Biochemical and Proteomic Analyses of Mycobacterial Heme Uptake

描述(申请人提供)：结核分枝杆菌(Mtb)，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病疗法。铁是结核分枝杆菌生存所必需的，它必须从宿主中隔离出来。这些拟议的研究集中在新发现的、新的分枝杆菌血红素-铁获取途径上。最近，一种Mtb胞内血红素降解蛋白MhuD已被鉴定，MhuD通过裂解血红素四吡咯环催化从血红素中释放铁。然而，Mtb mhuD缺失突变体并没有在存在血红素的情况下完全消除生长，这表明另一种未被发现的蛋白质也具有从血红素中释放铁的功能。我们认为，Mtb染料脱色过氧化物酶，Mtb DYP，可以从血红素中提取铁，而不需要裂解四个吡咯环，因为Mtb DYP与以这种方式发挥作用的大肠杆菌蛋白有密切的同源关系。此外，Mtb还含有分泌性包膜同源蛋白(Enc)，与Mtb DYP形成双基因操纵子。这些蛋白质被认为是通过Mtb DYP中延长的C末端延伸相互作用的，从而Mtb DYP被Mtb Enc纳米笼包裹。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末端尾巴，这可能表明Mtb BFRB也被Mtb Enc区隔。这项建议概述了一种实用的结构、生化和遗传学方法来表征Mtb DYP的血红素降解功能，以及它与Mtb Enc的相互作用。因此，这项研究的结果不仅将加深我们对Mtb血红素铁摄取和利用的理解，而且还将为开发创新的抗结核治疗药物提供新的候选者，Mtb DYP和Mtb Enc，它们没有真核同源物。 公共卫生意义：结核病(TB)的病原体结核分枝杆菌(Mtb)仍然是困扰世界人口的最致命的疾病之一，因此迫切需要开发新的抗结核病疗法。这项建议试图通过鉴定两种蛋白质Mtb DYP和Mtb Enc来应对这一挑战，这两种蛋白质没有真核同源物，并被认为参与了新的分枝杆菌血红素摄取途径。对这些蛋白质的结构、功能和基因研究的结果可能为发现一类全新的抗结核病疗法和潜在的候选疫苗奠定基础，这可能会对全球健康产生非同寻常的影响。

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.