Investigating the role of a cytosolic copper storage protein in Pseudomonas aeruginosa and the link to pathogenicity

铜对几乎所有生物体都是必不可少的，但也可能是有害的，因为它的氧化还原活性和结合其他金属的能力。这导致了体内平衡系统的进化，促进了铜作为真核生物和原核生物中许多重要酶的辅因子的使用。最近在Dennison和沃尔德龙的BBSRC资助的工作（BB/K 008439/1）中发现了细菌铜储存蛋白的新家族Csps（Nature 2015，525，140-143）。这些四螺旋束蛋白具有大量的Cys残基，使得能够结合许多Cu（I）离子。甲烷氧化菌从胞质中输出的csp为主要的甲烷氧化酶储存铜。胞质Csp 3存在于广泛的细菌中，包括病原体如铜绿假单胞菌。表达csp 3的细菌将铜隔离在其胞质溶胶中，从而防止毒性。细菌不被认为使用胞质铜酶，并且在任何生物体中Csp 3结合的铜的目的地仍然未知。铜的毒性被哺乳动物免疫系统用来对抗细菌病原体。细菌用铜稳态蛋白来抵御这种攻击，我们假设Csp 3在这个过程中很重要。铜绿假单胞菌是革兰氏阴性细菌和机会主义病原体，其具有已知参与促进感染的适应的铜酶，例如在囊性纤维化患者的肺中。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（CopA 1和CopA 2）。CopA 1对于铜耐受性是必需的，并且在缺乏copA基因的大肠杆菌菌株中异源表达赋予对铜毒性的抗性，如来自枯草芽孢杆菌的Csp 3的异源表达。CopA 1是小鼠感染所必需的，而两种CopA都是植物感染所必需的。我们将用一系列生物物理技术在体外对铜绿假单胞菌的Csp 3蛋白进行鉴定，确定它如何结合和释放Cu（I）。Csp 3在大肠杆菌中的抗毒性能力。将测试缺乏CopA的大肠杆菌菌株。将分析铜绿假单胞菌中Csp 3缺失如何影响铜分布，以确定其铜储存的目标。将研究csp 3缺失菌株的表型，包括毒力研究。正在测试的假设是，Csp 3为铜绿假单胞菌中目前未鉴定的靶标提供了细胞溶质铜的安全储存，并且它作为毒力因子。该项目符合“世界级基础生物科学”BBSRC DTP战略研究领域的职权范围。此外，我们正在开发的用于分析铜在细胞内分布的金属蛋白质组学方法属于“开发新的工作方式”DTP使能主题，并促进了Csps的最初发现（Nature 2015，525，140-143）。我们将开展世界一流的生物科学，不仅将提供细菌如何处理铜的理解，但将阐明这一过程如何使病原体克服宿主的防御引起感染。通过Csp 3对Cu（I）结合的生物化学和结构研究将有助于保持核心基础学科的实力，包括化学和结构生物学。突变体（基因缺失和定点突变）的生产和分析需要分子和细胞生物学核心领域的技能。在一个多学科的合作项目中，所有这些方法的结合将提供关于一类新的铜储存蛋白的功能的重要信息。此外，他们还将对Csp 3在致病性中的潜在作用提供机制性见解，这将有助于设计治疗铜绿假单胞菌感染的方法。

Copper is essential for almost all organisms yet can also be harmful due to its redox activity and ability to bind at sites for other metals. This has resulted in the evolution of homeostatic systems that facilitate copper's use as the cofactor for many important enzymes in both eukaryotes and prokaryotes. A new family of bacterial copper storage proteins, the Csps, were recently discovered (Nature 2015, 525, 140-143) in BBSRC-funded work (BB/K008439/1) by Dennison and Waldron. These four-helix bundle proteins possess a large number of Cys residues enabling the binding of many Cu(I) ions. Csps exported from the cytosol in methanotrophs store copper for the main methane-oxidising enzyme. Cytosolic Csp3s are present in a wide range of bacteria, including pathogens such as Pseudomonas aeruginosa. Csp3-expressing bacteria sequester copper in their cytosol thus preventing toxicity. Bacteria are not thought to use cytosolic copper enzymes, and the destination of Csp3-bound copper remains unknown in any organism.The toxicity of copper is used by the mammalian immune system to fight bacterial pathogens. Bacteria defend against this attack with copper homeostasis proteins and we hypothesise that Csp3s are important in this process. P. aeruginosa is a Gram negative bacterium and an opportunist pathogen that possesses copper enzymes known to be involved in adaptation that facilitates infection, for example in the lungs of patients with cystic fibrosis. 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 and heterologous expression in an Escherichia coli strain lacking its copA gene confers resistance to copper toxicity, as does heterologous expression of Csp3 from Bacillus subtilis. CopA1 is required for infection in mice, whilst both CopAs are needed for infection in plants. We will characterise the Csp3 protein from P. aeruginosa in vitro with an array of biophysical techniques, determining how it binds and releases Cu(I). The ability of Csp3 to prevent toxicity in the E. coli strain lacking CopA will be tested. How Csp3 deletion in P. aeruginosa influences copper distribution will be analysed to identify targets for its store of copper. The phenotype of the csp3-deletion strain will be investigated, including virulence studies. The hypotheses being tested are that Csp3 provides a safe store of cytosolic copper for currently unidentified targets in P. aeruginosa and that it acts as a virulence factor.This project fits within the remit of the 'world-class underpinning bioscience' BBSRC DTP strategic research area. Furthermore, the metalloproteomic approaches that we are developing to analyse the distribution of copper within a cell fall within the 'exploiting new ways of working' DTP enabling theme, and facilitated the initial discovery of the Csps (Nature 2015, 525, 140-143). We will carry out world-class bioscience that will not only provide understanding of how bacteria handle copper, but will elucidate how this process enables a pathogen to overcome a host's defences to cause infection. The biochemical and structural studies on Cu(I) binding by Csp3 will help maintain strength in core underpinning disciplines, including chemical and structural biology. The production and analysis of mutants (both gene-deletion and site-directed) requires skills in the core areas of molecular and cellular biology. The combination of all these approaches in a collaborative multi-disciplinary project will provide vital information about the function of a new class of copper storage proteins. Furthermore, they will also give mechanistic insight into the potential role of Csp3 in pathogenicity, which will help in devising approaches to treat P. aeruginosa infections.