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Title: Structural and functional characterisation of adhesin and effector proteins from pathogenic fungi
Author: Jones, Rhian
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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This PhD has focussed on characterising two proteins key to fungal infections; the Als adhesins and BEC1054. The Als (agglutinin-like sequence) family of surface adhesins are critical virulence factors from Candida albicans, contributing to biofilm formation and colonisation of a range of tissues in the human host. The broad tissue tropism of Als has been linked to a broadly specific peptide binding cavity (pbc) identified in crystal structures of Als N-terminal domains (NTs). Questions remained regarding the relative roles of the pbc and Als aggregative mechanisms in host cell binding. A series of targeted mutants generated in Als3 were characterised in vitro via NMR and in vivo (collaborator’s work) to delineate these adhesion mechanisms and demonstrate that the pbc alone mediates host binding. It was further demonstrated that sequence differences in the pbcs of Als1 and 3 underlie differences in Als substrate binding specificities. Solution based assays showed that Als3 binds to a wider range of peptide sequences; correlating with a larger pbc and additional interactions with peptides formed in Als-peptide co-crystal structures. We hypothesise that the broader binding specificity of Als3 correlates with the protein’s increased contribution to C. albicans adhesion and virulence in infection. Binding assays provided new insights into the peptide binding mechanism, and a robust system for future development of novel Als inhibitors. Following infection of barley, Blumeria graminis delivers effector proteins such as BEC1054 into the plant to perturb host immunity. The crystal structure of BEC1054 was solved, revealing an RNAse fold that lacks catalytic residues required for hydrolysis. Binding to nucleic acid has been demonstrated, and the binding site localised via mapping of chemical shift perturbations (CSPs) in NMR. Preliminary data have been acquired to test the theory that BEC could associate with ribosomes. Understanding the role of BEC1054 could provide important insights into effector-based modulation of plant immunity, an area that is currently poorly understood.
Supervisor: Cota, Ernesto Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral