Characterisation of a pair of copper storage proteins from pathogenic bacteria

Abstract

Neisseria gonorrhoeae is a pathogenic Gram-negative bacterium that causes human gonorrhoeal disease. Its genome encodes a putative periplasmic homologue of a new family of copper storage proteins (Csp) recently described in methanotrophic bacteria. Although members of this protein family are abundant in pathogenic bacteria, the presence of this periplasmic Csp in N. gonorrhoeae is almost unique. Most Csp genes in pathogenic bacterial genomes lack a signal peptide and are therefore presumed to be cytosolic. For example, the Csp protein encoded in genomes of Salmonella sp. lacks a signal sequence. However, the N. gonorrhoeae Csp possesses a putative Tat signal for targeting it to the periplasm. Salmonella enterica is an important global pathogen, consisting of more than 2500 various serovars that can be host-specific or can have a broad range of hosts, whereas N. gonorrhoeae is highly specialised for infecting humans. Antibiotic resistance in both of these bacteria, N. gonorrhoeae and S. enterica, further enhances their risk to human health. Copper is a redox active metal that is essential for several biological functions as a cofactor used by a number of copper-dependent enzymes. However, excess copper is toxic; thus, its homeostasis is carefully regulated through a system of protein transporters, sensors, trafficking proteins, and storage proteins. The Waldron lab is studying the form and function of these Csp proteins in pathogenic bacteria, as copper is known to play an important role in the innate immune system’s ability to fight infection. It is anticipated that a putative role for N. gonorrhoeae Csp1 and Salmonella Csp3 in defending these pathogens from attack by the immune system would make these proteins potential therapeutic targets for future antibiotics. This study explored the copper binding properties of Csp1 from N. gonorrhoeae and of Csp3 from S. enterica, in order to understand how they may be able to aid virulence, either through sequestration of excess copper, thereby reducing copper toxicity, or by storing copper during times of abundance and subsequent release of copper during copper deficiency. Copper binding by the Csp proteins was assessed, and the crystal structure of Salmonella Csp3 was determined. We concluded that Csps bind a large number of copper ions, likely as a storage mechanism, within a four-helix bundle structure that could be targeted in future drug discovery programmes.