Investigating copper storage in Pseudomonas aeruginosa and its role in pathogenicity.

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，并表现出对许多抗菌素的耐药性。铜绿假单胞菌有铜酶参与肺适应，促进感染。除了作为必需酶的辅助因子，铜也可能是有害的。铜的潜在毒性导致了体内平衡系统的进化，促进了这种金属的安全使用。哺乳动物的免疫系统利用铜毒性来对抗细菌病原体，细菌病原体利用铜稳态蛋白来抵御这种攻击。令人惊讶的是，我们对铜绿假单胞菌如何处理铜知之甚少。这种生物具有两种铜外排p型atp酶（CopA1和CopA2）。CopA1对铜的耐受性至关重要，而CopA2则需要将金属输送到需要铜的酶。CopA1是小鼠感染所必需的，而这两种copa都是植物感染所必需的。主要负责人（Nature 2015, 525:140）发现了一个新的细菌铜储存蛋白家族，Csps。这些四螺旋束结合了大量的Cu(I)离子，输出的Csp1将Cu(I)储存在甲烷氧化菌中，供主要的甲烷氧化酶使用。胞质Csp3s在细菌中更为普遍，包括病原菌如铜绿假单胞菌。表达csp3的细菌将铜隔离在其胞质中以防止毒性，但csp3结合铜的目的地在任何细菌中都是未知的。我们假设，在病原体中，除了为目前未知的靶标提供细胞质铜的安全储存外，Csp3s还可以作为毒力因子。铜绿假单胞菌的Csp3将在体外进行表征。这将涉及使用一系列生化和生物物理技术来研究Cu(I)的结合和释放。位点导向突变体的研究将识别对蛋白质关键特性最重要的残基。这项工作将通过确定装载Cu(I)的野生型（WT）蛋白的晶体结构以及具有最有趣性质的变体来进行。WT铜绿假单胞菌Csp3，以及潜在的位点定向突变体，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。从铜绿假单胞菌（小鼠感染所需）和枯草芽孢杆菌Csp3中异源表达CopA1，均在该菌株中重建了对铜毒性的抗性。csp3基因和其他铜稳态蛋白的表达研究将在铜绿假单胞菌菌株中进行，包括由第二和第三主管建立的铜绿假单胞菌国际参考小组的菌株，包括环境和临床（流行病）菌株，CF分离株和具有特定毒力特征的菌株。本文将研究铜绿假单胞菌csp3缺失突变体的表型，包括铜浓度对其生长的影响，并分析铜的分布以确定Cps3储存铜的靶点。将进行生物膜和感染模型（如人工痰培养基和人类细胞系）的毒力研究。产生的关键问题将在铜绿假单胞菌慢性肺部感染的自然吸入小鼠模型中得到解决。目前正在验证的假设是，Csp3在铜绿假单胞菌的胞质中储存和保护铜，可以将铜传递给目前尚未确定的关键酶，并在该生物的致病性中发挥重要作用。

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. P. 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 the safe use of this metal. Copper toxicity is exploited by the mammalian immune system to fight bacterial pathogens, who defend against this attack using copper homeostasis proteins. 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 whilst CopA2 is required for delivery of the metal to copper-requiring enzymes. CopA1 is necessary for infection in mice, whilst both CopAs are needed for infection in plants.The primary supervisor (Nature 2015, 525:140) has discovered a new family of bacterial copper storage proteins, the Csps. 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-expressing bacteria sequester copper in their cytosol preventing toxicity, 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.The Csp3 from P. aeruginosa will be characterised in vitro. This will involve using a range of biochemical and biophysical techniques to investigate Cu(I) binding and release. The study of site-directed mutants will identify residues that are most important for key properties of the protein. This work will be informed by determination of the crystal structure of the wild type (WT) protein loaded with Cu(I), and also variants with the most interesting properties. The ability of WT P. aeruginosa Csp3, and potentially also site-directed mutants, 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 Bacillus subtilis Csp3, both re-establish 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 P. aeruginosa strains, including those from the international reference panel of P. aeruginosa isolates, established by the secondary and third supervisors, which includes environmental and clinical (epidemic) strains, CF isolates and strains with specific virulence characteristics. The phenotype of a Csp3-deletion mutant of P. aeruginosa will be investigated including the effect of copper concentration on growth, with copper distribution analysed to identify targets for the Cps3 store of copper. Virulence studies in biofilm and infection models, such as artificial sputum medium and human cell lines, will be undertaken. Key questions generated will be addressed in a natural inhalation murine model of P. aeruginosa chronic lung infection.The hypotheses being tested are that Csp3 stores and protects copper in the cytosol of P. aeruginosa, can deliver the metal to key, currently unidentified, enzymes and plays an important role in the pathogenicity of this organism.