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Title: Engineering bacteriophages to enhance their potential use in therapy
Author: Grigonyte, Aurelija
ISNI:       0000 0004 8497 6069
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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The rise of antibiotic resistance (AMR) is one of the world's major health threats1. The infections caused by antimicrobial resistance bacteria are increasing faster than the introduction of new antibiotics2,3. The emergence of a multi-drug resistant bacteria has pushed towards bacteriophage therapy as one of the alternatives to antibiotics4. Bacteriophages are viruses that infect bacteria. Lytic phages have been examined as a potential therapy against drug resistant bacteria because they propagation involves killing of their bacterial host5-7. In addition to vast number of advantages as therapeutics, bacteriophages have several limitations i.e. narrow host range and pharmokinetics - not being able to get high enough phage concentration to the site of infection. A variety of methods have been used for the genetic modification of bacteriophage genomes to undercome bacteriophage limitations, including Yeast Artificial Chromosome (YAC), CRISPR/Cas systems and marker-based methods. However, no direct comparison has been carried to standardise the methods in question. This study aimed to establish the most efficient method by comparing effectiveness of CRISPR/Cas and marker-based systems (Chapter 3) followed by implementation of the method to address narrow host range and tissue binding specificity. I show that the most optimal engineering method is trxA marker-based method (Chapter 3). I then modify T7 tail fibers using the method established to retarget an alternative host, Bordetella pertussis (Chapter 4) and characterise them on E. coli expressing the receptor of interest as well as Bordetella pertussis and Bordetella bronchiseptica strains (Chapter 4). This uncovered an unexpected T7 infectivity of Bordetella bronchiseptica strains. Furthermore, I uncovered a novel way for T7 infection, a co-infection between T7 and genetically modified (chimeric phage) (Chapter 5). I hypothesised that dependence arose from T7 lack of host factor gene (trxA) and chimeric phage lack of infective tail fibers. I examined the co-dependency by inducing the bacterial lysis after secondary addition of either of the phages to verify that both phages were required to yield the lysis. I further demonstrate that phage genetic modification can allow phage to carry additional cargo, that can be used to kill bacteria in addition to the phages natural host. To do this I engineered phage T4 to express three different endolysins, and verify the ability of these phage to also lyse S. aureus, C. difficile and B. subtilis. Finally, I examined ways of making T7 phage tissue specific. This was achieved by genetically modifying T7 capsid, major and minor proteins, with small homing peptides that specifically bind to lung epithelial tissue (Chapter 7). This study provides an important leap forward in our understanding of bacteriophage engineering towards therapeutics. In so doing it also uncovers unexpected co-operative phage behaviour upon infection when exposed to limiting resources.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology ; RM Therapeutics. Pharmacology