Final Report Summary - HUMAN MICROBIOTA (Understanding and exploiting complex glycan metabolism in the human microbiota)
The human large intestine is colonized by a community of microbes, the microbiota, which has a significant impact on human health and nutrition. The major nutrients available to these organisms are dietary complex carbohydrates that are not metabolised by host enzymes in the small intestines. Dietary and nutraceutical strategies can, potentially, be deployed to encourage the dominance of beneficial microbes in the microbiota ensuring that this microbial ecosystem has a positive influence on human health. This approach, however, is greatly restricted by a critical lack of understanding of the mechanisms by which complex carbohydrates are metabolized by the microbiota. This project seeks to capitalize on the substantial genomic information on the human microbiota to understand the mechanisms by which complex carbohydrates are metabolized by this ecosystem. The work focussed on the characterization of the mechanisms utilised by prominent members of the microbiota (mainly Bacteroides spp) to access a range of dietary as well as host derived polysaccharides. Using transcriptomic data to identify the target proteins we have produced in recombinant form several hundred enzymes that degrade the major complex carbohydrates available to the microbiota. These include yeast mannan, plant xylans as well as the repertoire of plant pectic polysaccharides homogalacturonic acid, rhamnogalacturonan I, arabinan, galactan and rhamnogalacturonan II (RGII). Furthermore we also studied the breakdown of the prominent host glycans, heparan and chondroitin sulfate. The cellular location of key enzymes involved in these processes were determined in the cognate Bacteroides species. The role of the different enzymes in glycan degradation, particularly those acting on the surface of the bacteria, was established by deleting the respective genes and exploring the effect on growth of the mutant strains. Based on these data models for the degradation of the major dietary and host polysaccharides that are metabolised by the human microbiota were formulated. Except for the highly branched carbohydrate RGII, all the other complex carbohydrates are initially cleaved into large oligosaccharides by endo-acting glycanases on the bacterial surface. These products are imported into the periplasm where a range of endo- and acting-acting enzymes hydrolyse these oligosaccharides into their constituent monosaccharides. The enzymes that catalyse polysaccharide degradation are highly specific for their target complex carbohydrate revealing no evidence of catalytic redundancy in these complex degradative systems. The crystal structure of many of these enzymes in complex with their glycan substrates provide insight into the structural basis for specificity. Overall the data generated provide major new insights into the mechanism of glycan degradation by the gut microbiota and the models established will underpin the development of novel dietary strategies that are designed to maximize human health through manipulation of microbiota structure.