Structural And Biochemical Characterization Of A Novel Mycobacterial Heme Uptake

新型分枝杆菌血红素摄取的结构和生化特征

• 批准号: 8073064
• 负责人: Celia Goulding
• 金额: $ 33.07万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8073064
• 关键词: Accounting; Affect; Affinity; Amino Acid Substitution; Antitubercular Agents; Attenuated; Bacteria; Binding; Biochemical; Carrier Proteins; Collaborations; Communities; Data; Development; Disease; Environment; Genes; Genus Mycobacterium; Goals; Health; Heme; Heme Iron; Hemoglobin; Human; Investigation; Iron; Laboratories; Life; Light; Mass Spectrum Analysis; Membrane; Metals; Mind; Molecular; Mutation; Mycobacterium tuberculosis; Nuclear Magnetic Resonance; Pathway interactions; Play; Protein Sequence Homologs; Proteins; Proteome; Proteomics; Research; Resolution; Role; Siderophores; Source; Structure; System; Techniques; Transferrin; Tuberculosis; Tuberculosis Vaccines; base; drug development; extracellular; in vivo; killings; mutant; mycobacterial; mycobactins; novel; novel therapeutics; pathogen; prevent; therapeutic development; tool; tuberculosis drugs; uptake; yeast two hybrid system

DESCRIPTION (provided by applicant): Tuberculosis (TB) continues to kill millions of people around the world. New tools to prevent and treat this disease are urgently needed. Iron is an essential metal for all forms of life and most bacterial pathogens including mycobacteria must import iron from its host to survive. Hence iron acquisition pathways are well studied in mycobacteria as their components are essential to mycobacterial viability. Thus far, it is thought that iron uptake in mycobacteria is orchestrated by mycobactins that are capable of removing iron from human transferrin. However in humans, transferrin iron accounts for less than 1% of the body's total iron whereas heme iron can represent greater than 80%. Thus one may speculate that mycobacteria are capable of acquiring iron from human heme sources. Recent studies, which focus on mycobactin deficient mutants of Mycobacterium tuberculosis (Mtb) and BCG, suggest that there is a novel heme acquisition pathway in mycobacteria. Interestingly, BCG has an attenuated heme uptake pathway compared to Mtb. In addition, an Mtb proteome-wide approach has been undertaken in our laboratory to identify potential proteins involved in heme acquisition. We propose a putative pathway where heme is sequestered from human hemoglobin by a secreted hemophore, transferred across the membrane by heme transporters, and broken-down by cytosolic heme-degrading protein to release iron. This research will shed light on the molecular mechanism of heme transfer from humans to bacteria. In addition, we will investigate the affect each gene within this proposed pathway, has on mycobacterial heme uptake in vivo. The specific aims of this proposal are as follows: 1) Biophysical and biochemical investigation of the novel mycobacterial hemophore. 2) Explore the mechanism of heme transfer from host hemoglobin to hemophore to heme transporter. 3) Identification and characterization of other proteins involved in heme uptake. 4) Investigation into mycobacterial heme uptake system in Mtb. My proposed research focuses on gaining a comprehensive understanding of this novel mycobacterial heme uptake system on both molecular (single protein) and cellular levels. Interestingly, most of the proteins involved in this pathway have no close non-mycobacterial, protein sequence homologs, and additionally, the hemophore has a novel three-dimensional fold. With this in mind, the heme uptake pathway provides a number of good protein targets for the development of therapeutics against TB. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) continues to kill millions of people worldwide and is caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb). Iron is essential for all living species, and the TB community thought that Mtb acquired its iron in vivo solely via its iron siderophores; thus our identification of a novel heme acquisition pathway whereby iron is obtained via the uptake of human heme breaks this paradigm. In this proposal, we will characterize this mycobacterial specific heme uptake pathway at a molecular and cellular level, which will open new avenues for anti-TB drug development.

描述（由申请人提供）：结核病 (TB) 继续导致全世界数百万人死亡。迫切需要预防和治疗这种疾病的新工具。铁是所有生命形式的必需金属，包括分枝杆菌在内的大多数细菌病原体必须从宿主进口铁才能生存。因此，铁的获取途径在分枝杆菌中得到了充分研究，因为它们的成分对于分枝杆菌的活力至关重要。到目前为止，人们认为分枝杆菌中的铁吸收是由分枝杆菌素精心策划的，分枝杆菌素能够从人转铁蛋白中去除铁。然而，在人体中，转铁蛋白铁仅占体内总铁的不到 1%，而血红素铁则可占体内总铁的 80% 以上。因此，人们可以推测分枝杆菌能够从人类血红素来源中获取铁。最近的研究重点关注结核分枝杆菌 (Mtb) 和卡介苗的分枝杆菌素缺陷突变体，表明分枝杆菌中存在一种新的血红素获取途径。有趣的是，与 Mtb 相比，BCG 的血红素摄取途径减弱。此外，我们的实验室还采用了 Mtb 蛋白质组方法来识别参与血红素获取的潜在蛋白质。我们提出了一种假定的途径，其中血红素通过分泌的血红蛋白与人血红蛋白隔离，通过血红素转运蛋白跨膜转移，并被胞质血红素降解蛋白分解以释放铁。这项研究将揭示血红素从人类转移到细菌的分子机制。此外，我们将研究该途径中的每个基因对体内分枝杆菌血红素摄取的影响。该提案的具体目标如下：1）新型分枝杆菌血团的生物物理和生化研究。 2）探索血红素从宿主血红蛋白转移到血红素再到血红素转运蛋白的机制。 3) 参与血红素摄取的其他蛋白质的鉴定和表征。 4) 结核分枝杆菌血红素摄取系统的研究。我提出的研究重点是在分子（单一蛋白质）和细胞水平上全面了解这种新型分枝杆菌血红素摄取系统。有趣的是，参与该途径的大多数蛋白质没有紧密的非分枝杆菌蛋白质序列同源物，此外，血细胞具有新颖的三维折叠。考虑到这一点，血红素摄取途径为结核病疗法的开发提供了许多良好的蛋白质靶点。公共卫生相关性：结核病 (TB) 继续导致全世界数百万人死亡，它是由细菌病原体结核分枝杆菌 (Mtb) 引起的。铁对于所有生物物种都是必需的，结核病界认为结核菌在体内仅通过其铁铁载体获取铁；因此，我们发现了一种新的血红素获取途径，通过摄取人类血红素来获取铁，打破了这一范式。在本提案中，我们将在分子和细胞水平上表征这种分枝杆菌特异性血红素摄取途径，这将为抗结核药物的开发开辟新途径。