Chalkophore-mediated copper homeostasis in Staphylococcus lugdunensis

金属离子是许多酶的辅助因子，因此对功能性代谢是必不可少的。然而，由于其氧化还原电位，金属离子也具有内在毒性，细胞内金属稳态需要严格控制。金属稳态是人体先天免疫的重要靶点。可获得的微量金属，如铁（Fe）被主动限制在宿主内，以减少病原体在感染期间的增殖。相反，有针对性地增加吞噬溶酶体内的铜浓度可以杀死细菌。细菌产生多种金属载体以改善金属的获取。研究得最好的是铁结合的铁载体。然而，有孤立的报道称，除了铁，铁载体还可以结合铜。这可以允许解毒，因为化合物结合影响氧化还原特性。此外，它还保证了代谢活动所需的充足的cu离子供应。然而，在定植和疾病的背景下，铜结合金属团（白垩团）与病原体的相关性尚不充分了解。我们发现病原菌S. lugdunensis产生化合物Ulbactin F，并发现该分子是一种白垩体。负责的遗传位点编码生物合成系统以及假定的出口和进口，强烈表明该分子对金属稳态很重要。我建议在分子水平上对这一系统进行表征，并研究其生理相关性。我请求资助两个博士职位，使用高度协同的方法研究不同的主题。第一个博士生将研究Ulbactin F生物合成和膜转运与S. lugdunensis金属稳态的相关性。该学生将对野生型和Ulbactin F缺陷菌株进行基本分析，以确认该分子在细胞内金属水平上的作用。此外，学生将研究Ulbactin F的膜转运。我们在Ulbactin F基因座内发现了一种能量偶联因子（ECF）类型的abc转运蛋白。ecf转运体是微量营养获取系统。我们将在分子水平上研究ecf转运体的Ulbactin F结合特性。此外，我们将使用等基因突变体和ecf转运体的异源表达来验证其在Ulbactin F获取中的作用。第二名博士生将研究Ulbactin F的生理相关性。该学生将进行体外实验，以研究Ulbactin F的生产是否在铜离子限制条件下提供生长优势，或者该分子是否提供对铜离子的抗性。此外，学生将研究Ulbactin F生物合成的金属依赖调节，并使用离体和体内感染模型来研究该分子是否代表S. lugdunensis的毒力因子。总之，我相信，建议的实验将提供一个完整的画面的生物学的白垩菌Ulbactin F。

Metal ions are needed as cofactors for many enzymes and are therefore essential for a functional metabolism. However, due to their redox potential, metal ions do also harbour intrinsic toxicity and cytosolic metal homeostasis needs strict control. Metal homeostasis is an important target for human innate immunity. Availability of trace metals such as iron (Fe) is actively limited within the host to reduce pathogen proliferation during infection. In contrast, a targeted increase of copper concentration within phagolysosomes kills bacteria. Bacteria produce several metallophores to improve metal acquisition. Best studied are iron-binding siderophores. However, isolated reports exist describing that in addition to iron, siderophores can bind copper. This can allow detoxification, as compound binding impacts redox characteristics. Additionally, it ensures sufficient Cu-ion supply for metabolic activity. However, the relevance of copper-binding metallophores (chalkophores) for pathogens in the context of colonisation and disease is insufficiently understood. We found that the pathogen S. lugdunensis produces the compound Ulbactin F, and found the molecule to be a chalkophore. The responsible genetic locus encodes biosynthesis systems as well as putative exporters and importers, strongly suggesting that the molecule is important for metal homeostasis. I suggest to characterize this system on the molecular level and to study its physiological relevance. I request funding for two PhD positions to study distinct topics using highly synergistic approaches. The first PhD student will investigate the relevance of Ulbactin F biosynthesis and membrane transport on metal homeostasis of S. lugdunensis. The student will perform elementary analysis of wild type and Ulbactin F deficient strains to confirm the role of the molecule on intracellular metal levels. Additionally, the student will investigate membrane transport of Ulbactin F. We identified an ABC-transporter of the energy coupling factor (ECF) type within the Ulbactin F locus. ECF-transporters are trace nutrient acquisition systems. We will study the Ulbactin F binding properties of the ECF-transporter on the molecular level. Additionally, we will use isogenic mutants and heterologous expression of the ECF-transporter to validate its role in Ulbactin F acquisition. The second PhD student will study the physiological relevance of Ulbactin F. The student will perform in vitro experiments to investigate whether Ulbactin F production provides a growth advantage under copper ion-limiting conditions or if the molecule provides resistance to copper ions. Additionally, the student will study metal dependent regulation of Ulbactin F biosynthesis and use ex vivo and in vivo infection models to investigate whether the molecule represents a virulence factor of S. lugdunensis. Altogether, I am convinced that the suggested experiments will give a complete picture of the biology of the chalkophore Ulbactin F.