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Title: Glycan utilisation and resource allocation by prominent members of the human gut microbiota
Authors: Briggs, Jonathon
Issue Date: 2017
Publisher: Newcastle University
Abstract: The human gut microbiota (HGM) represents a diverse community of bacteria with cell numbers in the trillions. The HGM is presented with dietary glycans, primarily from plant material consumed in the human diet, and host glycans which allow the bacterial community to proliferate in the gut. As such carbohydrate active enzymes (CAZymes) and multi-protein glycan utilisation systems are commonly expressed by members of the HGM. The human genome encodes few CAZymes rendering the vast majority of carbohydrates consumed accessible to the HGM. During bacterial fermentation of glycans short chain fatty acids (SCFAs), acetate, butyrate and propionate, are released into the gut lumen and are utilised for energy by colonocytes. SCFAs have also been shown to repress genes relating to proliferation in cancerous gut cells and activation of Treg cells. Bacteroidetes have been shown to possess a unique glycan utilisation strategy involving degradation of the target glycan at the cell surface before transport into the periplasm where degradation is completed before fermentable substrates are transported into the cytoplasm. Bacteroides ovatus deploys a xylan utilisation system expressed from two loci which is capable of targeting the relatively simple glucuronoxylans (GX) and arabinoxylans (AXs), but also the highly complex glucuronoarabinoxylans (GAXs) from corn. Work presented in this thesis has contributed to our understanding of xylan degradation. The data showed that xylan degradation begins at the cell surface with endo-acting xylanases from family GH10, targeting the backbone of simple xylans, while a GH98 xylanase targets specific structures in complex GAXs generating oligosaccharides that are transported into the periplasm. Co-culturing B. ovatus alongside Bifidobactertium adolescentis on GX and AX demonstrated cross-feeding of xylooligosaccharides, allowing Bi. adolescentis access to previously inaccessible substrate. This interaction provides further evidence for use of xylans and xylooligosaccharides as prebiotic supplements to the human diet with the potential to enrich for both butyrate-producing Bifidobacterium and propionate producing Bacteroides. Bacteroides thetaiotaomicron is regarded as a glycan generalist with upwards of 88 polysaccharide utilisation loci (PULs), the majority of which target different glycans in the gut. Among these target glycans are pectic polysaccharides, homogalacturonan, galactan, arabinan, rhamnogalacturonan I (RGI) and rhamnogalacturonan II (RGII). Galactan, which represents a relatively simple polysaccharide, required only two glycoside hydrolases for complete degradation, a surface galactanase (GH53) and a periplasmic galactosidase (GH2). Arabinan required two surface endo-arabinanases (GH43), a periplasmic α-1,2-arabinofuranosidase (GH43) and α-1,3-arabinosidase (GH51) for the removal of sidechains and a final periplasmic α-1,5-arabinosidase (GH51) to complete degradation of the arabinan backbone. Remaining galactooligosaccharide sidechains appended to rhamnose of RGI were degraded by three galactosidases (two GH2s and a GH35), each displaying different substrate preferences. During B. thetaiotaomicron pectin utilisation a degree of surface degradation was shown to be required prior to import; each of the resulting oligosaccharides can be scavenged by a B. thetaiotaomicron mutant lacking the specific surface enzymes required for polysaccharide degradation. These data demonstrate cross-feeding is prevalent during B. thetaiotaomicron utilisation of pectic polysaccharides in the HGM. Fructans are particularly well described prebiotics found to be a bifidogenic growth substrate. B. ovatus and B. thetaiotaomicron are capable of utilising inulin- and levan-type fructans, respectively. Co-culture of B. ovatus and Bi. adolescentis on inulin demonstrates that the Bifidobacterium sp out competes the Bacteroides sp, despite B. ovatus being capable of utilising long and short inulin chains. Cross-feeding also occurs when Bi. longum or Bacteroides vulgatus is the oligosaccharide recipient. B. thetaiotaomicron growth on levan is able to support growth of Bi. adolescentis indicating levan oligosaccharides have probiotic potential. Cross-feeding has the potential to have a dramatic influence on how probiotics are processed, utilised and distributed in the gut and their contribution to the HGM food web must be considered when designing dietary supplements.
Description: PhD Thesis
Appears in Collections:Institute for Cell and Molecular Biosciences

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