Molecular determinants of Salmonella cell-envelope copper homeostasis

项目摘要 这项建议的目的是定义铜在细胞被膜中的动态平衡机制 病原体肠炎沙门氏菌。这种微生物是胃肠炎的重要和常见原因，如 还有，系统性感染。在酶的催化中心，铜作为氧化还原辅助因子是必需的。 然而，游离铜具有高度的活性和对细胞的危害性。铜，连同氧化爆发，是中心 在宿主与病原体的相互作用中，作为先天免疫反应的一部分。因此，氧化还原/铜的动态平衡 对细菌的毒性是必不可少的。虽然在鉴定细胞质铜方面取得了重大进展 动态平衡机制，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铜 在沙门氏菌细胞膜中的分布，并确定其与氧化还原应激反应的分子联系。 这一建议的目的是：1)量化铜在周质、细胞质和 外部隔室，同时定义周质铜吸收池的大小和特性。2)定义 CueP作为周质铜伴侣与不同靶交换金属的作用。我们会监控 CueP在体内的丰度，以及它的apo/holo平衡对周质铜变化的响应 级别。我们将确定CueP是如何从内外膜转运蛋白中获得铜的 膜，并将其输送到替代载体，以达到稳定的周质铜水平。CueP 将对几种周质铜酶的金属化参与进行评估。3)确定 ScsABCD系统在铜和氧化还原动态平衡界面上的作用。这些化合物的氧化还原活性 将测定酶并鉴定体内底物。Scsabcd活动与 铜与底物或ScsABCD酶之间的结合将被建立。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相结合 (金属熔剂建模、蛋白质组学、金属组学、体外宿主/病原体相互作用)。我们的方法是 系统阐明铜/氧化还原动态平衡的机制在一个重要的 人类病原体具有新颖性、时代性和创新性。

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.