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The Discovery and Application of Bacteriophage Receptor Binding Proteins

  • Author / Creator
    Simpson, David James
  • Bacteriophages are considered to be the most abundant and potentially the most diverse form of life on earth. Phage receptor binding proteins (RBPs), which allow phages to specifically target their host bacteria, consequently represent a massive diversity of bacterial targeting proteins. These RBPs can bind to bacteria with strong affinity and show considerable resistance to proteases and detergents. In recent years, a number of new technologies have been developed for pathogen detection through the attachment of RBPs to surfaces or beads. This thesis describes new techniques that take advantage of these proteins that act as surrogate antibodies. Recent years have seen a resurgence in bacteriophage research, often in the form of phage therapy, due to the recent rise in antibiotic resistance. However, bacteriophage RBPs remain difficult to identify based on homology alone due to their considerable diversity. While these proteins share a trimeric structure, they can be very dissimilar on the sequence level. In order to further exploit the use of RBPs, I have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. Briefly, the phage DNA is sheared and ligated into an expression library, this library is transferred to a nitrocellulose membrane where the colonies are induced to express the inserts, the cells are then lysed and the membrane is probed with the host bacteria. When the Salmonella enterica serovar Typhimurium phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. I then examined the Escherichia coli O157:H7 typing phage 1 using this assay and identified a previously undescribed RBP, Gp145. This general approach has the potential to assist in the identification of RBPs from other relevant bacteriophages. In previous studies the bacteriophage P22 RBP has also been shown to be able to reduce colonization of S. Typhimurium in chickens. In order to exploit this finding further, the protein was expressed in plants with the aim of creating an inexpensively produced selective antimicrobial feed. An elastin like polypeptide (ELP) tag was added to the P22 RBP to increase expression of the protein in Nicotiana benthamiana. This thesis demonstrates that the RBP containing plant extract was capable of capturing S. Typhimurium on a nitrocellulose membrane, and moderately reducing the ability of S. Typhimurium to colonize chickens. RBPs bind bacteria with high affinity, to make use of this trait in a diagnostics-based platform, the cellulose binding module CBM9, that enables proteins to bind to paper, was added to Gp9 and Gp145. The N-terminally tagged Gp9 and Gp145 constructs were spotted on paper and are able to capture S. Typhimurium and E. coli O157:H7, respectively on paper. Gp145 was further characterized and shown to bind to the lipopolysaccharide of E. coli O157:H7 and interestingly also binds to S. Typhimurium, likely through a protein receptor. Taken together, these results demonstrate that RBPs represent an exciting new technology for microbial detection and treatment which are inexpensive, easy to use and readily scalable.

  • Subjects / Keywords
  • Graduation date
    2016-06:Fall 2016
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R39S1KT36
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biological Sciences
  • Specialization
    • Microbiology and Biotechnology
  • Supervisor / co-supervisor and their department(s)
    • Szymanski, Christine (Biological Sciences)
  • Examining committee members and their departments
    • Stein, Lisa (Biological Sciences)
    • Pukatzki, Stefan (Medical microbiology and Immunology
    • Moineau, Sylvain (Département de biochimie, de microbiologie et de bio-informatique University of Laval)
    • Deyholos, Michael (Faculity of Biology University of British Columbia)
    • Dennis, Jon (Biological Sciences)