Mechanistic studies to develop a polysaccharide degradation signature (PDS) and its application in improving host health

项目总结/摘要 人类结肠含有大量的微生物，这些微生物统称为结肠微生物。 微生物组结肠微生物组中的微生物以拟杆菌门的细菌为主， 厚壁菌门在拟杆菌中，普雷沃氏菌属（Prevotella spp.）和拟杆菌属（Bacteroides spp.）大量存在于结肠环境中 并进化出了一种复杂的蛋白质机制，使它们能够从两种宿主体内获取能量， 饮食中的多糖和宿主衍生的聚糖，如粘蛋白。对基本机制至关重要 拟杆菌的多糖降解是多糖降解位点（PUL）或位点（普尔斯） 存在于它们的基因组中。普尔斯是由基因簇组成的，这些基因簇编码的蛋白质能够使 拟杆菌能够感知、运输和降解各种多糖，并将其转化为单位糖用于发酵。一 大蛋白，称为杂交双组分系统（HTCS），在大肠杆菌的普尔斯中是保守的。 拟杆菌通过感知多糖或其寡糖来启动表达 水解酶及其相关的转运蛋白。在这一建议中，我们证明， 拟杆菌HTCS含有传感器模块，其感测独特的多糖或其降解产物 在结肠环境中。因此，我们假设HTCS多肽中的不同传感器共同 可以作为个体结肠中多糖传感的代表。我们已指定此代理为 多糖降解特征或PDS。通过使用公开的3000多个HTCS序列， 可用的数据库，我们构建了一个系统发育树，似乎聚类传感器模块到 不同的分支在人类饮食中发现的宿主不可降解多糖中，如小麦、大麦、大米 是阿拉伯木聚糖。因此，我们使用阿拉伯木聚糖生长和转录组学分析来确定 分别靶向可溶性阿拉伯木聚糖和不溶性阿拉伯木聚糖降解的普尔斯在三个 人类结肠拟杆菌的成员。我们的数据显示，我们的系统发育树或PDS中的簇 可以与阿拉伯木聚糖传感和代谢相匹配。有趣的是，我们还发现类杆菌 代谢复合阿拉伯木聚糖的spp释放植物酚化合物阿魏酸， 化合物在用过的培养基中积累。已知阿魏酸具有抗氧化作用， 对抗机械感觉性脱发因此，我们将确定复合阿拉伯木聚糖的合生元 而代谢阿拉伯聚糖的拟杆菌有能力保护头发免受机械感觉的伤害 无菌斑马鱼的损失。这一观察结果的证实将使我们能够通过转录组学分析， 确定这种保护的潜在分子机制。此外，我们将使用生物化学和 结构分析，以完全表征人结肠癌细胞降解阿拉伯木聚糖的机制 拟杆菌我们还预计，我们对PDS的开发将允许合理操纵 这些多糖被个体的微生物组感知，以提供健康和营养益处。

PROJECT SUMMARY/ABSTRACT The human colon harbors a large number of microorganisms that collectively are referred to as the colonic microbiome. The microbes in the colonic microbiome are dominated by bacteria of the phyla Bacteroidetes and Firmicutes. Among the Bacteriodetes, Prevotella spp. and Bacteroides spp. abound in the colonic environment and have evolved a complex protein machinery that allows them to harvest energy from both host undegradable polysaccharides in the diet and host derived-glycans, such as mucin. Central to the mechanism underlying polysaccharide degradation by the Bacteroidetes is the Polysaccharide Degradation Locus (PUL) or Loci (PULs) present on their genomes. The PULs are composed of gene clusters that encode proteins that enable the Bacteroidetes to sense, transport, and degrade diverse polysaccharides to their unit sugars for fermentation. A large protein, known as the Hybrid Two Component System (HTCS), is conserved in the PULs of the Bacteroidetes and functions by sensing either a polysaccharide or its oligosaccharides to turn on the expression of the hydrolytic enzymes and their associated transporters. In this proposal, we demonstrate that indeed the Bacteroidetes HTCS contain sensor modules that sense unique polysaccharides or their degradative products in the colonic environment. Thus, we hypothesized that the diverse sensors in the HTCS polypeptides collectively can serve as a proxy for polysaccharide sensing in the colon of an individual. We have designated this proxy as the Polysaccharide Degradation Signature or PDS. By using more than 3000 HTCS sequences in the publicly available databases, we constructed a phylogenetic tree that appeared to cluster the sensor modules into different branches. Among host undegradable polysaccharides found in human diets, such as wheat, barley, rice and oats, is arabinoxylan. We, therefore, used growth on arabinoxylan and transcriptomic analysis to determine the PULs that target soluble arabinoxylan and insoluble arabinoxylan degradation, respectively, in three members of the human colonic Bacteroidetes. Our data showed that clusters in our phylogenetic tree or PDS can be matched to arabinoxylan sensing and metabolism. Interestingly, we also discovered that the Bacteroides spp that metabolize complex arabinoxylan release the plant phenolic compound ferulic acid and that the compound accumulates in the spent medium. Ferulic acid is known to have antioxidant effects and also to protect against mechanosensory hair loss. We will, therefore, determine whether a synbiotic of complex arabinoxylan and arabinoxylan-metabolizing Bacteroidetes has the capacity to confer protection against mechanosensory hair loss in germ-free zebrafish. Confirmation of this observation will allow us, through transcriptomics analysis, to determine the underlying molecular mechanisms for this protection. Furthermore, we will use biochemical and structural analyses to completely characterize the mechanism of arabinoxylan degradation by the human colonic Bacteroidetes. We also anticipate that our development of the PDS will allow rational manipulation of the polysaccharides sensed by an individual’s microbiome for health and nutritional benefits.