Revealing dynamics of individual mucin glycan utilisation by gut bacteria: The interaction between dietary- and mucin glycans and the intestinal bacteria

Human intestinal health is impacted by the interaction between bacteria residing in the intestine and available glycans from diet and host. Analysis of microbial composition in relation to glycan degradation and utilisation in the gut can help to understand the mechanisms practiced by the bacteria to use mucin glycans and reside in the mucosal surface of the gut.

For this purpose, a robust and reliable method was set up and validated during this thesis for the analysis of intact O- and N-glycans by MALDI-TOF MS and PGC-LC-MS/MS. Using this method, the impact of pasteurised bacteria A. muciniphila, B. fragilis, and R. gnavus on glycosylation in in vitro mucus produced by HT29-MTX-E12 cells revealed an increase in sialylated O-glycans and high-mannose N-glycans. Next, the degradation of glycans by prominent intestinal bacteria A. muciniphila, R. torques, and B. thetaiotaomicron was investigated by incubation of the bacteria in monocultures, co-cultures, and a synthetic community on porcine gastric mucin (PGM). R. torques favoured degradation of fucosylated O-glycans and A. muciniphila and B. thetaiotaomicron equally favoured degradation of fucosylated and non-fucosylated O-glycans. The three bacteria favoured degradation of first core 2 O-glycan structures relative to core 1 O-glycan structures. A. muciniphila, R. torques, and B. thetaiotaomicron partially degraded N-glycans and these were only completely degraded by the synthetic community. Enzyme extracts from the bacterial growth experiment were incubated with human milk oligosaccharides, sulphated glycan structures, and O-glycans removed from PGM and bovine submaxillary glands mucin (BSM) to investigate cleavage of specific glycan linkages and structures. Proteomics was performed to characterise produced enzymes and these results were linked together to reveal favoured cleavage of beta1-4 linked Gal, beta1-3 linked GlcNAc, alpha1-2 linked Fuc, and alpha2-6 linked Sia by A. muciniphila, degradation of all tested linkages except sulphate by R. torques, and favoured cleavage of beta1-4 linked Gal and alpha2-6 linked Sia by B. thetaiotaomicron. Incubation of a synthetic community, consisting of mucin glycan degraders and cross-feeders, on different mucin types revealed a high relative abundance of A. muciniphila, R. gnavus, R. torques, and Faecalibacterium duncaniae when grown on PGM. Addition of sialylated structures to PGM resulted in an increase in relative abundance of A. muciniphila and a decrease in relative abundance of Ruminococcus spp. Incubation of A. muciniphila, R. torques, and B. thetaiotaomicron on galacto-oligosaccharides (GOS) + PGM resulted in increased GOS degradation by A. muciniphila, decreased GOS degradation by R. torques, and minimal influence on GOS degradation by B. thetaiotaomicron. GOS and PGM O-glycans were simultaneously degraded by the co-cultures illustrating the need for a diverse bacterial community to degrade and utilise all available carbohydrate resources.

The results presented in this thesis show new insights in understanding the mechanisms of glycan degradation by prominent gut bacteria. The tested bacteria differently degrade and utilise mucin glycans and the bacteria demonstrate unique interactions in co-culture and as part of a synthetic community.