Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls.

Ingrida Vendele,Lisete M Silva,Maria Spyrou,Yan Liu,Janet A Willment,Wengang Chai,Mark H T Stappers,Angelina S Palma,Neil A R Gow,Ten Feizi,Gordon D Brown

doi:10.1371/journal.ppat.1007927

Abstract

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.

Highlights

Fungi are associated with a wide spectrum of diseases ranging from superficial skin and mucosal surface infections in immunocompetent people, to life-threatening systemic infections in immunocompromised patients [1, 2]
Immune recognition of fungal pathogens involves binding of specific cell wall components by pathogen recognition receptors (PRRs) and subsequent activation of immune defences
Immune reactivity of fungal cell surfaces was not correlated with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions

Summary

Introduction

Fungi are associated with a wide spectrum of diseases ranging from superficial skin and mucosal surface infections in immunocompetent people, to life-threatening systemic infections in immunocompromised patients [1, 2]. Innate immunity is the primary defence mechanism against fungal infections and involves host Pattern Recognition Receptors (PRRs) that recognise specific Pathogen-Associated Molecular Patterns (PAMPs), which are mostly located within the cell wall [7,8,9]. These receptor-ligand interactions are the primary origin of all immune responses and they promote expression and secretion of various chemokines and cytokines that results in recruitment of neutrophils, macrophages and other immune cell types to the site of infection, which leads to containment and clearance of the pathogen and the activation of protective longer term adaptive immunity [9,10,11,12].

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Conclusion