Characterisation of the fungal ligands, structure and function of the C-type lectin receptor, MelLec

The incidence of fungal infections has increased significantly over the last 30 years mainly because of the HIV/AIDS pandemic and invasive medical interventions. Superficial mycoses caused mainly by dermatophytes affect approximately 25% of world population while invasive fungal infections lead to more than 50% mortality. There is an urgent need to fully understand the mechanisms that our immune system employs to fight fungal infections as this will aid in the development of diagnostic tools and novel treatments.
Protection against fungal pathogens is initially provided by the innate immune system via the recognition of the conserved microbial-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). One of the families of PRRs that are critical for antifungal immunity are the C-type Lectin Receptors (CLRs). We have identified a novel CLR, the Melanin sensing C-type Lectin receptor (MelLec), that plays an important role in antifungal immunity as it recognises 1,8-dihydroxynaphthalene (DHN)-melanin in conidial spores of Aspergillus fumigatus. Unlike the other essential CLRs in antifungal immunity, murine MelLec is expressed in non-myeloid cells and is ubiquitously expressed by CD31+ endothelial cells. Interestingly, murine MelLec is also expressed by a sub-population of CD31+ cells that unusually co-express the epithelial marker, EpCAM (CD31+EpCAM+). These double-positive cells are only detected in the mouse lung and liver and their function is unknown. Using MelLec knock out mice this receptor was demonstrated to be required for protection against disseminated infection with A. fumigatus and in humans, a single nucleotide polymorphism of this receptor negatively affects the inflammatory responses and significantly increases susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. In this project, we will further explore the role of this MelLec in antifungal immunity and determine if ligands in addition to melanin are recognised.
The biosynthetic pathway of DHN-melanin is well characterised in A. fumigatus and by screening mutants we determined that the ligand heptaketide napthopyrone (YWA1) of MelLec was synthesised by polyketide synthases during the very first step of the pathway. We have shown that a purified form of YWA1 can inhibit a soluble fusion protein Fc-MelLec from binding to A. fumigatus conidia. Polyketide synthases are involved in the generation of many other fungal secondary metabolites, and in this project, using our Fc-MelLec in flow cytometry assays, we have shown that secondary metabolites of many fungal species (e.g. A. fumigatus, Penicillium species) are ligands for this receptor. Cellular responses induced by MelLec following recognition of fungal secondary metabolites will be elucidated in vitro using appropriate transfected and primary human and mouse cell lines. Responses to be examined include, for example, MelLec-mediated uptake of secondary metabolites (by flow cytometry and ELISA based methods) and the production of cytokines and chemokines. Given the huge impact of mycotoxins on human disease, we shall explore the role of MelLec in the carcinogenic activities of fungal secondary metabolites, using established in vivo models of hepatocarcinogenesis.
In this project, we will also characterise the CD31+EpCAM+ cells that express MelLec. To do this, we will isolate the CD31+EpCAM+ cells from mouse lungs and then we will perform single-cell sorting and single-cell RNA sequencing. Since these cells express MelLec, which recognises 1, 8-DHN melanin of A. fumigatus, we might find that they play a significant role in anti-fungal immunity or immunity in general. Also, because the expression of MelLec in CD31+EpCAM+ cells is tissue specific, these cells might have a distinct function on the lung which might be currently unknown.
In sum, this project will significantly advance our knowledge of the role and function of MelLec in immunity and disease.