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Title: Infection mechanism of filamentous bacteriophage fd : interaction between E. coli F-pilus and phage protein pIII
Author: Deng, L. W.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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Gene 3 protein (pIII), a minor coat protein located at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. An introduction and the experimental materials and methods are described in Chapters 1 and 2, respectively. Chapter 3 describes the construction and expression of the N-terminal di-domain and individual domains of pIII protein. The plaque-forming assay in vivo was used to investigate the ability of the isolated pIII domains to interact with F-pilus. Chapter 4 presents the investigation of the structure of F pilus, including displaying fragments of F pilus subunit (pilin) by means of phage display technology, expression of pilin subunit and X-ray fibre-diffraction of F pilus. Site-directed alanine mutagenesis of the second N-terminal domain of pIII in the phage as well as the evaluation of their infectivity are described in Chapter 5. Chapter 6 describes the establishment of a competitive ELISA assay in vitro in order to analyse all the mutated phages. Combining the results from two assays and mapping out the affected residues on the 3D structure, a region located at the outer rim of pIII-D2 domain was implicated as the F pilus binding epitope. Second generation mutagenesis and evaluation of these mutants were performed in order to define the interface region more closely (Chapter 7). Furthermore, several pIII proteins each containing an alanine mutation were isolated to investigate whether any major disruption of protein integrity occurs. Finally, Chapter 8 places this work in perspective and offers ideas for further work.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available