Structural And Biochemical Characterization Of A Novel Mycobacterial Heme Uptake

描述（由申请人提供）：结核病（TB）继续在世界各地杀死数百万人。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，大多数细菌病原体（包括分枝杆菌）必须从其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很好的研究，因为它们的组分对分枝杆菌的生存力是必不可少的。到目前为止，认为分枝杆菌中的铁摄取是由能够从人转铁蛋白中去除铁的分枝杆菌素协调的。然而，在人类中，转铁蛋白铁占身体总铁的不到1%，而血红素铁可以占80%以上。因此，可以推测分枝杆菌能够从人类血红素来源获得铁。近年来，针对结核分枝杆菌（Mtb）和卡介苗（BCG）的分枝杆菌素缺陷突变体的研究表明，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具有减弱的血红素摄取途径。此外，在我们的实验室已经进行了Mtb蛋白质组范围的方法，以确定潜在的蛋白质参与血红素收购。我们提出了一个假定的途径，血红素是从人类血红蛋白的分泌血细胞，通过血红素转运蛋白跨膜转移，并打破了胞质血红素降解蛋白释放铁螯合。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，我们将调查在这个拟议的途径中的每个基因的影响，在体内分枝杆菌血红素摄取。本研究的具体目的如下：1）对分枝杆菌血团进行生物物理和生物化学研究。2)探讨血红素从宿主血红蛋白到血红素载体再到血红素转运蛋白的转运机制。3)血红素摄取相关蛋白质的鉴定与表征。4)结核分枝杆菌血红素摄取系统的研究。我建议的研究重点是在分子（单蛋白）和细胞水平上全面了解这种新型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没有密切的非分枝杆菌，蛋白质序列同源物，此外，血团有一个新的三维折叠。考虑到这一点，血红素摄取途径为开发抗TB的治疗剂提供了许多良好的蛋白质靶点。公共卫生相关性：结核病（TB）继续在全世界杀死数百万人，并且由细菌病原体结核分枝杆菌（Mtb）引起。铁对所有生物物种都是必不可少的，结核病界认为结核分枝杆菌仅通过其铁载体在体内获得铁;因此，我们鉴定了一种新的血红素获得途径，通过摄取人类血红素获得铁，打破了这种范式。在本研究中，我们将在分子和细胞水平上对这种分枝杆菌特异性血红素摄取途径进行表征，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.