Genomic and metabolomic foundations of human-microbial symbiosis in the gut

描述（由申请人提供）：人类肠道微生物群主要由拟杆菌门和厚壁菌门组成，并为我们提供了我们不必自行进化的生理特性，包括处理否则难以消化的膳食多糖的能力。这项拨款更新的重点是描述微生物群的关键物种的基因组和“代谢组学”特征，它们对衰老/共享行为的营养基础的操作原则，以及我们从与它们的共生关系中获得的生理益处。我们使用无菌（GF）小鼠定植了糖溶性厌氧菌拟杆菌（B. theta），对体内一种重要的人类肠道共生体进行了首次报道的全基因组转录分析。基于功能基因组和质谱的代谢组学研究表明，B. theta的适应性觅食行为如何在面对膳食多糖变化时帮助稳定微生物群。我们现在建议系统地增加这种非生物小鼠模型的复杂性，以检查我们已经测序的人类来源的拟杆菌门（B. theta， B. vulgatus， B. distasonis），史密斯甲烷杆菌（人类结肠中主要的产甲烷古菌）和厚壁菌门成员（直肠真杆菌，E. eligens）之间的相互作用。目的1。描述拟杆菌门-古细菌共生关系的基因组和代谢基础及其对宿主生物学的影响如下：(i)产生一个完整的、带注释的M. smithii基因组（第一个被测序的甲烷预防细菌物种）；（ii）定植GF小鼠，喂食标准的富含多糖的食物，富含果聚糖的食物，或含有B. theta和/或M. smithii的高脂肪西方饮食；（iii）使用定制的GeneChips对两种微生物进行体内全基因组转录谱分析；（iv）利用激光捕获微解剖结肠上皮和小鼠基因芯片同时分析宿主的转录反应；(v)基于转录组数据集进行代谢组的二氧化硅重建，并使用结果指导基于假设的微生物代谢途径对共生关系的贡献的生化和遗传分析；（vi）评估我们使用普通白僵菌和异象白僵菌的研究结果的普遍性。目标2。描述厚壁菌门对营养共享的贡献，在一个简化的人类微生物群3组分模型中，该模型由该分支的一个重要成员、拟杆菌门成员和一个产甲烷菌组成。我们将用E. rectale（人类结肠微生物群的一员，我们目前正在对其基因组进行测序）、B. theta和M. smithii定植GF小鼠，从而建立一个初始群落。然后将微生物和宿主的转录和代谢反应作为群落成员和饮食的功能进行评估。根据结果，我们将研究含有拟杆菌、史密斯芽孢杆菌和直肠芽孢杆菌的选择组合的联合体的性质。eligens。这些研究应该为监测和控制微生物群的功能提供新的方法，以促进人类健康。

DESCRIPTION (provided by applicant): The human gut microbiota is dominated by the Bacteroidetes and Firmicutes divisions of Bacteria, and provides us with physiological traits we have not had to evolve on our own, including the ability to process otherwise indigestible dietary polysaccharides. This grant renewal focuses on characterizing the genomic and "metabolomic" features of the microbiota's keystone species, the operating principles that underlie their nutrient for aging/sharing behaviors, and the physiological benefits we accrue from our mutualistic (symbiotic) relationship with them. We have used germ-free (GF) mice colonized with Bacteroides thetaiotaomicron (B. theta), a saccharolytic anaerobe, to conduct the first reported genome-wide transcriptional profiling of a prominent human gut symbiont in vivo. Functional genomic and mass spectrometry-based metabolomic studies showed how B. theta's adaptive foraging behavior could help stabilize the microbiota in the face of changes in dietary polysaccharides. We now propose to systematically add complexity to this gnotobiotic mouse model to examine interactions between human-derived Bacteroidetes we have sequenced (B. theta, B. vulgatus, B. distasonis), Methanobrevibacter smithii (a dominant methanogenic archaeon in the human colon), and members of Firmicutes (Eubacterium rectale, E. eligens). Aim 1. Characterize the genomic and metabolic underpinnings of Bacteroidetes-Archaeal mutualism and its impact on host biology as follows: (i) produce a finished, annotated M. smithii genome (first Methanobrevibacter species to be sequenced); (ii) colonize GF mice fed standard polysaccharide-rich chow, a chow enriched for fructans, or a high fat Western diet with B. theta and/or M. smithii; (iii) perform whole-genome transcriptional profiling of both microbial species in vivo using custom GeneChips; (iv) simultaneously profile host transcriptional responses using laser capture microdissected colonic epithelium and mouse GeneChips; (v) perform in silica reconstructions of the metabolome based on the transcriptome datasets and use the results to direct hypothesis-based biochemical and genetic analyses of the contributions of microbial metabolic pathways to the mutualism; (vi) assess the generality of our findings using B. vulgatus and B. distasonis. Aim 2. Characterize the contributions of the Firmicutes to nutrient sharing in a simplified 3-component model of the human microbiota composed of a prominent member of this division, a member of Bacteroidetes, plus a methanogen. We will create an initial community by colonizing GF mice with E. rectale (a member of the human colonic microbiota whose genome we are currently sequencing), B. theta and M. smithii. Microbial and host transcriptional and metabolic responses will then be assessed as a function of community membership and diet. Based on the results, we will examine the properties of consortia containing selected combinations of Bacteroides spp., M. smithii, and E. rectale/E. eligens. These studies should provide new approaches for monitoring and manipulating the functions of the microbiota to promote human health.