This interdisciplinary project bridges world-leading labs with expertise in bacterial cell biology and infection. Novel therapies are needed to overcome increasing antibacterial resistance. Pseudomonas aeruginosa is the key Gram negative bacterium causing opportunistic pathogen infections, particularly in patients with cystic fibrosis (CF), and expresses resistance to many antimicrobials. Pseudomonas aeruginosa has copper enzymes involved in adaptation in the lung that facilitate infection. As well as being required as the cofactor for essential enzymes, copper can also be harmful. The potential toxicity of copper has resulted in the evolution of homeostatic systems facilitating safe use of this metal. Copper toxicity is exploited by the mammalian immune system to fight bacterial pathogens, who defend against attack using copper homeostasis proteins.
Novelty, Timelines and Hypothesis
A new family of bacterial copper storage proteins, the Csps, have been discovered (Nature 2015, 525, 140) by the Dennison lab. These four-helix bundles bind large numbers of Cu(I) ions, and an exported Csp1 stores Cu(I) in methanotrophs for the main methane-oxidising enzyme. Cytosolic Csp3s are more widespread in bacteria, including pathogens such as P. aeruginosa. Csp3 expression allows copper to be safely sequestered in the cytosol, but the destination of Csp3-bound copper remains unknown in any bacterium. We hypothesise that in pathogens, as well as providing a safe store of cytosolic copper for currently unidentified targets, Csp3s can act as virulence factors.
Experimental Approach and Objectives
The Csp3 from P. aeruginosa will be characterised in vitro. This will involve using biochemical and biophysical techniques to investigate Cu(I) binding and release. The ability of P. aeruginosa Csp3 to restore copper tolerance in Escherichia coli lacking its copper-efflux pump CopA will be tested. Heterologous expression of CopA1 from P. aeruginosa, required for infection in mice, and Csp3 from Bacillus subtilis, confer resistance to copper toxicity in this strain. Expression studies of the csp3 gene, and those for other copper-homeostasis proteins, will be carried out in environmental and clinical (epidemic) P. aeruginosa strains, CF isolates and strains with specific virulence characteristics. A copper-dependent phenotype will be explored for the Csp3-deletion mutant of P. aeruginosa. Virulence studies in biofilm and infection models, such as artificial sputum medium and human cell lines, will be undertaken. Key questions generated can be addressed in a natural inhalation murine model of P. aeruginosa chronic lung infection. All knowledge obtained will help in devising new treatments, particularly for patients with CF.
This project will deliver training in the analysis of copper proteins in vitro, as well as the microbial ecology, molecular biology, immunology and in-cell techniques necessary to study bacterial pathogens. Approaches required to investigate how a pathogen infects different model hosts will be taught. The student will learn how to produce publication-quality data and will contribute to the preparation of manuscripts describing their work, resulting in co-authorship of papers.
The student will be based at Newcastle University in Prof Chris Dennison’s lab (https://www.ncl.ac.uk/camb/staff/profile/christopherdennison.html#background
) and the project involves collaboration with Prof Anthony De Soyza (https://www.ncl.ac.uk/icm/people/profile/anthonyde-soyza.html#background
) and Prof Craig Winstanley (https://www.liverpool.ac.uk/infection-and-global-health/staff/craig-winstanley/
Informal enquiries can be made to [email protected]
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 can be found on our website: http://www.dimen.org.uk/
C. Dennison, S. David (PhD student), and J. Lee (PhD student), Bacterial copper storage proteins. J. Biol. Chem. 2018, 293, 4616-4627.
Y. Hilliam (PhD student), M. P. Moore (PhD student), I. L. Lamont, D. Bilton, C. S. Haworth, J. Foweraker, M. J. Walshaw, D. Williams, J. L. Fothergill, A. De Soyza, and C. Winstanley, Eur. Respir. J. 2017, 49:1602108.
N. Vita, S. Platsaki (PhD student), A. Basle, S. J. Allen, N. G. Paterson, A. T. Crombie, J. C. Murrell, K. J. Waldron and C. Dennison, A four-helix bundle stores copper for methane oxidation. Nature 2015, 525, 140-143.