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Title: Structures of epitopes on filamentous bacteriophage virions and the viral interaction with bacterial pili
Author: Darton, Nicholas John
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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In this thesis the structural effect of introducing a disulfide constraint into phage-displayed epitopes has been studied by means of chemical modification and limited proteolysis. It is shown that when peptide epitopes were engineered into disulfide-constrained loops on hybrid virion they form tight folds less susceptible to proteinases, held in place by the disulfide constraint. The binding of HIV-1 neutralising antibodies to an HIV-1 epitope displayed on phage was found to improve substantially following the introduction of this disulfide constraint. In these experiments, a possible method, based on protein ligation using intein technology, was also explored to circumvent the limiting effect that phage assembly has on the size and nature of peptides that can be displayed on the virion. Filamentous bacteriophages infect bacteria by means of attaching to an extracellular filamentous pilus. Type-IV pili, which mediate the infection of Pseudomonas aeruginosa strain K (PAK) by the Pfl bacteriophage, are also found on the surface of other human pathogenic bacteria including Neisseria gonorrhoea and Vibrio cholerae. The pathogenicity of these bacteria is known to be dependent on bacteriophage infection. Type-IV pili are also found to be important for bacterial motility and host colonisation. There is therefore a particular interest in the structure and biology of type-IV pili, as they may provide a useful therapeutic target. The structure and function of the PAK pilus was investigated by X-ray diffraction, electron microscopy, mass spectrometry, velocity gradient centrifugation, chemical modification, computer modelling and ELISA. The resulting findings allowed detailed mapping of the intra-assembly contacts between the pilin protein subunits in the pilus. This provided crucial information for developing a final model of the PAK pilus, based on 5 pilin subunits per turn in a helical assembly, which shed light on its interaction with mammalian tissues and bacteriophage Pfl virions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available