DiMeN Doctoral Training Partnership: Investigating a cytosolic copper storage protein in Pseudomonas aeruginosa and its role in pathogenicity

Summary: Copper homeostasis in a bacterial pathogen is linked to infection mechanisms. Copper is used by the host to fight bacteria and the pathogen is able to resist this chemical attack. Pseudomonas aeruginosa requires copper for essential enzymes that are involved in infection, for example during adaptation within the lungs of patients with cystic fibrosis (CF). Interestingly, P. aeruginosa possesses an unstudied member of a new family of bacterial copper storage proteins (the Csps) that we have recently discovered (http://www.nature.com/nature/journal/v525/n7567/full/nature14854.html). Many Csps, including the P. aeruginosa protein are cytosolic, an unexpected finding considering that bacteria are not though to use copper in this compartment due to its toxicity. Understanding the role of a Csp in copper homeostasis in P. aeruginosa, and its influence on pathogenesis, will be addressed in this multidisciplinary project using an array of biochemical, biophysical, microbiological, genomic, molecular biology, cell biology and metalloproteomic techniques.

Detailed project description: Copper is essential for almost all organisms yet can also be harmful due to its redox activity and ability to bind to sites for other metals. The potential toxicity of copper has resulted in the evolution of homeostatic systems that facilitate the use of this metal ion as the cofactor for many important enzymes in both eukaryotes and prokaryotes. Copper toxicity is exploited by the mammalian immune system to fight bacterial pathogens. Bacteria defend against this attack using copper homeostasis proteins. P. aeruginosa is a Gram negative bacterium and an opportunist pathogen, particularly in patients with CF, and possesses copper enzymes known to be involved in adaptation in the lung that facilitates infection.

A new family of bacterial copper storage proteins, the Csps, were recently discovered (http://www.nature.com/nature/journal/v525/n7567/full/nature14854.html) by Dennison and Waldron (third supervisor based at Newcastle University). These proteins are four-helix bundles possessing many Cys residues enabling the binding of a large number of Cu(I) ions. Csps exported from the cytosol in methanotrophs store copper for the main methane-oxidising enzyme. Cytosolic Csp3s are present in a wide range of 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. In pathogens, we hypothesise that Csp3s are important in defence against copper attack by a host’s immune system.

Despite the importance of copper for the pathogenicity of P. aeruginosa surprisingly little is known about how this organism handles this metal ion. P. aeruginosa possesses two copper-effluxing P-type ATPases (CopA1 and CopA2). CopA1 is essential for copper tolerance and infection in mice, whilst both CopA1 and CopA2 are required for infection in plants. A role for CopA2 in copper protein biosynthesis has been suggested but not proven. Heterologous expression of CopA1 in an Escherichia coli strain lacking its copA gene confers resistance to copper toxicity as does heterologous expression of Csp3 from Bacillus subtilis.

We will characterise the Csp3 protein from P. aeruginosa in vitro with an array of biochemical and biophysical techniques, determining how it binds and releases Cu(I). The ability of Csp3 to prevent toxicity in the CopA deletion strain of E. coli and competition between Csp3 and CopA for cytosolic copper will be studied. How Csp3 deletion in P. aeruginosa influences copper distribution will be analysed using metalloproteomics to identify targets for its store of copper. The phenotype of the Csp3-deletion strain will be investigated, including virulence studies in biofilm and infection models with Winstanley at the University of Liverpool (http://www.nature.com/articles/ncomms5780). Key questions generated as a result of these experiments will be addressed in a natural inhalation murine model of P. aeruginosa chronic lung infection. The hypotheses being tested are that Csp3 provides a safe store of cytosolic copper for currently unidentified targets and can act as a virulence factor in P. aeruginosa.