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

项目摘要/摘要 人的结肠含有大量的微生物，这些微生物统称为结肠 微生物组。结肠微生物群中的微生物主要是拟杆菌门的细菌和 菲米库斯。在细菌中，普雷沃特氏菌(Prevoella spp.)和拟杆菌属(Bacteroids spp.)在结肠环境中大量存在 并进化出一种复杂的蛋白质机制，使它们能够从两个不可降解的宿主中获取能量 饮食中的多糖和宿主衍生的多糖，如粘蛋白。是潜在机制的核心 拟杆菌对多糖的降解是多糖降解位点(PUL)或PULS(PULS) 存在于它们的基因组中。PULs是由基因簇组成的，这些基因簇编码的蛋白质使 拟杆菌能感应、运输和降解不同的多糖为单位糖以供发酵。一个 大型蛋白质，被称为混合双组分系统(HTCS)，在PULS中保守 类杆菌及其功能是通过感知多糖或其低聚糖来开启表达 水解酶及其相关的转运体。在这项提案中，我们确实证明了 类杆菌HTC含有感测独特的多糖或其降解产物的传感器模块 在结肠环境中。因此，我们假设HTCS多肽中的不同感受器共同作用 可以作为个体结肠中多糖感知的代理。我们已将此代理指定为 多糖类降解特征或PDS。通过使用公开的3000多个HTCS序列 在现有的数据库中，我们构建了一个系统发育树，似乎将传感器模块聚为 不同的分支机构。在人类饮食中发现的宿主不可降解的多糖中，如小麦、大麦、大米 燕麦，是阿拉伯木聚糖。因此，我们使用阿拉伯木聚糖生长和转录分析来确定 分别以可溶性阿拉伯木聚糖和不溶性阿拉伯木聚糖降解为目标的PULS在三个 人类结肠类杆菌的成员。我们的数据显示，在我们的系统发育树或PDS中的集群 可以匹配阿拉伯木聚糖的感官和新陈代谢。有趣的是，我们还发现类杆菌 代谢复合阿拉伯木聚糖的SPP释放植物酚类化合物阿魏酸，并 化合物在废弃的介质中积累。众所周知，阿魏酸具有抗氧化作用，还可以保护 防止机械性脱发。因此，我们将确定一种复杂的阿拉伯木聚糖的合生体 而代谢阿拉伯木聚糖的类杆菌具有保护头发免受机械感觉的能力。 无菌斑马鱼的损失。证实这一观察结果将使我们能够通过转录组分析， 确定这种保护的潜在分子机制。此外，我们将使用生化和 人结肠对阿拉伯木聚糖降解机理的结构分析 拟杆菌。我们还预计，我们开发的PDS将允许合理地操纵 个人微生物群感受到的对健康和营养有益的多糖。

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.