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，其促进感染。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这种金属安全使用的稳态系统的发展。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这种攻击。令人惊讶的是，人们对铜绿假单胞菌如何处理铜知之甚少。该生物体具有两种铜流出P型ATP酶（CopA1和CopA2）。CopA1是铜耐受性所必需的，而CopA2是将金属输送到需要铜的酶所必需的。CopA1是小鼠感染所必需的，而两种CopA都是植物感染所必需的。主要主管（Nature 2015，525：140）发现了细菌铜储存蛋白的新家族，Csps。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。表达Csp3的细菌将铜隔离在其胞质溶胶中以防止毒性，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，以及提供一个安全的存储细胞溶质铜目前未确定的目标，Csp3s可以作为毒力因子。这将涉及使用一系列生物化学和生物物理技术来研究Cu（I）的结合和释放。定点突变体的研究将确定对蛋白质的关键特性最重要的残基。这项工作将通过测定负载Cu（I）的野生型（WT）蛋白质的晶体结构以及具有最有趣特性的变体来了解。将测试WT铜绿假单胞菌Csp3以及潜在的定点突变体恢复缺乏其铜外排泵CopA的大肠杆菌中的铜耐受性的能力。来自铜绿假单胞菌（小鼠感染所需的）和枯草芽孢杆菌Csp 3的CopAl的异源表达都重新建立了该菌株对铜毒性的抗性。将在铜绿假单胞菌菌株中进行csp3基因和其他铜稳态蛋白的表达研究，包括来自由二级和三级监督员建立的铜绿假单胞菌分离株国际参考样本组的研究，其中包括环境和临床（流行）菌株、CF分离株和具有特定毒力特征的菌株。将研究铜绿假单胞菌的Csp3缺失突变体的表型，包括铜浓度对生长的影响，分析铜分布以鉴定铜的Cps3储存的靶标。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。产生的关键问题将在自然吸入铜绿假单胞菌慢性肺部感染的小鼠模型中得到解决。正在测试的假设是，Csp3在铜绿假单胞菌的胞质溶胶中储存和保护铜，可以将金属传递给目前尚未鉴定的关键酶，并在这种生物体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.