Molecular determinants of Salmonella cell-envelope copper homeostasis

项目摘要 本提案的目标是确定铜稳态的机制，在细胞被膜的细胞， 肠道沙门氏菌这种微生物是胃肠炎的重要和常见原因， 以及全身感染。在酶的催化中心中，铜作为氧化还原辅因子是必需的。 然而，游离Cu是高度反应性的并且对细胞有害。铜，沿着氧化爆发，是中心 作为先天免疫反应的一部分。因此，氧化还原/Cu稳态是 对细菌的毒性至关重要。虽然在鉴定细胞质Cu方面取得了重大进展， 由于缺乏稳态机制，因此缺乏对细胞包膜如何处理和 分配铜，同时保持相关的氧化还原平衡。我们的目标是定义和建模Cu 分布在沙门氏菌细胞包膜，并确定其与氧化还原应激反应的分子联系。 本提案的目的是：1）量化周质、细胞质和细胞质之间的铜通量和平衡 外部区室，同时定义周质Cu库的大小和身份。2)定义 CueP作为周质Cu分子伴侣与各种靶交换金属的作用。我们会监察 CueP在体内的丰度，以及它的apo/holo平衡，对周质Cu的变化的反应 程度.我们将确定CueP如何从内外膜转运蛋白中获得Cu 膜并将其递送至替代载体以实现周质Cu的稳态水平。CueP 将评估几种周质铜酶代谢的参与。3)确定 ScsABCD系统在铜和氧化还原稳态界面的作用。氧化还原活性的这些 将测定酶并鉴定体内底物。ScsABCD活性与 将建立Cu与底物或ScsABCD酶之间的结合。为了实现这些目标， 两个具有互补专业知识的实验室的联合努力将使用多种方法的组合 （金属通量建模、蛋白质组学、金属组学、体外宿主/病原体相互作用）。我们的方法来 系统地阐明了一个重要的包膜中Cu/氧化还原稳态的机制， 人类病原体是新颖、及时和创新的。

Project Summary The goal of this proposal is to define the mechanisms of Cu homeostasis in the cell envelope of the pathogen Salmonella enterica. This organism is an important and frequent cause of gastroenteritis, as well as, systemic infections. Cu is required as a redox co-factor in the catalytic centers of enzymes. However, free Cu is highly reactive and deleterious to cells. Cu, along with the oxidative burst, is central in host-pathogen interactions as part of the innate immune response. As such, redox/Cu homeostasis is essential for bacterial virulence. While there has been significant progress in identifying cytoplasmic Cu homeostatic mechanisms, there is a lack of understanding of how the cell envelope handles and distributes Cu, whilst maintaining the associated redox balance. Our goal is to define and model the Cu distribution in the Salmonella cell envelope and identify its molecular links with the redox stress response. The aims of this proposal are: 1) Quantify Cu fluxes and equilibria among periplasmic, cytoplasmic and external compartments while defining the size and identity of the periplasmic Cu sink pool. 2) Define the role of CueP as the periplasmic Cu chaperone exchanging the metal with various targets. We will monitor CueP in vivo abundance, as well as its apo/holo equilibria, in response to changes in periplasmic Cu levels. We will determine how CueP obtains Cu from membrane transporters in the inner and outer membranes and delivers it to alternative carriers to achieve steady state levels of periplasmic Cu. CueP participation in the metallation of several periplasmic cuproenzymes will be assessed. 3) Determine the role of the ScsABCD system at the interface of Cu- and redox-homeostasis. The redox activity of these enzymes will be determined and in vivo substrates identified. The relation between ScsABCD activity and Cu binding to substrates or among ScsABCD enzymes will be established. To achieve these aims, the joint efforts of two laboratories with complementing expertise will use a combination of approaches (modeling of metal fluxes, proteomics, metallomics, in vitro host/pathogen interaction). Our approach to systematically elucidate the mechanisms of Cu/redox homeostasis in the envelope of an important human pathogen is novel, timely and innovative.