The biochemical mechanism of microbial glycan utilisation by Bacteroides species

Abstract

The human gastrointestinal tract harbours the most highly diverse ecosystem known to date, the Human Gut Microbiota (HGM). Bacteroides species, prominent and persistent members of the HGM, are highly adapted foragers of recalcitrant dietary, host and microbial glycans. Bacteroides encode co-regulated gene clusters, termed Polysaccharide Utilisation Loci (PUL), for the sensing, capture, import and degradation of complex polysaccharides. Glycan breakdown fuels the HGM and has beneficial effects on host health via maintenance of microbial diversity and production of Short Chain Fatty Acids, which account for 10 % of the hosts daily calorific intake. Microbial glycans are resistant to gastrointestinal digestion and thus may be a significant carbon source for the HGM. Commensal bacteria and fermented foodstuffs are both viable sources of microbial glycans for gut Bacteroides. We have screened a library of human gut Bacteroidetes to identify species capable of utilising exopolysaccharides harvested from monocultures of Lactic Acid Bacteria. Comparative proteomics revealed Glycoside Hydrolases (GH) associated with a PUL and two Carbohydrate Active enZyme (CAZyme) clusters are up-regulated in response to growth of Bacteroides fragilis NCTC 9343 on an exopolysaccharide harvested from Bifidobacterium animalis IPLA R1 monocultures. Characterisation of a GH2 (BF9343_0813) and GH29 (BF9343_2997) has revealed activity against a range of exopolysaccharides, suggesting cross-reactivity of the catabolic machinery. We have biochemically characterised the GH66 (BT3087), GH31 (BT3086) and GH97 (BT4581) encoded within Bacteroides thetaiotaomicron VPI-5482 dextran PUL – the first discrete PUL targeting a microbial glycan to be characterised. We propose a model for dextran utilisation by B. thetaiotaomicron. Cyclic b-1,2-glucans are osmoregulated periplasmic glucans which occur in almost all pathogenic and symbiotic proteobacteria, including Escherichia coli. Human gut Bacteroides have been screened for their ability to utilise b-1,2-glucans, a subclass of exopolysaccharide, as a sole carbon and energy source. Bacteroides fragilis NCTC 9343 was capable of utilising both linear and cyclic b-1,2-glucans as a nutrient source. We have biochemically characterised the GH3 (BF9343_4062), GH43_28 (BF9343_4058) and GH144 (BF9343_4059) 3 encoded within predicted PUL 52 by B. fragilis and report a new activity for the GH43 family. We have also biochemically characterised the GH144, Fjoh_3523, encoded by the environmental Bacteroidetes, Flavobacterium johnsoniae UW101