Vitamin B12 trafficking and selectivity in gut bacteria

维生素 B12 在肠道细菌中的运输和选择性

• 批准号: 10447917
• 负责人: Romila Nina Mascarenhas
• 金额: $ 10万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-06 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447917
• 关键词: Address; Affinity; Anabolism; Animal Model; Bacteria; Bacteroides thetaiotaomicron; Bacteroidetes; Binding; Biochemical; Biochemical Reaction; Biological Assay; Carbon; Carrier Proteins; Cells; Cobalamin; Communities; Complex; Corrinoids; Crystallization; Disease; Ecosystem; Environment; Enzymes; Equilibrium; Escherichia coli; Fluorescence; Gene Expression Regulation; Genes; Gram-Negative Bacteria; Health; Human; Human Genome; Individual; Individual Differences; Kinetics; Lipoproteins; Measures; Membrane; Membrane Transport Proteins; Metabolic; Metabolic Pathway; Metabolism; Metagenomics; Methylmalonyl-CoA Mutase; Molecular; Molecular Chaperones; Nitrogen; Operon; Oxidation-Reduction; Periplasmic Binding Proteins; Phase; Play; Predisposition; Process; Proteins; Reaction; Research; Resources; Ribonucleotide Reductase; Role; Shapes; Signal Transduction; Site; Specificity; Structure; Surface; System; Testing; Therapeutic; Thermodynamics; Vitamin B 12; Vitamins; biophysical techniques; cobamamide; cofactor; cost; dysbiosis; fitness; gut bacteria; gut microbiome; insight; interest; microbial; microbial community; microbial composition; microbiome; microbiota; paralogous gene; preference; protein protein interaction; protein transport; symbiont; trafficking; treatment response; uptake

Project Summary The human gut microbiome is inhabited by trillions of bacteria that encode over 150-fold more genes than the human genome itself. The inter-individual differences in microbial composition can be significant, and the factors contributing to this diversity are not well understood. Metagenomic studies suggest that the microbiome might play an important role in determining an individual’s predisposition to disease and responses to treatments. The paucity of understanding how cofactors and other factors influence microbial composition limit strategies to rationally alter it for therapeutic purposes. Much of our current understanding of the factors that shape the gut microbial flora composition derives form studies on how bacteria generate energy, maintain redox balance and acquire carbon and nitrogen. The enzymatic reactions that support these metabolic processes often rely on cofactors that are in short supply. Vitamin B12 is an example of one such cofactor that is essential for many bacteria that are unable to biosynthesize it and lack parallel B12-independent metabolic pathways to circumvent its absence. So, one approach to the targeted manipulation of the gut microbiome is via altering the levels of available corrinoids. In this proposal, I seek to elucidate the corrinoid selectivity of transport systems to provide needed insights into how gut bacteria compete with each other and their hosts for a critical resource in a complex ecosystem. My studies will focus on Bacteroidetes thetaiotaomicron, a common gut bacterium, which lacks the genes required for de novo synthesis of vitamin B12 but encodes multiple B12-dependent enzymes. 5’-Deoxyadenosylcobalamin is the active cofactor form that is utilized by some B12 dependent enzymes and is synthesized by BtuR in B. thetaiotaomicron. The chaperone and catalytic activities are uncharacterized and will be addressed in Aim 1. It also encodes three copies of the outer membrane B12-transporter BtuB with each system displaying a different preference for corrinoid derivatives. The bacterium also possesses additional transport machinery that is not observed in E. coli, a model organism in which studies on B12 transport in gram-negative bacteria have been focused. Using a combination of biochemical and biophysical approaches, I propose to elucidate the mechanism of B12 transport by the B12-uptake (Btu) system in Aim 2. The kinetic and thermodynamic studies in Aims 1 and 2 will define the selectivity of the Btu proteins for cobamides and provide insights into protein-protein interactions. Combined with the structures determined in Aims 1 and 2, my studies will furnish mechanistic insights into how a precious and rare cofactor is relayed from the environment across two layers of bacterial membranes to support the metabolic needs of a common gut bacterium.

项目概要 人类肠道微生物群中栖息着数万亿个细菌，这些细菌编码的基因比人类肠道微生物多 150 倍。 人类基因组本身。微生物组成的个体差异可能很大，并且 造成这种多样性的因素尚不清楚。宏基因组研究表明 微生物组可能在确定个体的疾病易感性和 对治疗的反应。缺乏对辅助因子和其他因素如何影响微生物的了解 成分限制策略以合理地改变它以达到治疗目的。我们目前的大部分理解 影响肠道微生物菌群组成的因素来源于对细菌如何产生的研究 能量，维持氧化还原平衡并获取碳和氮。支持的酶促反应 这些代谢过程通常依赖于供应不足的辅助因子。维生素 B12 是一个例子 一种这样的辅助因子，对于许多无法生物合成它并且缺乏平行的细菌来说是必需的 独立于 B12 的代谢途径可避免其缺失。因此，一种实现目标的方法 肠道微生物群的操纵是通过改变可用类咕啉的水平来实现的。在这个提案中，我寻求 阐明转运系统的类咕啉选择性，为肠道细菌如何作用提供所需的见解 在复杂的生态系统中，彼此及其宿主争夺关键资源。我的学业将 重点关注 Bacteroidetes thetaiotaomicron，一种常见的肠道细菌，它缺乏 de 所需的基因 维生素 B12 的新合成，但编码多种 B12 依赖性酶。 5’-脱氧腺苷钴胺素 是一些 B12 依赖性酶利用的活性辅因子形式，由 BtuR 合成 B.thetaiotamicron。伴侣和催化活性尚未表征，将在 目标 1. 每个系统还编码外膜 B12 转运蛋白 BtuB 的三个副本 对类咕啉衍生物表现出不同的偏好。该细菌还具有额外的 在大肠杆菌中未观察到的转运机制，大肠杆菌是一种模式生物，其中对 B12 转运的研究 革兰氏阴性菌受到关注。结合使用生物化学和生物物理 方法，我建议阐明 Aim 中 B12 吸收 (Btu) 系统的 B12 运输机制 2. 目标 1 和 2 中的动力学和热力学研究将定义 Btu 蛋白的选择性 考巴酰胺并提供对蛋白质-蛋白质相互作用的见解。结合确定的结构 在目标 1 和 2 中，我的研究将为珍贵而稀有的辅助因子如何传递提供机制见解 从环境中穿过两层细菌膜来支持细菌的代谢需求 常见的肠道细菌。