Biochemical and Proteomic Analyses of Mycobacterial Heme Uptake

描述（由申请人提供）：结核分枝杆菌（Mtb）是结核病（TB）的病原体，仍然是困扰世界人口的最致命的疾病之一，因此迫切需要新的抗结核治疗方法。铁对结核分枝杆菌的生存至关重要，结核分枝杆菌必须将其与宿主隔离。这些拟议的研究围绕着新发现的，新的分枝杆菌血红素-铁获取途径。最近，一种胞浆血红素降解蛋白MhuD已被表征，其中MhuD通过切割血红素四吡啶环催化血红素释放铁。然而，Mtb mhuD缺失突变体并没有完全消除血红素存在下的生长，这表明另一种未被发现的蛋白质也起着从血红素中释放铁的作用。我们提出，Mtb染料脱色过氧化物酶（Mtb Dyp）可以在不切割四吡啶环的情况下从血红素中提取铁，因为Mtb Dyp与大肠杆菌蛋白有密切的同源性，并以这种方式起作用。此外，Mtb还含有一个分泌的包封蛋白同源物（Enc），与Mtb Dyp形成一个双基因操纵子。这些蛋白被认为通过Mtb Dyp中延长的c端延伸相互作用，其中Mtb Dyp被Mtb Enc纳米笼封装。有趣的是，我们还观察到Mtb铁结合铁蛋白Mtb BfrB也含有一个延长的c端尾部，这可能表明Mtb BfrB也被Mtb Enc区隔化。本建议概述了一种实用的结构，生化和遗传方法来表征Mtb Dyp的血红素降解功能。因此，这项研究的结果不仅将进一步加深我们对Mtb血红素铁摄取和利用的理解，而且将为Mtb Dyp和Mtb Enc提供新的候选物，这些候选物没有真核同源物，用于开发创新的抗结核治疗药物。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is still one of the most deadly diseases afflicting the world's population, therefore novel anti-TB therapeutics are urgently needed. Iron is an essential for Mtb survival, which it must sequester from its host. These proposed studies center around the newly discovered, novel mycobacterial heme-iron acquisition pathway. Recently, an Mtb cytosolic heme-degrading protein, MhuD, has been characterized, whereby MhuD catalyzes the release of iron from heme by cleavage of the heme tetrapyrrole ring. However, an Mtb mhuD deletion mutant did not completely abolish growth in the presence of heme, suggesting that another undiscovered protein is also functioning to release iron from heme. We propose that Mtb dye-decolorizing peroxidase, Mtb Dyp, may abstract iron from heme without the cleavage of the tetrapyrrole ring, as Mtb Dyp is a close homolog to E. coli proteins which function in this manner. Furthermore, Mtb also harbors a secreted encapsulin homolog (Enc), forming a two-gene operon with Mtb Dyp. These proteins are proposed to interact via an extended C-terminal extension in Mtb Dyp, whereby Mtb Dyp is encapsulated by Mtb Enc nano-cage. Interestingly, we have also observed that an Mtb iron-binding ferritin protein, Mtb BfrB, also contains an extended C-terminal tail, and may indicate that Mtb BfrB is also be compartmentalized by Mtb Enc. This proposal outlines a pragmatic structural, biochemical and genetic approach to characterize the heme-degrading function of Mtb Dyp, along with its interaction with Mtb Enc. Thus results from this study will not only further our understanding of Mtb heme-iron uptake and utilization but will also provide new candidates, Mtb Dyp and Mtb Enc, which have no eukaryotic homologues, for the development of innovative anti-TB therapeutics. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is still one of the most deadly diseases afflicting the world's population therefore there is an urgent need to develop novel anti-TB therapeutics. This proposal seeks to address this challenge through characterizing two proteins, Mtb Dyp and Mtb Enc, which have no eukaryotic homologues and are proposed to be involved in the novel mycobacterial heme uptake pathway. The results from this structural, functional, and genetic study of these proteins may set the stage for the discovery of an entirely new class of anti-TB therapeutics and potential vaccine candidates, which has the potential to have an extraordinary impact on global health.