Investigating the role of a cytosolic copper storage protein in Pseudomonas aeruginosa and the link to pathogenicity

Copper is essential for almost all organisms yet can also be harmful due to its redox activity and ability to bind at sites for other metals. This has resulted in the evolution of homeostatic systems that facilitate copper's use as the cofactor for many important enzymes in both eukaryotes and prokaryotes. A new family of bacterial copper storage proteins, the Csps, were recently discovered (Nature 2015, 525, 140-143) in BBSRC-funded work (BB/K008439/1) by Dennison and Waldron. These four-helix bundle proteins possess a large number of Cys residues enabling the binding of many 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 Pseudomonas aeruginosa. Csp3-expressing bacteria sequester copper in their cytosol thus preventing toxicity. Bacteria are not thought to use cytosolic copper enzymes, and the destination of Csp3-bound copper remains unknown in any organism.

The toxicity of copper is used by the mammalian immune system to fight bacterial pathogens. Bacteria defend against this attack with copper homeostasis proteins and we hypothesise that Csp3s are important in this process. P. aeruginosa is a Gram negative bacterium and an opportunist pathogen that possesses copper enzymes known to be involved in adaptation that facilitates infection, for example in the lungs of patients with cystic fibrosis. 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 and heterologous expression in an Escherichia coli strain lacking its copA gene confers resistance to copper toxicity, as does heterologous expression of Csp3 from Bacillus subtilis. CopA1 is required for infection in mice, whilst both CopAs are needed for infection in plants.

We will characterise the Csp3 protein from P. aeruginosa in vitro with an array of biophysical techniques, determining how it binds and releases Cu(I). The ability of Csp3 to prevent toxicity in the E. coli strain lacking CopA will be tested. How Csp3 deletion in P. aeruginosa influences copper distribution will be analysed to identify targets for its store of copper. The phenotype of the csp3-deletion strain will be investigated, including virulence studies. The hypotheses being tested are that Csp3 provides a safe store of cytosolic copper for currently unidentified targets in P. aeruginosa and that it acts as a virulence factor.

This project fits within the remit of the 'world-class underpinning bioscience' BBSRC DTP strategic research area. Furthermore, the metalloproteomic approaches that we are developing to analyse the distribution of copper within a cell fall within the 'exploiting new ways of working' DTP enabling theme, and facilitated the initial discovery of the Csps (Nature 2015, 525, 140-143). We will carry out world-class bioscience that will not only provide understanding of how bacteria handle copper, but will elucidate how this process enables a pathogen to overcome a host's defences to cause infection. The biochemical and structural studies on Cu(I) binding by Csp3 will help maintain strength in core underpinning disciplines, including chemical and structural biology. The production and analysis of mutants (both gene-deletion and site-directed) requires skills in the core areas of molecular and cellular biology. The combination of all these approaches in a collaborative multi-disciplinary project will provide vital information about the function of a new class of copper storage proteins. Furthermore, they will also give mechanistic insight into the potential role of Csp3 in pathogenicity, which will help in devising approaches to treat P. aeruginosa infections.