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Title: Peptide aptamer selection for antifungal drug discovery and diagnostics
Author: Grice, Christopher Martin
ISNI:       0000 0004 6423 3161
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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The demand for more effective fungal diagnostics and therapeutics is becoming increasingly urgent with increases in incidence of fungal diseases, antifungal resistance and lack of rapid diagnosis resulting in high mortality rates. The research described in this thesis evaluates the Aspergillus fumigatus pH-signalling receptor PalH (which non-redundantly regulates processing of the transcription factor PacC, and is essential for pathogenicity), as a viable therapeutic target. To assess intracellular modulation of PalH functionality, a novel proof-of-principle library of peptide aptamers (PAs), constrained within an inert thioredoxin A (TrxA) scaffold, was constructed for the expression and isolation of anti-PalH PAs using a S. cerevisiae membrane two-hybrid (YMTH) assay. Three PAs demonstrating significant binding to PalH were recovered. In parallel, peptide antigens representing the four PalH surface exposed regions, were synthesised to permit the generation of murine monoclonal antibodies (mAbs) to PalH. Both anti-PalH PAs and anti-PalH mAbs were found to modulate PalH functionality towards a gain-of-function alkaline-mimicking phenotype. These findings demonstrate that therapeutic modalities having the ability to modulate PalH functionality, are accessible by such means and availability of such reagents can assist further characterisation of PalH mode of action. To gain insight into a proposed role for PalH oligomersation in pH signalling and pathogenicity, co-immunoprecipitation of differentially tagged PalH variants derived from A. nidulans diploid analysis, confirmed constitutive presence of an oligomer regardless of environmental pH. Finally, the YMTH assay was further exploited to isolate novel interactors of PalH which could serve as alternative therapeutic targets. The proteins CipC (antibiotic response-like protein), COMPASS complex subunit Sdc1, and a hypothetical protein, were isolated as PalH-interacting moieties impacting pH-dependent activities in A. fumigatus.
Supervisor: Bignell, Elaine ; Armstrong-James, Darius Sponsor: Biotechnology and Biological Sciences Research Council ; Pfizer Ltd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral