Investigating copper storage in Pseudomonas aeruginosa and its role in pathogenicity

背景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。需要新的治疗方法来克服日益增长的抗菌素耐药性。铜绿假单胞菌是引起条件致病菌感染的主要革兰氏阴性菌，尤其是囊性纤维化患者，并对多种抗菌药表现出耐药性。铜绿假单胞菌有铜酶参与肺部的适应，促进感染。铜不仅是必需酶的辅因子，也可能是有害的。铜的潜在毒性导致了促进这种金属安全使用的动态平衡系统的演变。哺乳动物的免疫系统利用铜中毒来对抗细菌病原体，细菌病原体使用铜稳态蛋白来防御攻击。新颖性、时间线和假设丹尼森实验室发现了一个新的细菌铜储存蛋白家族--CSPs(自然2015,525,140)。这些四螺旋束结合了大量的铜(I)离子，出口的Csp1将铜(I)储存在甲烷氧化酶的甲烷氧化菌中。胞液中的Csp3在细菌中分布更广，包括铜绿假单胞菌等病原体。Csp3的表达使铜可以安全地隔离在细胞质中，但Csp3结合的铜的目的地在任何细菌中仍然未知。我们假设，在病原体中，Csp3除了为目前未确定的靶标提供安全的胞液铜储备外，还可以作为毒力因子。实验方法和目的研究铜绿假单胞菌Csp3的体外特性。这将涉及使用生化和生物物理技术来研究铜(I)的结合和释放。铜绿假单胞菌Csp3在缺乏铜外排泵COPA的大肠杆菌中恢复铜耐受性的能力将被测试。小鼠感染所需的铜绿假单胞菌CopA1和枯草芽孢杆菌Csp3的异源表达使该菌株对铜中毒具有抵抗力。Csp3基因和其他铜稳态蛋白的表达研究将在环境和临床(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。铜绿假单胞菌Csp3缺失突变体的铜依赖表型将被探索。将进行生物被膜和感染模型的毒力研究，如人工痰培养基和人类细胞系。产生的关键问题可以在自然吸入的铜绿假单胞菌慢性肺部感染的小鼠模型中得到解决。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.