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

基本信息

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
项目类别：
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

项目摘要

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。人类基因组本身的微生物组成差异可能很大，并且宏基因组研究表明，造成这种多样性的因素尚不清楚。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了解辅助因子和其他因素如何影响微生物。我们目前的大部分理解都限制了合理改变其成分的策略。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反应。这些代谢过程通常依赖于供应短缺的辅助因子，维生素 B12 就是一个例子。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因此，一种解决目标的方法是通过独立于 B12 的代谢途径来规避它。在本提案中，我寻求通过改变可用类咕啉的水平来操纵肠道微生物组。阐明转运系统的类咕啉选择性，为肠道细菌如何发挥作用提供所需的见解我的研究将与其他研究对象及其宿主竞争复杂生态系统中的关键资源。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。是一些 B12 依赖性酶所利用的活性辅因子形式，由 BtuR 合成 B. thetaiotaomicron 的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其他特性。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 转运的研究结合使用生物化学和生物物理，革兰氏阴性细菌受到关注。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 蛋白的选择性钴酰胺并结合所确定的结构提供对蛋白质-蛋白质相互作用的见解。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。