Dietary and Microbial Reprogramming of Intestinal Microbiota-Produced Metabolites

项目摘要 人类结肠容纳了复杂的微生物群落，称为肠道微生物群，其具有未映射的 代谢能力。细菌代谢途径处理饮食的成分，如氨基酸，并产生一个阵列 代谢物的不确定性。这种微生物生态系统产生的许多代谢物被人类宿主吸收， 被宿主酶修饰，并最终由肾脏排出。当肾脏衰竭时，这些溶质积累， 包括在维持高浓度的患者的血浆中以非常高的水平发现的“尿毒症”溶质的显著部分 透析这些化合物在个体患者之间可以有很大的差异，但在一段时间内相对稳定。 这可能反映了肠道微生物群组成的个体间差异。其中一些分子 已被调查并与肾脏病患者的不良健康结果有关。然而，对于大多数这些化合物， 无论是负责其形成的生化途径，还是其对宿主的生物学效应， 阐明。本申请集中于源自酪氨酸、4-氨基丁酸和酪氨酸的普遍高浓度尿毒症溶质。 乙基苯基硫酸盐（4-EPS）和对甲酚硫酸盐（PCS），以及4-羟基苯基丙酸硫酸盐、酪氨酸 一种与尿毒症无关但对了解肠道内酪氨酸利用生态位很重要的代谢物 生态系统研究的目标是（i）确定肠道微生物群中负责 产生微生物代谢物4-乙基苯酚和对甲酚，它们是4-EPS和PCS的前体;（ii） 阐明这些分子对与尿毒症疾病相关的宿主生物学方面的影响;以及（iii）研究两种 微生物群重编程的不同策略，目的是降低宿主中的尿毒症溶质水平。Aim 1采用 预测微生物代谢途径的两种方法，一种使用计算/机器学习方法， 第二种方法使用比较基因组学与细菌代谢组表型相结合。基因预测将是 使用基因缺失或异源表达进行遗传验证。在Aim 2中，使用gnotobiotic小鼠作为平台， 研究微生物代谢产物转化为循环溶质，以及溶质水平如何受饮食和 微生物群的其他成员。同位素标记的氨基酸被用来追踪尿毒症溶质的饮食基质 产品.目的3利用野生型与突变型细菌定殖的知菌小鼠，它们在存在或不存在突变方面不同。 不存在4-EPS或PCS，以检查代谢物对宿主生物学的影响。动脉血栓形成的变化， 将评估与尿毒症疾病相关的认知功能。目标4的重点是重新编程微生物群， 产生有害的尿毒症溶质。单一菌株靶向重编程或基于复杂聚生体的微生物群 使用多种可培养细菌的重建将作为降低尿毒症的补充策略进行测试。 小鼠中的溶质水平。饮食改变或基于益生菌的微生物群消融将用于增强 重编程疗法。

PROJECT SUMMARY The human colon houses a complex community of microbes, known as the gut microbiota, which possesses unmapped metabolic capabilities. Bacterial metabolic pathways process components of diet, like amino acids, and produce an array of ill-defined metabolites. Many of the metabolites produced by this microbial ecosystem are absorbed by the human host, modified by host enzymes, and ultimately excreted by the kidneys. When the kidneys fail, these solutes accumulate and comprise a significant portion of the "uremic" solutes found at very high levels in the plasma of patients maintained on dialysis. These compounds can vary widely between individual patients, yet are relatively stable over time within an individual, potentially reflecting inter-individual differences in gut microbiota composition. A few of these molecules have been investigated and linked to poor health outcomes in renal patients. For most of these compounds, however, neither the biochemical pathways responsible for their formation nor their biological effects on the host have been elucidated. This application is focused on the prevalent high concentration uremic solutes derived from tyrosine, 4- ethylphenylsulfate (4-EPS) and p-cresolsulfate (PCS), as well as 4-hydroxyphenylpropionic acid sulfate, a tyrosine metabolite not associated with uremia but important in understanding the tyrosine-utilization niche within the gut ecosystem. The goals of the research are to (i) determine the genes and species within the gut microbiota responsible for production of the microbial metabolites 4-ethylphenol and p-cresol that serve as precursors to 4-EPS and PCS; (ii) elucidate the effects of these molecules on aspects of host biology relevant to uremic illness; and (iii) investigate two distinct strategies for microbiota reprogramming with a goal of lowering uremic solute levels in a host. Aim 1 employs two approaches to predict microbial metabolic pathways, one using a computational/machine learning approach and a second method using comparative genomics combined with bacterial metabolomic phenotyping. Gene predictions will be genetically validated using gene deletion or heterologous expression. In Aim 2, gnotobiotic mice are used as a platform to investigate the conversion of microbial metabolites into circulating solutes, and how solute levels are affected by diet and other members of the microbiota. Isotopically labeled amino acids are used to trace dietary substrates to uremic solute products. Aim 3 leverages gnotobiotic mice colonized by WT versus mutant bacteria, which differ in the presence or absence of 4-EPS or PCS, to examine the effect of the metabolite on host biology. Changes in arterial thrombosis and cognitive function relevant to uremic illness will be assessed. The focus of Aim 4 is to reprogram the microbiota to reduce production of harmful uremic solutes. Single strain targeted reprogramming or complex consortium-based microbiota reconstitution using a diverse array of culturable bacteria will be tested as complementary strategies for lowering uremic solute levels in mice. Dietary modifications or antibiotic-based ablation of the microbiota will be used to augment the reprogramming therapies, respectively.