During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the β-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.

29During the course of fungal infection, pathogen recognition by the innate immune system is 30 critical to initiate efficient protective immune responses. The primary event that triggers 31 immune responses is the binding of Pattern Recognition Receptors (PRRs), which are 32 expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns 33 (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs 34 in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). 35However, the precise spatial distribution of the ligands that induce immune responses within 36 the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three 37 murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate 38 recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and the β-1,3 39 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall. We show that 40 epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried 41 in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic 42 relationships between fungi, and that Fc-lectin binding discriminated between mannosides 43 expressed on different cell morphologies of the same fungus. We also demonstrate CTL 44 epitope differentiation during different phases of the growth cycle of Candida albicans and that 45 MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans 46 displayed deeper in the cell wall. These immune receptor maps of fungal walls therefore reveal 47 and capable of changing its composition and organization when adapting to different growth 56 niches and environmental stresses. Differences in the composition of the cell wall lead to 57 differential immune recognition by the host. Understanding how changes in the cell wall 58 composition affect recognition by PRRs is likely to be of major diagnostic and clinical 59 relevance. Here we address this fundamental question using four soluble immune receptor-60 probes which recognize mannans and β-glucan in the cell wall. We use this novel methodology 61 to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers 62 of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces 63 did not correlate with relatedness of different fungal species, and mannan-detecting receptor-64 probes discriminated between cell surface mannans generated by the same fungus growing 65 under different conditions. These studies demonstrate that mannan-epitopes on fungal cell 66 surfaces are differentially distributed within and between the cell walls of fungal pathogens. 67 68 receptor-ligand interactions are the primary origin of all immune responses and they promote 82 expression and secretion of various chemokines and cytokines that results in recruitment of 83 neutrophils, macrophages and other immune cell types to the ...