Bacteriophage engineering as a therapeutic strategy to target antibiotic resistant enterococci

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About the ProjectThis project will build on our preliminary work to develop phage therapeutics targeting Vancomycin-Resistant-Enterococci (VRE), which are opportunistic pathogens causing both community and hospital-acquired infections (Al-zubidi et al., 2019). Due to the emergence of pathogens multi-resistant to antibiotics, there is a pressing need for novel antimicrobial strategies. Bacteriophages are highly suitable as therapeutic agents because they target specific pathogens within complex bacterial communities without causing a microbiome imbalance. Bacteriophages recognise specific structures at the bacterial cell surface and usually display a narrow host range, sometimes limited to a few strains. To circumvent this potential issue, phages isolated from the environment are combined to produce "phage cocktails" with an extended host range. An alternative approach is to carry out phage engineering to modify the receptor binding proteins, to generate tailored therapeutics.The specific objectives of the project are the following:1) Characterising the receptor binding proteins of phages targeting enterococci. We recently isolated virulent phages targeting Enterococcus faecalis. Three phages displayed high sequence identity but distinct host ranges. One phage was shown to recognise decorations of the Enterococcal Polysaccharide Antigen (EPA), variable between strains. Using the expertise of the primary supervisor in protein-bacterial cell wall interactions, we propose to investigate how phage receptor proteins recognise the EPA. We will use both bacterial mutants and in vitro interaction assays with recombinant receptor binding proteins and purified cell wall fragments to elucidate the molecular mechanism underpinning surface recognition by phages. 2) Engineering recombinant virulent phages with altered host range. Recombinant phages encoding distinct or combined receptor binding proteins will be assembled in vitro and "rebooted" using golden gate assembly to modify or expand their host range of. Using error-prone PCR, we will explore the possibility to generate phages with extended host range.3) Exploring the therapeutic potential of recombinant phages. The antimicrobial activity of phages against planktonic cultures and biolfilms will be tested. We will also investigate how phages eradicate infections in the context of host-pathogen interaction using the zebrafish experimental model of infection during mono-or polymicrobial infections.This work will exploit the transformative strategy recently described that allows the production of tailored-made phage genomes that can be assembled using golden gate assembly. The project will generate new therapeutic agents to treat bacterial infection caused by VRE, accounting for a large proportion of nosocomial infections.The project will involve a multidisciplinary approach encompassing microbiology, cell wall biochemistry, synthetic biology and host-pathogen interactions.