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The understanding and characterisation of gut phages

Primary Supervisor: Professor Martha Clokie, Department of Genetics and Genome Biology

Secondary supervisor: Andrew Millard and Dr Ed Galyov

PhD project title: The understanding and characterisation of gut phages

University of Registration: University of Leicester

Project outline:

Characterising the defence strategies of gut pathogens against bacteriophages

Background

Healthcare associated infections are a major problem for patients and a global health challenge. Clostridium difficile infection (CDI) is particularly problematic, for example the UK has ~20,000 infectious diarrhoeal cases every year, and progressive complications can lead to death in ~3% of infected patients. This observed high morbidity and mortality, is attributed to the emergence of severe and antibiotic-resistant strains, and inadequate treatment options. Conventional treatment of CDI is limited to three antibiotics, and there are problems with efficacy, cost and with the observation that they can trigger dysbiosis (microbial imbalance) that leads to recurrent infection. The related Clostridium perfringens is also of concern both to humans and animals where it causes food poisoning in humans and necrotic enteritis in animals. Salmonella, another gut pathogen is associated with contaminated food, and is becoming increasingly resistant to antibiotics.

To effectively control CDI, C. perfingens and Salmonella novel specific antimicrobials are urgently needed, preferably those that will target the releant pathogen and not disrupt the ‘commensal’ microbiota. Previously, we have isolated set of bacteriophages (phages) that target C. difficile, C. perfringens and Salmonella, and these phages have significant therapeutic promise (1-3). It is clear however bacteria from these genera have multiple ways of evading phages. These defence methods need to be understood within the context of using phages as therapeutics. In this PhD the student will determine how bacteria most commonly develop resistance to phages. They will subject key bacterial strains to phages and determine rates of receptor-based resistance, CRISPR based resistance and of lysogeny. They will also determine the impact of the phages on the rest of the gut microbiota.

Hypothesis

The overall hypothesis to be tested is that C. difficile, C. perfingens and Salmonella have multiple ways of becoming resistant to phages, and that all methods contribute to phage evasion in these bacterial species.

Methodology and approaches

Objective 1. To identify all genes involved in phage resistance student will use transposon mutagenesis to create a library of relevant bacterial strains or use exsisting libraries, with at least one fatal mutation per gene. They will subject the library to phages identify all the genes involved with phage resistance. They will use cutting edge bioinformatic analysis approaches to determine key genes of interest.

Objective 2. From the set of mutants generated above the receptor-based mutants will be confirmed by adsorption assays.

Objective 3. Rates of lysogeny will be determined subjecting bacteria to high dosages of phages as described in 1, and then using PCR to identify how often phages have integrated. Rates for our key phages will be tested in different strain backgrounds.

Objective 4. To determine the importance of the CRISPR systems the student will take strains of C. difficile that have active CRISPR regions and use PCR to determine how the CRISPRs respond to phage infection.

Objective 5. To establish how phages impact gut microbiota the phages under study will be introduced in in vitro and in animal model systems and the resulting bacteria analysed using metagenomic approaches.

This PhD will obtain an assessment of how frequently different phage defence systems are deployed. Data from this basic science will inform future healthcare translation.

References:

  1. Nale et al. (2016), Antimicrob Agents Chemother, 60:968; 2 Nale, et al. (2018) Antibiotics 7, 13; 3 Shan, et al. (2018) Scientific Reports 8, 5091

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Animal Health and Welfare & Microbial Food Safety. Understanding the Rules of Life: Immunology & Microbiology. Integrated Understanding of Health: Pharmaceuticals

  • Techniques that will be undertaken during the project:
  • Molecular biology; Creation of transposon libraries
  • Genome sequencing for bacteria and phages and metagenomes, so DNA extraction and library preparation
  • Microbiology; Plaque assays, phage titration and bacterial culture, working in with in vitro model systems
  • Bioinformatic analysis and comparative analysis of genomes and metagenomes

Contact: Professor Martha Clokie, University of Leicester