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

铜对几乎所有的生物都是必不可少的，但由于其氧化还原活性和与其他金属结合的能力，也可能是有害的。这导致了动态平衡系统的进化，促进了铜在真核生物和原核生物中作为许多重要酶的辅因子的使用。最近，Dennison和Waldron在BBSRC资助的工作(BB/K008439/1)中发现了一个新的细菌铜储存蛋白家族CSPs(自然2015,525,140-143)。这些四股螺旋束蛋白具有大量的半胱氨酸残基，能够与许多铜(I)离子结合。从甲烷氧化菌中的胞浆中输出的CSP为主要的甲烷氧化酶储存铜。胞液Csp3存在于广泛的细菌中，包括铜绿假单胞菌等病原体。表达CSP3的细菌将铜隔离在胞浆中，从而防止毒性。细菌不被认为使用胞质铜酶，Csp3结合的铜在任何生物体中的目的地尚不清楚。铜的毒性被哺乳动物的免疫系统用来对抗细菌病原体。细菌通过铜稳态蛋白来防御这种攻击，我们假设Csp3在这个过程中是重要的。铜绿假单胞菌是一种革兰氏阴性杆菌，是一种机会性病原体，具有铜酶，已知铜酶参与适应，促进感染，例如在囊性纤维化患者的肺部。令人惊讶的是，人们对铜绿假单胞菌是如何处理铜的知之甚少。该菌具有两种出铜的P型ATPase(CopA1和CopA2)。CopA1是铜耐受所必需的，在缺乏CopA基因的大肠杆菌中异源表达可以抵抗铜毒，枯草芽孢杆菌Csp3的异源表达也是如此。小鼠的感染需要CopA1，而植物的感染需要两种CopA。我们将在体外用一系列生物物理技术表征铜绿假单胞菌的Csp3蛋白，确定它是如何结合和释放铜(I)的。将测试Csp3在缺乏COPA的大肠杆菌菌株中防止毒性的能力。将分析铜绿假单胞菌中Csp3缺失对铜分布的影响，以确定其铜储备的目标。将对csp3缺失菌株的表型进行调查，包括毒力研究。正在测试的假设是，Csp3为目前尚未确定的铜绿假单胞菌靶标提供了安全的胞液铜储存，并作为毒力因子发挥作用。该项目符合BBSRC DTP战略研究领域的“世界级支撑生物科学”的范围。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方法属于‘探索新的工作方式’DTP使能主题，并促进了CSP的初步发现(《自然》2015,525,140-143)。我们将进行世界级的生物科学，这不仅将提供对细菌如何处理铜的了解，还将阐明这一过程如何使病原体能够克服宿主的防御而导致感染。Csp3与铜(I)结合的生化和结构研究将有助于保持其在包括化学和结构生物学在内的核心学科中的优势。突变的产生和分析(基因缺失和定点定位)需要分子和细胞生物学核心领域的技能。所有这些方法的结合将在一个协作的多学科项目中提供关于一类新的铜储存蛋白功能的重要信息。此外，它们还将提供对Csp3在致病性中的潜在作用的机械性洞察，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.