Sialic acid metabolism in gut microbes

Bell, Andrew (2019) Sialic acid metabolism in gut microbes. Doctoral thesis, University of East Anglia.

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Abstract

Sialic acids are nine carbon monosaccharides that play a crucial role in many biological processes. In the gastrointestinal tract they are found at the terminal position of mucin glycans, and as such are a highly sought-after nutrient source for members of the gut microbiota. This is typically through the release of Neu5Ac and its subsequent metabolism. The gut symbiont Ruminococcus gnavus encodes an intramolecular transsialidase which releases 2,7-anhydro-Neu5Ac from mucin glycans instead of Neu5Ac. Here, we established protocols for the synthesis, detection and quantification of 2,7anhydro-Neu5Ac, which has been a limiting factor in the study of this sialic acid derivative. We used this compound to demonstrate that the full 11 gene nan cluster the IT-sialidase is part of is dedicated to 2,7-anhydro-Neu5Ac metabolism. To investigate the metabolic pathway in more detail, the proteins encoded by the cluster were recombinantly expressed in E. coli and characterised using a range of biochemical and biophysical approaches. The predicted SAT2 transporter protein, RgSBP, was found to be uniquely specific to 2,7-anhydro-Neu5Ac, which once inside the cell is converted by a novel oxidoreductase enzyme, RgNanOx, to Neu5Ac. This is subsequently catabolised to ManNAc and pyruvate by the action of a sialic acid aldolase, RgNanA, which is structurally and biochemically similar to previously described sialic acid aldolases. Using gnotobiotic mice we confirmed that this cluster was important for R. gnavus colonisation and spatial location in the mucus layer in vivo, and our bioinformatic analysis showed that the cluster was found in very few species within the gut microbiota. This was supported by in vitro analysis using a simulated colon model showing that 2,7-anhydroNeu5Ac supplementation promotes growth of R. gnavus in a complex microbial community. We also showed that the presence of R. gnavus can limit the outgrowth of S. Typhimurium post-antibiotic treatment by reducing the levels of free and bound Neu5Ac, by converting it to 2,7-anhydro-Neu5Ac which these pathogens cannot utilise. The data presented in this work unravel a unique metabolic pathway for the utilisation of 2,7-anhydro-Neu5Ac and highlight its potential in novel therapeutic strategies.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Katherine Whittaker
Date Deposited:	14 Feb 2020 14:35
Last Modified:	24 Jan 2023 01:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/74202
DOI:

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