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Phages as the ultimate puppeteers of bacterial metabolism

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

Secondary supervisor: Dr Hasan Yesilkaya

PhD project title: Phages as the ultimate puppeteers of bacterial metabolism

University of Registration: University of Leicester

Project outline:

Bacteriophages, or phages are bacterial viruses, and are the most abundant and diverse biological entities on Earth. Throughout the biosphere they outnumber their bacterial hosts by at least 10 to 1 and strikingly, the majority of the functions of most genes encoded by phages remains unknown. What is known is that their impact on life ranges from the maintenance of healthy microbiota to the promotion of biodiversity in soil and oceans. Phages also have a plethora of potential practical uses, from the treatment of infections caused by antibiotic resistant bacteria to preventing antibiotic resistance build up within food animals and increasing the shelf life of food products to reduce global food waste.

To optimise and extend the applications of phages within food security and disease settings, it is important to understand how different phages impact their host bacteria, particularly how they manipulate their metabolism. Our lab and work of others have shown using metabolomic profiling experiments that phages can profoundly alter the metabolites that bacteria express [1-2] and reviewed and contextualised in our review [3]. However, our understanding of the underlying molecular and genetic mechanisms by which this happens is really limited. To unravel this during this exciting PhD project, the student will work in an interdisciplinary way both in a phage-focused laboratory, in an analytical chemistry lab, and within a group focused on determining how metabolism impacts bacterial phenotype.

Aims and Objectives

In this project, we aim to test the hypothesis that particular phage types target specific bacterial pathways and determine the mechanistic basis for such targeting, which will help us better develop phages for therapy.

Specific Objectives are:

  1. Determine how phages impact bacterial metabolism by using RNASeq and qPCR and analytical chemistry in order to probe infected transcriptomes
  2. By comparative analysis of transcriptomic data, identify phage specific infection strategies for the phages tested, for example establish if phages that are equally aggressive impact the same metabolic pathways
  3. Make bacterial knockout mutants of whole or partial metabolic pathways targeted by phages and determine how phages interact with these bacterial mutants
  4. Establish the impact of a) infected cells and b) phage resistant mutants on bacterial virulence and eukaryotic host response in a range of models including biofilm generation, capsule synthesis, and virulence.

In order to answer these objectives, in this project we will use Klebsiella and it’s phages as a model system. We will study the impact of our diverse, well characterised Klebsiella phage collection on its bacterial host metabolism. Klebsiella are Gram-negative bacteria, some of which cause diseases and others are found in the environment as free-living microbes. They have diverse metabolic adaptive capabilities including the ability to fix nitrogen. K. pneumoniae can also cause many diseases including pneumonia, urinary tract infection, bacteremia, and septicaemia in humans and animals, showing that the microbe is environmentally adept very likely due to its inherent metabolic flexibility. Worryingly, many strains are antibiotic resistant thus pose a serious health threat, hence it is one of the few pathogens for which there is an urgent need for effective antibiotics as declared by the WHO.

Importance

This project aims to provide a fundamental understanding of the relationship of specific phages to their bacterial hosts and in doing so to compare how the host takeover strategies differ between phages, and how the subsequent behaviour of the infected bacteria is affected when challenged with different phages. This dataset and outputs from the thesis would provide the proof of a novel method by which to assess phages for therapeutic and industrial applications.

References:

  1. Howard-Varona, C., Lindback, M.M., Bastien, G.E. et al. Phage-specific metabolic reprogramming of virocells. ISME J 14, 881–895 (2020). https://doi.org/10.1038/s41396-019-0580-z
  2. De Smet, J., Zimmermann, M., Kogadeeva, M. et al. High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection. ISME J 10, 1823–1835 (2016).https://doi.org/10.1038/ismej.2016.3
  3. Francesca E. Hodges, Thomas Sicheritz-Pontén, and Martha R.J. Clokie. PHAGE.Mar 2021.16-25.http://doi.org/10.1089/phage.2020.0041

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Animal health and welfare & Microbial Food Safety & Understanding the Rules of Life: Microbiology & Systems Biology

Techniques that will be undertaken during the project:

  • Maintenance and handling of bacterial cultures and bacteriophage stocks
  • Bacteriophage enumeration and phenotypic characterisation
  • Transcriptional profiling by RNAseq and quantitative reverse transcriptase real time PCR assays.
  • Analytical Chemistry using Gas Chromatography Mass Spectrometry
  • Targeted mutation and associated molecular techniques
  • In vivo assays using mouse and/or Galleria mellonella

Contact: Professor Martha Clokie, University of Leicester