Investigating copper storage in Pseudomonas aeruginosa and its role in pathogenicity.

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. P. 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 the safe use of this metal. Copper toxicity is exploited by the mammalian immune system to fight bacterial pathogens, who defend against this attack using copper homeostasis proteins. Surprisingly little is known about how P. aeruginosa handles copper. This organism possesses two copper-effluxing P-type ATPases (CopA1 and CopA2). CopA1 is essential for copper tolerance whilst CopA2 is required for delivery of the metal to copper-requiring enzymes. CopA1 is necessary for infection in mice, whilst both CopAs are needed for infection in plants.

The primary supervisor (Nature 2015, 525:140) has discovered a new family of bacterial copper storage proteins, the Csps. 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-expressing bacteria sequester copper in their cytosol preventing toxicity, 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.

The Csp3 from P. aeruginosa will be characterised in vitro. This will involve using a range of biochemical and biophysical techniques to investigate Cu(I) binding and release. The study of site-directed mutants will identify residues that are most important for key properties of the protein. This work will be informed by determination of the crystal structure of the wild type (WT) protein loaded with Cu(I), and also variants with the most interesting properties. The ability of WT P. aeruginosa Csp3, and potentially also site-directed mutants, 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 Bacillus subtilis Csp3, both re-establish 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 P. aeruginosa strains, including those from the international reference panel of P. aeruginosa isolates, established by the secondary and third supervisors, which includes environmental and clinical (epidemic) strains, CF isolates and strains with specific virulence characteristics. The phenotype of a Csp3-deletion mutant of P. aeruginosa will be investigated including the effect of copper concentration on growth, with copper distribution analysed to identify targets for the Cps3 store of copper. Virulence studies in biofilm and infection models, such as artificial sputum medium and human cell lines, will be undertaken. Key questions generated will be addressed in a natural inhalation murine model of P. aeruginosa chronic lung infection.

The hypotheses being tested are that Csp3 stores and protects copper in the cytosol of P. aeruginosa, can deliver the metal to key, currently unidentified, enzymes and plays an important role in the pathogenicity of this organism.