MRC DiMeN Doctoral Training Partnership: Understanding recognition of enterococcal cell surface by bacteriophages to produce genetically modified phages as novel therapeutics

Background
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 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.

Aim and objectives
We propose to 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.
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 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” after transformation of L-form bacteria 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.

Experimental strategy
The project will generate new therapeutic agents to treat bacterial infection caused by VRE, accounting for a large proportion of nosocomial infections. It will involve a multidisciplinary approach encompassing microbiology, cell wall biochemistry, synthetic biology and host-pathogen interactions.
The project will rely on experimental strategies available in the supervisors labs to explore protein-cell wall interactions (Gonzalez-Delgado et al., Nature Chemical Biology, 2020 16(1):24-30), phage activity (Al Zubidi et al., Infection and Immunity, 2019, 87(11):e00512-19) and virulence in the context of the zebrafish model of infection (Prasjnar et al., Infection and Immunity 2013 81(11):4171-4179).
The project will also exploit the transformative strategy recently described that allows the production of tailored-made phage genomes that can be “rebooted” in bacterial L-forms (Kilcher et al., PNAS, 2018 115(3):567-572).

For more info about the supervisor”s research:
http://www.stafford.group.shef.ac.uk/
https://mesnagelab.weebly.com/


Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.

Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards

Further information on the programme and how to apply can be found on our website:
https://bit.ly/3lQXR8A