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Title: Acanthamoeba Mannose-Binding Protein: Structural and functional characterisation of a therapeutic target for Acanthamoeba keratitis
Authors: Banjo, Taiwo Abayomi
Supervisors: Wallis, Russell
Heaphy, Shaun
Award date: 20-Apr-2018
Presented at: University of Leicester
Abstract: Acanthamoeba mannose-binding protein (AcMBP) is a virulence factor of the free-living amoeba, Acanthamoeba castellanii. It is crucial for the development of Acanthamoeba keratitis (AK), a corneal infection that often causes blindness. AK is associated with contact lens use and contaminated water sources. Therapeutic unresponsiveness is attributed to similarities in the biological processes that Acanthamoeba shares with humans and its ability to form drug-resistant cysts. I aimed to characterise AcMBP as a basis for developing future drugs against Acanthamoeba. To start with, I carried out morphological studies on the two well-known life stages of Acanthamoeba and characterised a third stage: the protocysts. Mature cysts and protocysts could not interconvert directly, but always excysted to trophozoites. This is important because Acanthamoeba can potentially be trapped as protocysts, which are likely to be more susceptible to drugs. I also studied Acanthamoeba adhesion towards various surfaces and cytopathic activities towards cells (including human corneal epithelial cells). Whilst AcMBP was important for adhesion, it is not the only receptor involved. To gain structure/function information, I expressed the extracellular portion of AcMBP and three truncated fragments. AcMBP is a Ca2+-dependent lectin (~100 kDa) that binds to mannose. Ca2+ is essential for lectin activity and stability. The extracellular fragment is monomeric, indicating that trimerisation, shown previously, depends on the membrane-spanning and/or intracellular regions. Bioinformatics revealed that lectin activity is almost certainly located in a DUF 4114 domain (~10 kDa, DUF: domain of unknown function). N-terminal fragments, including the DUF4114 domain did not bind to mannose-Sepharose, suggesting that part of the cysteine-rich domain is also important. AcMBP bound to a variety of mammalian glycans so may have more than one lectin activity. Although attempts to crystallise AcMBP were unsuccessful, future structural analysis will be useful for defining the domains and determining how it binds to mannose.
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Leicester Theses
Theses, Dept. of Infection, Immunity and Inflammation

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