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A mucus hydrogel covers the intestinal epithelium, protecting the host from passing food and resident gut microbiota. The mucus consists of a highly organized glycoprotein network, mainly produced by goblet cells. In the distal colon, where microbial abundance is highest, goblet cells continuously secrete mucus, which “grows” by expansion, forming a gradient from an inner nearly sterile layer to an outer loose layer where bacteria reside. This pushes microbes away from the host epithelium, thereby reducing the risk of infection and inflammation.

The gut microbiota is predominantly composed of bacteria and is strongly influenced by diet. Individuals in industrialized societies exhibit reduced microbial diversity compared to those in non-industrialized societies – a difference partly attributed to the Western-style diet (WSD), which is low in dietary fiber and high in simple sugars and saturated fat. In mice fed a WSD, microbiota diversity is reduced, and the mucus barrier is weakened, as seen by a slower mucus growth rate and increased bacterial penetration, which raises the risk for harmful microbial interactions. Similar microbial and mucus alterations are observed in patients with inflammatory bowel disease (IBD). While mucus properties are microbiota-dependent, the underlying regulatory mechanisms are still largely elusive. This thesis aims to clarify these mechanisms by studying the colonic mucus barrier in a WSD environment, with both human and mouse microbiota. Moreover, the effects of structures previously untested for their mucus-influencing properties during WSD feeding are evaluated in mice.

In study 1, the impact of diet-induced changes in the human microbiota on the colonic mucus barrier was studied by transplanting fecal samples into microbiota-depleted mice. Healthy participants increased their fiber intake over three months, and their microbiota was collected before and after the intervention. When transplanted into mice, only the high-fiber-derived microbiota maintained mucus growth under WSD feeding and reduced the pathogen load during intestinal infection. The bacterial taxon Blautia was enriched in the high-fiber group, and Blautia coccoides emerged as a key regulator of mucus integrity through the production of the short-chain fatty acids (SCFAs) acetate and propionate. These metabolites were shown to stimulate mucus growth via the free-fatty acid receptor 2 (Ffar2), revealing a previously unrecognized mechanism by which microbial metabolites directly impact mucus integrity.

In studies 2 and 3, the mucus-influencing effects of bovine milk-derived casein glycomacropeptide (CGMP) and human milk oligosaccharide (HMO) structures were examined in WSD-fed mice. CGMP is a glycosylated protein found in cheese whey, while HMOs are breast milk components with over 200 known structures. Both GCMP and HMOs share structural similarities with mucin glycans and may act as decoy substrates for bacterial degradation under low-fiber conditions. In study 2, CGMP structures improved mucus growth rate, where specifically, a highly sialylated CGMP (HSA) increased propionate levels and the relative abundance of Bifidobacteria, a bacterium previously linked to mucus maintenance. In study 3, specific HMO structures enhanced mucus integrity and improved mucus penetrability in a mouse model for IBD. HMO-dependent mucus modulation could be linked to changes in bacterial composition, increased SCFA levels and glycan-targeting enzyme activities. These findings emphasize the structure-specific effects of CGMPs and HMOs and their distinct modulation of microbiota–mucus interactions.

In summary, this thesis reveals new insights into how microbial metabolites regulate the mucus barrier in the Western gut and highlights novel strategies to be exploited for treating mucus-associated disorders such as IBD.

Environmental factors, such as antibiotics and diet, have a profound influence on the composition and function of the gut microbiota. While antibiotics consistently disrupt microbial communities, dietary influences are more variable, with some nutrient compositions promoting beneficial microbial activity while others drive dysbiosis. Yet, the subsequent microbiota-mediated effects on mucus function remain incompletely characterised. The colonic mucus layer forms a critical barrier between the gut microbiota and the intestinal epithelium, protecting against infection and inflammation. However, changes in the gut environment can shift microbial metabolism toward degradation of mucin O-glycans, compromising this barrier. To investigate how environmentally-shaped microbiota influence mucus function, human-to-mouse faecal microbiota transplantation (FMT) and ex-vivo mucus function analyses were used throughout this thesis. 

Antibiotics are known to markedly disrupt the gut microbiota, with repeated exposure linked to reduced microbial diversity. However, the consequences for mucus barrier function have remained unclear. To address this, microbiota from individuals with a history of repeated antibiotic exposure were transplanted into microbiota-depleted mice. Recipient mice exhibited significantly reduced mucus growth rate and increased mucus penetrability relative to controls. These functional changes were accompanied by enrichment of known mucin utilisers, including Akkermansia muciniphila and Bacteroides fragilis, together with a distinct metabolite profile. These findings demonstrate that long-term repeated antibiotic exposure can substantially shift microbial composition and function, with direct consequences for mucus barrier integrity. 

Dietary fibre availability shapes bacterial metabolism, with fibre fermentation supporting short-chain fatty acid (SCFA)-mediated mucus barrier function and fibre deficiency promoting mucin degradation. While the effects of the Western-style diets (WSDs) are well described, the consequences of low-carbohydrate diets (LCDs), which are also typically low in fibre, remain unclear. To address this, mice were fed a LCD or transplanted with LCD-shaped human microbiota, resulting in reduced mucus growth rate but unchanged mucus penetrability compared to controls. This indicates that LCD-associated microbiota alter mucus dynamics without fully compromising barrier function, illustrating that not all low-fibre diets exert equally detrimental effects.

Finally, to examine the microbiota-mediated benefits of fibre, FMT experiments were performed using microbiota from individuals who increased their fibre intake for three months. Despite consuming a low-fibre diet, mice receiving the high fibre-shaped human microbiota maintained mucus growth rates comparable to chow-fed controls and exhibited reduced pathogen burden in an intestinal infection model. Both human and mouse microbiota showed increased abundance of Blautia associated with fibre supplementation, and Blautia coccoides was identified as a key species promoting mucus growth through secretion of SCFAs. Functional profiling further revealed an enhanced capacity for dietary fibre degradation relative to mucin-glycans, illustrating how fibre shapes a microbiota capable of preserving mucus barrier integrity. Together, these findings demonstrate a microbiota-mediated protective effect against the otherwise harmful effects of a WSD.

Overall, this thesis advances understanding of how environmental pressures shape microbial communities and thereby influence colonic mucus barrier function. These insights provide a foundation for future mechanistic studies of microbiota–mucus interactions and highlight opportunities to leverage diet and microbial therapeutics to strengthen mucus barrier integrity.