside macrophages leading to serious infection even at relatively low infectious burdens. By contrast, A. niger appears to rely on extracellular germination, and rapid hyphal growth to establish infection. Despite these differences in the mechanism of infection between the species, galactofuranose mutant strains of both A. fumigatus and A. niger display attenuated pathogenesis. However, deficiency in this cell wall component has a stronger impact on A. niger, which is dependent on rapid extracellular hyphal growth. In conclusion, we uncover differences in the interaction of the two fungal species with innate immune cells, noticeable from very early stages of infection, which drive a divergence in their route to establishing infections. Show less
Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the... Show moreAmong opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A. niger. We found that both species evoke similar immune cell migratory responses, but A. fumigatus is more efficiently phagocytized than A. niger. Though efficiently phagocytized, A. fumigatus conidia retains the ability to germinate and form hyphae from inside macrophages leading to serious infection even at relatively low infectious burdens. By contrast, A. niger appears to rely on extracellular germination, and rapid hyphal growth to establish infection. Despite these differences in the mechanism of infection between the species, galactofuranose mutant strains of both A. fumigatus and A. niger display attenuated pathogenesis. However, deficiency in this cell wall component has a stronger impact on A. niger, which is dependent on rapid extracellular hyphal growth. In conclusion, we uncover differences in the interaction of the two fungal species with innate immune cells, noticeable from very early stages of infection, which drive a divergence in their route to establishing infections. Show less
Among opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the... Show moreAmong opportunistically pathogenic filamentous fungi of the Aspergillus genus, Aspergillus fumigatus stands out as a drastically more prevalent cause of infection than others. Utilizing the zebrafish embryo model, we applied a combination of non-invasive real-time imaging and genetic approaches to compare the infectious development of A. fumigatus with that of the less pathogenic A. niger. We found that both species evoke similar immune cell migratory responses, but A. fumigatus is more efficiently phagocytized than A. niger. Though efficiently phagocytized, A. fumigatus conidia retains the ability to germinate and form hyphae from inside macrophages leading to serious infection even at relatively low infectious burdens. By contrast, A. niger appears to rely on extracellular germination, and rapid hyphal growth to establish infection. Despite these differences in the mechanism of infection between the species, galactofuranose mutant strains of both A. fumigatus and A. niger display attenuated pathogenesis. However, deficiency in this cell wall component has a stronger impact on A. niger, which is dependent on rapid extracellular hyphal growth. In conclusion, we uncover differences in the interaction of the two fungal species with innate immune cells, noticeable from very early stages of infection, which drive a divergence in their route to establishing infections. Show less
