Evidence for an ABC-type riboflavin transporter system in pathogenic spirochetes.
Evidence for an ABC-type riboflavin transporter system in pathogenic spirochetes.
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DOI：
10.1128/mbio.00615-12
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发表时间：
2013-02-12
期刊：
影响因子：
6.4
通讯作者：
Norgard MV
中科院分区：
文献类型：
--
作者：
Deka RK;Brautigam CA;Biddy BA;Liu WZ;Norgard MV
细菌转运蛋白参与许多必需营养素和代谢物的转运。然而，许多关键的细菌运输系统仍有待确定，包括那些参与核黄素（维生素B2）运输的系统。致病性螺旋体缺乏核黄素生物合成途径，这意味着依赖于从宿主获得核黄素。利用可能的配体结合成分的结构和功能特征，我们已经确定了致病性螺旋体中的abc型核黄素运输系统。从三种不同的螺旋体中克隆了这些系统的假定的脂蛋白配体结合成分，在大肠杆菌中高表达，并纯化到均匀性。三种纯化重组蛋白的溶液均为亮黄色。紫外可见光谱显示这些蛋白可能是黄素蛋白；电喷雾电离质谱和薄层色谱证实它们含有核黄素。梅毒螺旋体梅毒螺旋体编码的蛋白（TP0298）的1.3-Å晶体结构表明，该蛋白的折叠与abc型转运体的配体结合成分相似。该结构还揭示了核黄素结合位点的其他重要细节。螺旋体基因组的比较生物信息学分析，加上实验验证，促进了这种新的abc型核黄素转运系统的发现。我们将配体结合成分称为核黄素摄取转运体A （RfuA）。综上所述，致病螺旋体似乎已经进化出一种abc型运输系统（RfuABCD），以便在宿主环境中生存，特别是在人类宿主环境中。梅毒仍然是一个公共卫生问题，但对致病细菌知之甚少。这是因为梅毒螺旋体仍不能在实验室中培养。相反，白僵菌必须在实验兔身上培养，这一限制造成了许多无法克服的实验障碍。苍白球绦虫的细胞包膜是苍白球绦虫与人类宿主之间的物理和功能界面，对其结构和功能的进一步了解受到严重限制。阐明T. pallidum细胞包膜的一种方法是确定其膜脂蛋白的三维结构，这些分子具有许多关键的生存功能。在这里，我们描述了一个以前未知的运输系统，T. pallidum使用它来进口核黄素，这是生物体生存所必需的营养物质。此外，我们发现这种转运系统存在于其他致病性螺旋体中。这是对这种新型细菌核黄素转运系统的首次描述。
Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B2). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein’s fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host. Syphilis remains a public health problem, but very little is known about the causative bacterium. This is because Treponema pallidum still cannot be cultured in the laboratory. Rather, T. pallidum must be cultivated in laboratory rabbits, a restriction that poses many insurmountable experimental obstacles. Approaches to learn more about the structure and function of T. pallidum’s cell envelope, which is both the physical and functional interface between T. pallidum and its human host, are severely limited. One approach for elucidating T. pallidum’s cell envelope has been to determine the three-dimensional structures of its membrane lipoproteins, molecules that serve many critical survival functions. Herein, we describe a previously unknown transport system that T. pallidum uses to import riboflavin, an essential nutrient for the organism’s survival. Moreover, we found that this transport system is present in other pathogenic spirochetes. This is the first description of this new type of bacterial riboflavin transport system.