Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791371
Title: Mechanistic insights into N-glycan degradation by the human gut microbiota
Author: Briliūtė, Justina
ISNI:       0000 0004 8502 0229
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2019
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Abstract:
The gut microbiota, a dense microbial community present in the human large intestine, plays a significant role in the maintenance of human health. Apart from digestion of complex carbohydrates, these beneficial mutualists compete with invading pathogens and are involved in the modulation of human immune system. Despite being home to hundreds of different species of bacteria, two bacteria phyla dominate the human gut - Gram-positive Firmicutes and Gram-negative Bacteroidetes. Bacteroides spp., prominent members of this microbial ecosystem, degrade a vast range of dietary and host-derived glycans by utilizing a complex trans-envelope machinery known as Sus-like systems encoded by co-regulated clusters of genes known as polysaccharide utilization loci (PUL). Each Bacteroides spp. can have >100 PULs, each specialised in targeting and degrading different glycans, with majority of glycans having 1 to 2 dedicated PULs. Glycans are the major nutrients available to the human colonic microbiota and come from both dietary and host sources. While dietary plant glycans are the most prominent in terms of quantity, the ability to access host mucin O-glycans appears to be important for gut survival and is a trait shared by many members of the microbiota. Various N-glycosylated dietary and host proteins are also found in the human gut in significant quantities. These include antibodies and the terminal domains of mucins, however, it is currently unclear whether these N-glycans are also accessed as nutrients by the gut microbiota and if so, how they are broken down. In this study, several prominent Bacteroides spp. were shown to be capable of utilising complex Nglycans as a sole carbon source. Transcriptomic analysis identified the genes upregulated in one of these, B. thetaiotaomicron, during the growth on complex N-glycans. These include 16 predicted carbohydrate-active enzymes encoded by multiple discrete loci, some of which, such as BT0455-0461, do not fit classical PUL model due to the lack of SusC/D pair, SGBP and a regulator. Biochemical characterisation of the activated enzymes provided an insight into their role in N-glycan breakdown. Four GH20-family β-hexosaminidases with overlapping specificities were characterised and were found to employ a novel CBM domain to bind N-glycan structures. The crystal structure of one of these GH20s, BT0459GH20, suggests this glycosidase could utilize a novel strategy to target N-glycan substrates. The combination of the cellular localization, gene deletion analysis and biochemical characterization data allowed us to propose a model for N-glycan degradation where the structure is degraded sequentially and requires a cooperative activity of numerous glycosidases encoded by multiple discrete loci. The N-glycan degradation begins at the cell surface with a cleavage of the complex Nglycan structures off the protein backbones by endo-glycosidase BT1044GH18 . This step was found to be critical in complex bi-antennary N-glycan utilization. The liberated N-glycan structures are then imported into the cell where they are sequentially broken down into monosaccharides by the specialised exo-glycosidases. Variants of this intricate N-glycan utilization apparatus were identified in a number of other members of Bacteroides spp., suggesting this model of degradation could be widely employed by the human gut Bacteroides.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.791371  DOI: Not available
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