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

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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.