Corrinoid Response of the Human Gut Microbiota

描述(由申请人提供)： 人体肠道微生物区系是一种代谢器官，其细胞组成是由选择和竞争的动态过程决定的。微生物群落组成的变化影响我们获取能量的能力、对感染的敏感性、处理外来生物物质，并与克罗恩病和其他代谢紊乱有关。这个项目探索了一个意想不到的发现：一个与维生素B12相关的小分子家族(统称为Corrinoid)在体内调节人类肠道微生物之间的竞争和选择方面发挥着关键作用。我提议的实验整合了诺生菌小鼠模型，定义了人类肠道来源细菌共生体的序列，以及一种识别肠道定植所需微生物基因的新技术，以解决一个重要的预测：如果这种多样化的小分子菜单的产生、修饰和消费影响人类肠道微生物区系的组成和功能特性，则有可能通过直接操纵皮质激素代谢和/或其分子靶标来诊断和改善涉及微生物区系的病理生理状态。 尽管几十年来一直被研究为人体酶的关键辅因子，但B12是唯一一种完全由微生物合成的维生素：人类肠道微生物区系产生的各种Corrinoid的作用尚不清楚。这项提案提出了一项计划，通过两个特定的目标来剖析皮质激素交换在人类肠道相关细菌共生体中的作用。目的1将确定一种重要的人类肠道共生菌(Thetaiotaomicron)对外源Corrinoid的反应。人体肠道微生物区系的很大一部分缺乏合成这些小分子的能力；B.thetaiotaomicron提供了一个模型，用于确定这些共生体在体内如何感知和对Corrinoid做出反应。我的初步研究表明，B.thetaiotaomicron在远端肠道的适合性取决于其基因组中编码的三个皮质激素反应位点中的一个，而这一要求受到群落环境的调节。这些研究结合了遗传学和生物化学方法，将确定人类肠道共生体是否可以区分不同的Corrinoid，将为识别和解释其他基因组中的Corlinid反应位点提供实验基础，并将提供活体Corrinoid景观的微生物生物标记物。 在远端肠道中，Corrinoid可以来自两个来源：肠道微生物的局部生物合成和饮食消费。目的2将明确膳食皮质激素摄入对体内微生物区系结构和功能的作用。定植于已知全基因组序列的特定微生物群的诺生菌小鼠，提供了一个独特的机会来确定饮食对皮质醇交换的影响。这种影响将通过分析饮食中摄入B12对微生物群落结构、基因表达、健康决定因素和皮质醇产生的影响来衡量。来自确定的微生物群落的结果将与对不同饮食B12摄入量的诺生菌小鼠移植的未分离的人类肠道微生物区系的独立培养研究进行比较。总之，这些实验将产生一种机械上的理解，即小分子的交换如何受到群落组成和宿主饮食的影响，以塑造人类肠道微生物区系的结构和功能。 我的专业是生态学和进化生物学(普林斯顿大学学士)和微生物学和分子遗传学(哈佛大学博士)。我的初步研究最近发表在《细胞宿主和微生物》杂志上，描述了一种定量大规模并行测序技术，该技术允许第一个基因筛查剖析哺乳动物宿主中人类共生体的建立。本提案中描述的实验建立在这项工作的一些意想不到的结果的基础上，并整合了两个关键培训领域(独立于培养的微生物生态学和灵知生物畜牧业)，这将扩大我作为独立调查员的职业目标中可以追求的问题范围。戈登实验室是获得这些专业知识的理想培训环境：该实验室设在华盛顿大学的跨学科基因组科学中心，并与美国最大的大学相关灵知生物动物设施之一相连。这份提案还包括一份详细的职业发展计划和时间表，其中包括一个由两名资深和两名年轻教职员工组成的指导委员会，我定期与他们会面讨论和提供建议，三名科学顾问/合作者是这些研究领域的国际知名专家，以及我从杰弗里·戈登那里得到的极好的支持和指导。这份申请书中包括了这些个人的支持函。 人类肠道微生物区系贡献了一个巨大的遗传库，在其起源、组成和可塑性方面与我们自己的不同：我们的肠道微生物对健康和疾病的贡献已经受到越来越多的关注，但塑造这个微生物器官的因素却知之甚少。拟议的实验将剖析一类关键但基本上未被探索的小分子在这一过程中的作用，并将为定制我们的皮质激素消费提供基础，以最大限度地发挥肠道微生物区系对人类健康的贡献。

DESCRIPTION (provided by applicant): The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. Variation in microbial community composition influences our capacity for energy harvest, sensitivity to infection, processing of xenobiotics, and is implicated in Crohn's disease and other metabolic disorders. This project explores an unexpected finding: a family of small molecules related to vitamin B12 (collectively termed corrinoids) plays a key role in mediating competition and selection between human gut microbes in vivo. My proposed experiments integrate gnotobiotic mouse models, defined consortia of sequenced human gut-derived bacterial symbionts, and a new technique for identifying microbial genes required for gut colonization to address an important prediction: if the production, modification, and consumption of this diverse menu of small molecules influences the composition and functional properties of the human gut microbiota, it may be possible to diagnose and ameliorate pathophysiologic states involving the microbiota by directed manipulation of corrinoid metabolism and/or their molecular targets. Although studied for decades as a critical cofactor for human enzymes, B12 is the only vitamin that is synthesized exclusively by microbes: the role of the diverse corrinoids produced by the human gut microbiota is unexplored. This proposal presents a plan to dissect the role of corrinoid exchange in human gut-associated bacterial symbionts through two Specific Aims. Aim 1 will determine the response of a prominent human gut symbiont (Bacteroides thetaiotaomicron) to exogenous corrinoids. A significant portion of the human gut microbiota lacks the ability to synthesize these small molecules; B. thetaiotaomicron provides a model for determining how such symbionts sense and respond to corrinoids in vivo. My preliminary studies show that B. thetaiotaomicron fitness in the distal gut hinges upon one of the three corrinoid-responsive loci encoded in its genome, and that this requirement is modulated by community context. These studies integrate genetic and biochemical approaches and will establish whether a human gut symbiont can distinguish between different corrinoids, will produce an experimental foundation for identifying and interpreting corrinoid-responsive loci in other genomes, and will provide microbial biomarkers of the corrinoid landscape in vivo. In the distal gut, corrinoids can come from two sources: local biosynthesis by gut microbes and dietary consumption. Aim 2 will define the role of dietary corrinoid consumption on microbiota structure and function in vivo. Gnotobiotic mice, colonized with defined microbial consortia whose complete genome sequences are known, provide a unique opportunity to determine the influence of diet on corrinoid exchange. This influence will be measured through analysis of the effect of dietary B12 consumption on microbial community structure, gene expression, fitness determinants, and corrinoid production. Results from defined microbial communities will be compared with culture-independent studies of a transplanted unfractionated human gut microbiota in gnotobiotic mice that vary in their dietary B12 consumption. Together, these experiments will produce a mechanistic understanding of how the exchange of small molecules is influenced by community composition and host diet to shape the structure and function of the human gut microbiota. My training is in ecology and evolutionary biology (B.A., Princeton University) and microbiology and molecular genetics (Ph.D., Harvard University). My preliminary studies, recently published in Cell Host and Microbe, describe a quantitative massively parallel sequencing technique that allowed the first genetic screen to dissect the establishment of a human symbiont in a mammalian host. The experiments described in this proposal build on some unexpected results from this work, and integrate two key training areas (culture-independent microbial ecology and gnotobiotic animal husbandry) that will expand the range of questions I can pursue in my career goal as an independent investigator. The Gordon lab is an ideal training environment to gain this expertise: the lab is housed in an interdisciplinary Center for Genome Sciences at Washington University and connected with one of the largest university-associated gnotobiotic animal facilities in the country. This proposal also includes a detailed career development plan and timeline that centers on a mentoring committee of two senior and two younger faculty members that I meet with on a regular basis for discussion and advice, three scientific consultants/collaborators who are internationally known experts in these research areas, and the excellent support and mentoring I've received from Jeffrey Gordon. Letters of support from each of these individuals are included in this application. The human gut microbiota contributes an enormous genetic repository distinct from our own in terms of its origin, its composition, and its plasticity: the contribution of our gut microbes to health and disease has received increased attention, but the factors that shape this microbial organ are poorly understood. The proposed experiments will dissect the role of a critical but largely unexplored class of small molecules in this process, and will provide the foundation for tailoring our corrinoid consumption to maximize the contribution of the gut microbiota to human health.