Structural And Biochemical Characterization Of A Novel Mycobacterial Heme Uptake

描述(申请人提供)：结核病(TB)继续在世界各地导致数百万人死亡。迫切需要新的工具来预防和治疗这种疾病。铁是所有生命形式所必需的金属，包括分枝杆菌在内的大多数细菌病原体必须从宿主输入铁才能生存。因此，在分枝杆菌中，铁的获取途径得到了很好的研究，因为它们的成分对分枝杆菌的生存至关重要。到目前为止，人们认为分枝杆菌的铁摄取是由分枝杆菌菌素调控的，分枝杆菌菌素能够从人类转铁蛋白中去除铁。然而，在人类中，转铁蛋白铁占人体总铁的不到1%，而血红素铁可以占到80%以上。因此，人们可能会推测，分枝杆菌能够从人类的血红素来源中获取铁。最近的研究主要集中在结核分枝杆菌(Mtb)和卡介苗(BCG)的Mycobactin缺陷突变体上，表明在分枝杆菌中存在一种新的血红素获取途径。有趣的是，与结核分枝杆菌相比，卡介苗的血红素吸收途径减弱。此外，我们的实验室已经采用了Mtb蛋白质组范围的方法来识别与血红素获取有关的潜在蛋白质。我们提出了一种可能的途径，其中血红素被分泌的血细胞从人类血红蛋白中隔离，通过血红素转运体跨膜转移，并被胞浆中的血红素降解蛋白分解以释放铁。这项研究将阐明血红素从人类转移到细菌的分子机制。此外，我们还将研究这一途径中的每个基因对体内分枝杆菌血红素摄取的影响。本研究的具体目的如下：1)对新型分枝杆菌血细胞进行生物物理和生化研究。2)探讨了血红素从宿主血红蛋白到血细胞再到血红素转运体的转移机制。3)与血红素吸收有关的其他蛋白质的鉴定和鉴定。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)和细胞水平上全面了解这一新的分枝杆菌血红素摄取系统。有趣的是，大多数参与这一途径的蛋白质都没有紧密的非分枝杆菌蛋白质序列同源，此外，血细胞有一个新的三维折叠。考虑到这一点，血红素吸收途径为抗结核疗法的发展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，由细菌病原体结核分枝杆菌(Mtb)引起。铁对所有活着的物种都是必不可少的，结核病社区认为结核分枝杆菌在体内只通过其铁铁载体获得铁；因此，我们发现了一种新的血红素获得途径，通过吸收人类血红素获得铁打破了这一范式。在这项提案中，我们将在分子和细胞水平上表征这种分枝杆菌特异性的血红素吸收途径，这将为抗结核药物的开发开辟新的途径。

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.