Investigating copper storage in Pseudomonas aeruginosa and its role in pathogenicity
Lead Research Organisation:
Newcastle University
Department Name: Translational and Clinical Res Institute
Abstract
Background
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 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.
Organisations
People |
ORCID iD |
| Christopher Duncan (Primary Supervisor) | |
| Catherine Hatton (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| MR/N013840/1 | 01/10/2016 | 30/09/2025 | |||
| 2306762 | Studentship | MR/N013840/1 | 01/10/2019 | 31/03/2023 | Catherine Hatton |