Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.
Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.
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DOI：
10.1371/journal.ppat.1011650
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发表时间：
2023-09
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
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结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是结核病的病原体，对人类健康构成严重威胁。随着耐药性结核分枝杆菌菌株的出现，迫切需要新的治疗方法。由于铁对Mtb的生长和存活至关重要，Mtb获得宿主铁的机制代表了有吸引力的治疗靶点。Mtb通过Mtb铁载体依赖性和血红素铁摄取途径清除宿主铁。虽然多项研究描述了血红素和铁-铁载体的进口和出口的apo-siderophores跨内膜，很少有人知道他们的运输跨周质和细胞壁环境。Mtb FecB和FecB 2被预测为与宿主铁获得有关的周质结合蛋白;然而，它们的确切作用还不清楚。本研究试图区分FecB和FecB 2在结核分枝杆菌铁获取中的作用。Mtb FecB和FecB 2的晶体结构分别被确定为2.0 μ m和2.2 μ m分辨率，并且显示出不同的配体结合口袋。用血红素和来自Mtb和其他物种的细菌铁载体进行了FecB和FecB 2的体外配体结合实验，揭示了FecB和FecB 2都结合血红素，而只有FecB结合Mtb侧载体铁-羧基分枝杆菌素（Fe-cMB）。随后的FecB结构导向突变鉴定了一个单一的谷氨酸残基-Glu 339-，其显著有助于Fe-CMB结合。通过耻垢分枝杆菌和Mtb下拉试验证实了FecB在Mtb铁载体介导的铁获取途径中的作用，该试验揭示了FecB与分枝杆菌铁载体输出和输入机制成员之间的相互作用。同样，下拉法与FecB 2证实了其在血红素摄取揭示与潜在的内膜血红素进口商的相互作用的作用。由于配体的偏好和蛋白质的合作伙伴，我们的数据表明，结核杆菌FecB起着铁载体依赖的铁和血红素的收购途径的作用，此外，我们确认，结核杆菌FecB 2参与血红素摄取。结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是结核病的病原体，长期以来一直对全世界的人类健康构成威胁，并且仍然是导致死亡的主要传染性原因之一。了解结核分枝杆菌如何在人类宿主中生存和繁殖，使我们能够确定潜在的治疗途径。由于铁对Mtb生存至关重要，Mtb从宿主获得铁的机制是特别有吸引力的途径。在这里，我们提出的证据表明，两个结核分枝杆菌预测周质结合蛋白，FecB和FecB 2，参与铁的摄取机制。配体结合实验表明，FecB 2特异性结合血红素，而FecB可以结合血红素和Mtb铁载体羧基分枝杆菌素（CMB）在其载脂蛋白和铁结合形式。我们还确定了一个FecB残基参与铁-CMB识别使用结构功能分析。最后，使用免疫共沉淀，我们确定FecB蛋白相互作用的合作伙伴，将FecB的铁载体依赖性铁的收购途径，我们确认结核病FecB 2参与血红素摄取。这些实验提供了关于一个知之甚少但必不可少的Mtb生存机制的新的重要信息，最终可能允许新的抗Mtb治疗。
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue—Glu339—that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has long presented a threat to human health across the world and remains one of the leading infectious causes of death. Understanding how Mtb can survive and flourish in human hosts allows us to identify potential avenues for therapeutics. As iron is essential to Mtb survival, the mechanisms by which Mtb acquires iron from the host is a particularly attractive avenue. Here, we present evidence that two Mtb predicted periplasmic binding proteins, FecB and FecB2, are involved in iron uptake mechanisms. With ligand-binding experiments, we show that FecB2 specifically binds heme, while FecB can bind heme and the Mtb siderophore carboxymycobactin (cMB) in both its apo and iron bound forms. We also identify a FecB residue involved in ferric-cMB recognition using structure function analysis. Lastly, using co-immunoprecipitation, we identify FecB protein interaction partners that place FecB in the siderophore-dependent iron acquisition pathway and we confirm that Mtb FecB2 is involved in heme uptake. These experiments provide new and important information about a poorly understood, but essential Mtb survival mechanism, which ultimately may allow for novel anti-Mtb therapeutics.
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结核分枝杆菌的外塞蛋白从人体结合蛋白中去除铁，并将铁捐赠给结核分枝杆菌细胞壁中的霉菌素。
影响因子：
15.3
作者：
Gobin, J;Horwitz, MA
通讯作者：
Horwitz, MA
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4
作者：
Ganne, Geraldine;Brillet, Karl;Schalk, Isabelle J.
通讯作者：
Schalk, Isabelle J.
影响因子：
4.6
作者：
Agarwal, Shubhangi;Dey, Sanjay;Dasgupta, Jhimli
通讯作者：
Dasgupta, Jhimli
影响因子：
2.9
作者：
Chao, Alex;Goulding, Celia W.
通讯作者：
Goulding, Celia W.