Project description:Fungal cell walls undergo continual remodeling during hyphal growth, development, infection and adaptation to environmental stress. Cell wall remodeling generates 1,3-glucan fragments by diverse endo-glycosyl hydrolases (GH), which are well-known pathogen-associated molecular patterns (PAMPs). How fungal pathogens evade plant immunity triggered by1,3-glucan fragments and associated GH proteins is not known. Here, we report a novel mechanism of immune evasion underlying the suppression of 1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. An exo-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze 1,3-glucan and laminarin into glucose to prevent 1,3-glucan-triggered plant immunity, but also acts as a PAMP, independent of its hydrolase activity. Surprisingly, M. oryzae engages an elongation factor 1 alpha protein (EF1 to interact and co-localize with Ebg1 in the apoplast to suppress Ebg1-triggered immunity. Since both Ebg1 and EF1 are widely distributed in fungi, their interaction may be a conserved mechanism whereby fungal pathogens evade plant immunity and safeguard cell wall remodeling during infection.
Project description:The cell wall provides a major physical interface between fungal pathogens and their mammalian host. This extracellular armour is critical for fungal cell homeostasis and survival. Yet essential cell wall moieties, such as β-1,3-glucan, are recognised as pathogen-associated molecular patterns (PAMPs) that activate immune-mediated clearance mechanisms. We have reported that the opportunistic human fungal pathogen, Candida albicans, masks β-1,3-glucan following exposure to lactate, hypoxia or iron depletion. However, the precise mechanism(s) by which C. albicans masks β-1,3-glucan have remained obscure. Here, we performed proteomic analysis of cell walls from C. albicans cells grown in hypoxia or lactate compared to glucose-grown controls to identify mechanisms driving β-1,3-glucan masking.
Project description:The cell wall provides a major physical interface between fungal pathogens and their mammalian host. This extracellular armour is critical for fungal cell homeostasis and survival. Yet essential cell wall moieties, such as β-1,3-glucan, are recognised as pathogen-associated molecular patterns (PAMPs) that activate immune-mediated clearance mechanisms. We have reported that the opportunistic human fungal pathogen, Candida albicans, masks β-1,3-glucan following exposure to lactate, hypoxia or iron depletion. However, the precise mechanism(s) by which C. albicans masks β-1,3-glucan have remained obscure. Here, we performed proteomic analysis of supernatants harvested from C. albicans cells grown in hypoxia or lactate compared to glucose-grown controls to identify mechanisms driving β-1,3-glucan masking.
Project description:The cell wall is a structure involved in important stages of fungal growth and morphogenesis. Several studies in the literature have shown how perturbations at the cell wall-level trigger dramatic effects on growth (e.g. Horiuchi, 2009). Despite the importance of fungal cell walls and despite the great advances made in the field, there are still missing pieces in our understanding of cell wall dynamics in filamentous fungi. Some cell wall biosynthetic genes, for example, are still uncharacterized (for a detailed inventory of Aspergillus nidulans cell wall-related genes, see de Groot et al., 2009). The chief polysaccharides in the cell wall of the model organism A. nidulans are β-glucans (β-1,3-, β-(1,3;1,4)- and β-1,6-glucans), chitin and α-1,3-glucans. While much is known about the chitin and α-1,3-glucan biosynthetic genes in A. nidulans (Horiuchi et al., 1999; Fujiwara et al., 2000; Ichinomiya et al., 2002 and 2005; Takeshita et al., 2006; Yoshimi et al., 2013), no characterization is yet available for the celA gene (ANIA_08444) encoding a putative mixed-linkage glucan synthase (de Groot et al., 2009). Recently, a study on A. fumigatus has characterized Tft1, an enzyme shown to be responsible for the production of β-(1,3;1,4)-glucans in this organism (Samar et al., 2015). Deletion of Tft1 causes no obvious phenotype in A. fumigatus and a modest increase in virulence. To characterize the role of β-(1,3;1,4)-glucans in the growth and development of filamentous fungi, we here sought to provide transcriptomics data of an A. nidulans strain showing reduced expression of the gene encoding the putative mixed linkage glucan synthase celA.
Project description:Acanthamoeba castellanii (Ac) and macrophages share structural, morphological, physiological and biochemical similarities, including the ability to phagocyte a myriad of microorganisms in the environments they live. Whereas there is voluminous information on phagocytic receptors of macrophages, for Ac, the understanding of how the recognition of extracellular microorganisms works still awaits elucidation. Recently, our group described mannose-binding proteins expressed on the surface of the trophozoites. However, as soluble mannose did not inhibit entirely the process, other interactions might be possible. In the present work, we aimed to characterize the potential Ac proteins able to recognize the polysaccharide β-1,3-glucan on fungal surfaces. Our data demonstrate that Ac could bind curdlan or laminarin on its surface as detected by Dectin-1-Fc and fluorescent conjugate, suggesting the presence of β-1,3-glucan binding molecules. Optical tweezers detected higher adhesion affinity of laminarin or curdlan coated beads to A. castellanii (characteristic time of 46.9 s and 43.9 s, respectively) in comparison control beads (BSA or dextran-coated). In agreement, a H. capsulatum (Hc) G217B having β-1,3-glucan as the most external layer strongly adhered to Ac (characteristic time of 5.3 s), whereas Hc G186A, an α-1,3-glucan expressing strain, displayed much lower adhesion forces (characteristic time of 83.6 s). The specificity of our system was confirmed with addition of soluble β-1,3-glucan, which inhibited dramatically the adhesion of Hc G217B to Ac (characteristic time of 38,5 s). By indirect ELISA, the biotinylated extract of Ac showed higher binding to Hc G217B surface than Hc G186A, as similar results were observed when using Dectin-1-Fc. By interaction assays, association rates to Ac and RAW macrophages were twice higher for Hc G217B when compared to Hc G186A. Inhibitions with mannose, or its combinations with curdlan or laminarin demonstrated inhibitions higher than 50% during Ac and Hc G217B interaction. For RAW macrophages, the combinations mannose + laminarin and mannose + curdlan had inhibition of 64.4% and 51.5%, respectively, in the interaction with Hc G217B. The killing assay show that for A. castellanii, there was a decrease in the number of viable fungi when either laminarin and curdlan were added, which is similar to results observed with macrophages, suggesting the participation of this receptor for fungal entrance and survival within phagocytes. Proteomics identified several proteins with the capacity to bind β-1,3-glucans, including a membrane integral component (L8HDD6) displaying a legume lectin domain and also belonging to the Concanavalin A-like lectin/glucanase domain superfamily. By the demonstrated binding specificity of this receptor, our data reinforce other pathways of fungal recognition and suggests to a possible parallel or even divergent evolution, between A. castellanii and macrophages.
Project description:Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular spaces of plant tissues. Fungal hyphae are surrounded by an inner, insoluble cell wall layer and an outer, soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, implicating different functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic GH17 family, is not active on fungal walls, but releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose substituents and can scavenge reactive oxygen species via oxidative self-degradation. Additionally, exogenous application of β-GD leads to enhanced fungal colonization in barley. Our data highlights the hitherto undescribed capacity of this often-overseen fungal EPS layer to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.
Project description:The outer layers of the fungal cell wall are the first barrier facing external challenges. In theinvasive morphotype of the airborne pathogen Aspergillus fumigatus, i.e. conidia, the outer cell wall consists of α-(1,3)-glucan, melanin and proteinaceous rodlets made up by the RodA hydrophobin. The hydrophobicity of the RodA rodlets facilitates air dispersal of conidia and progression within lungs. When conidia reach the alveolae, these are phagocytosed by host cells and killed upon germination, which requires melanin and rodlets elimination to occur. The dormant condition of the conidia protects them against activation of the host killing pathways. The outer cell wall of conidia is thus essential for maintaining the viability of the fungus. To study the role of α-(1,3)-glucan, melanin, and RodA rodlets in conidia protection, multiple mutants without two or the three major components of the outer layer were constructed and analyzed. Deletions led to the exposure of new molecules on the conidial surface. Multiple gene deletions did not always lead to logical additivity of the phenotypes. Unexpected compensatory cell wall surface modifications were indeed observed, such as the synthesis of the mycelial virulence factor galactosaminogalactan, the presence of chitin and glycoproteins or specific changes in permeability. In spite of significant modifications, sensitivity to killing by phagocytic host cells of the multiple mutants involving melanin changed little compared to the sensitive parent strain lacking melanin (ΔpksP), further indicating the importance of melanin in protecting conidia against killing by monocytes.
Project description:Dectin1 controls the recruitment of TLR9 to β-1,3 glucan beads containing phagosomes. We sought to determine whether Dectin-1 also plays a role in controlling TLR9 dependent gene expression.
Project description:Immune inertness of Aspergillus fumigatus conidia, an airborne fungal pathogen, is attributed to its surface rodlet-layer made up of RodAp, a protein belonging to the hydrophobin family, characterized by eight conserved cysteine residues forming four disulfide bonding. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet-layer. Here we extended our study in comparing ccrp-mutation with RODA deletion on their mutant conidial surface organization, permeability and immunoreactivity. Western blot using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Upon immunolabelling, ccrp-mutant conidia showed strong positivity for -(1,3)-glucan and weak positivity for -(1,3)-glucan, which was reverse in ∆rodA conidia, and the parental strain conidia were negative; all of their conidial cell wall permeability was similar. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants, although lower in number, showed more similarities with the parental strain, but a significant difference with the RODA deletion mutant strain. Further, ccrp-mutant conidia are less immunostimulatory compared to ∆rodA conidia. Together, our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of rodlet-layer on the conidial surface, and (ii) point-mutation could be an alternative strategy to study the proteins involved in the organization of fungal cell wall.