Characterization of the Suf Fe-S Cluster Biosynthesis Pathway Under Stress

描述（由申请人提供）：铁对细菌病原体的生长至关重要，因为血红素和铁硫（Fe-S）簇需要铁。为了对抗感染，人类宿主使用两种利用铁依赖性的一般策略：铁隔离以减少病原体的生物可利用铁，并通过产生氧化应激来攻击病原体的铁金属蛋白。我们的长期目标是表征细菌病原体利用的遗传和生化系统，以在压力下保持细胞内铁稳态。本提案的目的是确定Suf途径在铁饥饿和氧化应激期间构建Fe-S簇的生化机制。subfabcdse操纵子在细菌暴露于氧化应激和铁饥饿时被激活以构建必需的Fe-S簇。Suf通路在许多细菌性病原体如志贺氏菌和结核分枝杆菌中是保守的。志贺氏菌每年造成1100万人死于细菌性痢疾，其中大多数是5岁以下儿童。由于当志贺氏菌进入其致病生命周期的细胞内阶段时，该suf操纵子被转录，因此它可能对志贺氏菌的发病机制很重要。结核分枝杆菌是结核病的病原体，每年直接导致200万人死亡。在结核分枝杆菌中，suf基因是必不可少的，因为suf基因的缺失对结核分枝杆菌和相关分枝杆菌是致命的。尽管它很重要，但体内Suf功能的分子细节仍不清楚。SufS酶是一种半胱氨酸脱硫酶，为Fe-S簇组装提供硫，而SufA、SufB、SufC、SufD和SufE的功能尚不完全清楚。我们的目标是(1)表征硫从SufS转移到Fe-S簇组装的最终目的地的逐步路径；(2)确定Suf操纵子中Fe-S簇组装的位点；(3)确定Fe-S簇组装过程中多蛋白SufBCD复合物中SufC atp酶活性的功能。我们将使用蛋白质化学、生物无机化学、分子生物学和微生物遗传学的方法来完成我们在简单的模式生物大肠杆菌中的目标。Lay声明：破坏细菌铁稳态是哺乳动物细胞在感染期间限制细菌生长的关键机制，因为铁是许多致病菌必需的营养物质。我们认为，利用新型抗生素靶向Suf Fe-S簇生物发生途径可能是一种帮助宿主防御破坏细菌铁稳态的策略。我们的建议旨在表征Suf途径，以便我们可以设计针对Suf蛋白的特异性抑制剂，这些抑制剂将作为一类新的抗生素

DESCRIPTION (provided by applicant): Iron is critical for growth of bacterial pathogens due to the need for iron in heme and iron-sulfur (Fe-S) clusters. To combat infection, the human host uses two general strategies that exploit this iron dependence: iron sequestration to minimize bioavailable iron for the pathogen and attack of the pathogen's iron metalloproteins via generation of oxidative stress. Our long-term goal is to characterize the genetic and biochemical systems utilized by bacterial pathogens to preserve intracellular iron homeostasis during stress. The objective of this proposal is to determine the biochemical mechanisms used by the Suf pathway to build Fe-S clusters during iron starvation and oxidative stress. The sufABCDSE operon is activated in bacteria to build essential Fe-S clusters during exposure to oxidative stress and iron starvation. The Suf pathway is conserved in many bacterial pathogens such as Shigella and Mycobacterium tuberculosis. Shigella is responsible for the deaths of 11 million people each year due to bacillary dysentery, the majority of which are children under the age of five. The suf operon may be important for Shigella pathogenesis since it is transcribed as Shigella enters the intracellular stage of its pathogenic lifecycle. M. tuberculosis is the causative agent of tuberculosis and directly causes 2 million deaths each year. In M. tuberculosis, the suf genes are essential since deletion of the suf genes is lethal in M. tuberculosis and related Mycobacteria. Despite its importance, the molecular details of in vivo Suf function are still unclear. The SufS enzyme is a cysteine desulfurase that provides sulfur for Fe-S cluster assembly, while the functions of SufA, SufB, SufC, SufD, and SufE are not fully known. Our Aims are to (1) characterize the step-by-step path of sulfur transfer from SufS to its ultimate destination for Fe-S cluster assembly; (2) identify the site(s) of Fe-S cluster assembly in the Suf operon; and (3) determine the function of SufC ATPase activity in the multi-protein SufBCD complex during Fe-S cluster assembly. We will use methods in protein chemistry, bioinorganic chemistry, molecular biology, and microbial genetics to accomplish our Aims in the facile model organism Escherichia coli. Lay Statement: Disruption of bacterial iron homeostasis is a key mechanism used by mammalian cells to limit bacterial growth during infection because iron is an essential nutrient for many pathogenic bacteria. We believe that targeting the Suf Fe-S cluster biogenesis pathway with novel antibiotics could be a strategy for assisting the host defenses in disrupting bacterial iron homeostasis. Our proposal is designed to characterize the Suf pathway so that we might design specific inhibitors against the Suf proteins that will act as a new class of antibiotics