Investigating copper storage in Pseudomonas aeruginosa and its role in pathogenicity

这个跨学科的项目将世界领先的实验室与细菌细胞生物学和感染方面的专业知识联系起来。需要新的疗法来克服日益增加的抗菌素耐药性。铜绿假单胞菌是引起机会性病原体感染的关键革兰氏阴性菌，特别是在囊性纤维化（CF）患者中，并对许多抗菌药物表现出耐药性。铜绿假单胞菌具有参与肺适应的铜酶，从而促进感染。除了作为必需酶的辅因子外，铜也是有害的。铜的潜在毒性导致了促进安全使用这种金属的稳态系统的发展。铜毒性被哺乳动物免疫系统利用来对抗细菌病原体，细菌病原体利用铜稳态蛋白来防御攻击。 新奇、时间线和假设Dennison实验室已经发现了细菌铜储存蛋白的新家族Csps（Nature 2015，525，140）。这些四螺旋束结合大量的Cu（I）离子，并且输出的Csp 1在甲烷氧化菌中为主要的甲烷氧化酶储存Cu（I）。胞质Csp3在细菌中更普遍，包括病原体如铜绿假单胞菌。Csp3表达允许铜被安全地隔离在胞质溶胶中，但Csp3结合铜的目的地在任何细菌中仍然未知。我们假设，在病原体中，以及提供一个安全的存储细胞溶质铜目前尚未确定的目标，Csp3s可以作为毒力因子。 实验方法和目的将在体外表征来自铜绿假单胞菌的Csp 3。这将涉及使用生物化学和生物物理技术来研究Cu（I）的结合和释放。将测试铜绿假单胞菌Csp 3恢复缺乏其铜外排栗CopA的大肠杆菌中的铜耐受性的能力。来自铜绿假单胞菌的CopA1（小鼠感染所需）和来自枯草芽孢杆菌的Csp 3的异源表达赋予该菌株对铜毒性的抗性。将在环境和临床（流行）铜绿假单胞菌菌株、CF分离株和具有特定毒力特征的菌株中进行csp3基因和其他铜稳态蛋白的表达研究。将探索铜绿假单胞菌Csp3缺失突变体的铜依赖性表型。将在生物膜和感染模型中进行毒力研究，如人工痰培养基和人类细胞系。产生的关键问题可以在铜绿假单胞菌慢性肺部感染的自然吸入小鼠模型中解决。所有获得的知识将有助于设计新的治疗方法，特别是对CF患者。

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.