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Title: Use of C-type lectin receptor probes and human monoclonal antibodies to map the dynamics of the fungal cell wall
Author: Raziunaite, Ingrida
ISNI:       0000 0004 7432 0007
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2018
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In this thesis I investigate the repertoire and distribution of immune reactive epitopes in the cell wall of fungal pathogens. Using recombinant Fc-coupled C-Type Lectin Receptor (CLR) probes that recognise fungal mannans and human B cell-derived monoclonal antibodies (mAbs), these epitopes were mapped to the cell surface during a wide variety of culture conditions. The rationale for this study was to determine how the expression of these immunologically relevant epitopes is represented in different fungi and within a single species of fungus growing under different conditions in different morphological forms. During the course of fungal infection, pathogen recognition by the host innate immune system is critical to initiate protective immune responses. This is mediated by Pattern Recognition Receptors (PRRs) which are expressed at the surface of host immune cells and bind to Pathogen-Associated Molecular Patterns (PAMPs) located on the fungal cell wall. Differences in the composition of the cell wall lead to differential immune recognition by the host. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of CLRs which are critical for anti-fungal immune responses. The precise distribution of mannose components in different Candida species and within the cell walls of specific fungal species remains largely undefined. This project therefore addressed this fundamental question by using recombinant CLR-Fc fusion probes and human anti-Candida mAbs for the analysis of the variability of fungal cell wall mannosides. CLR-Fc probes and mAbs were generated in mammalian cell expression systems and then characterised in terms of their binding profiles to fungal cell surfaces under varying growth parameters by employing confocal microscopy, gold-labelling with transmission electron microscopy, flow cytometry and carbohydrate microarray analyses. I show that immune relevant epitopes can either be diffused or clustered, superficial or buried deep in the Candida albicans cell wall. 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. In addition, the growth cycle of C. albicans was accompanied by time-dependent masking and unmasking of particular PAMPs. This work also demonstrated that human mAbs could be attractive targets for the design of novel immunotherapeutic approaches or used as basic research tools for the characterisation of the fungal cell wall. In conclusion, this study demonstrates that the fungal cell surface is ordered but also highly complex and dynamically changing, making immune recognition a challenging process. These findings suggest that the wide range of mannan-detecting CLRs may operate both singly and in combination to provide a redundant monitoring system that can cope with the variable and changing range of mannan epitopes represented in the cell walls of fungal pathogens. This type of cell wall PAMP variation carries significant impact on the design of future fungal diagnostics, vaccines and therapeutics.
Supervisor: Gow, Neil A. R. ; Brown, Gordon Douglas Sponsor: University of Aberdeen ; Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Lectins ; Monoclonal antibodies ; Fungal cell walls