Cross-feeding interactions of gut symbionts driven by human milk oligosaccharidesand mucins

The co-evolution of the human host and the gut microbiota has led to bacterial adaption to forage on host-produced glycans such as human milk oligosaccharides (HMOS) and mucins. In early life, the HMOS that are present in mother milk contribute to the establishment of a healthy gut microbiota. Furthermore, mucins that cover the intestinal lining create a stable niche for bacterial colonization throughout a person’s life. Due to the highly complex nature of these host-secreted glycans, bacteria equipped with specific glycan-degrading enzymes can exploit them as substrate for growth. Subsequently, the glycan-degrading bacteria can drive microbial networks via cross-feeding. The glycan-foraging microbial population exerts a large influence on the host physiology, by influencing the immune, metabolic, and neurological development early in life, and by conferring colonisation resistance throughout life. The work described in this thesis aims to improve our understanding of the metabolic dependencies between the milk- and mucin-degrading microbes (<em>Akkermansia muciniphila</em>, <em>Bifidobacterium</em> spp. and <em>Bacteroides</em> spp.) and butyrate-producing bacteria (<em>Anaerostipes caccae</em>, <em>Eubacterium hallii</em>, and <em>Faecalibacterium prausnitzii</em>). Bacteria-derived butyrate is the preferred energy source for host epithelial cells and is associated with a range of beneficial effects including enhancement of colonic barrier function, increased satiety, and protection against inflammation, and cancer. In the introductory chapter, the role of human milk and mucin glycans in fostering the gut microbial network is discussed. As such, the molecular mechanisms of glycan-foraging by key microbial species and the potential butyrate-inducing interaction among gut symbionts is presented. In Chapter 2 and 3, the role of HMOS as selective substrates for microbial growth that drive the establishment of the infant gut microbiota was investigated. At this developmental stage, HMOS promote the dominance of bifidobacteria from the <em>Actinobacteria </em>phylum. Upon weaning, the gut microbiota shifts towards an adult gut microbiota composition that is predominantly comprised of bacteria from the <em>Firmicutes </em>and <em>Bacteroidetes </em>phyla. In chapter 2, the interaction between a HMOS-degrader, <em>Bifidobacterium infantis </em>(<em>Actinobacteria </em>phylum) and a butyrogenic non-HMOS-degrader, <em>Anaerostipes caccae </em>(a member of the <em>Lachnospiraceae</em> from the <em>Firmicutes</em> phylum) was studied. <em>Anaerostipes caccae </em>in monoculture was not able to metabolise lactose or HMOS but its growth and concomitant butyrate production were detected in co-cultures with <em>Bifidobacterium infantis</em>. <em>Anaerostipes</em> <em>caccae</em> was sustained by cross-feeding on the monosaccharides, lactate and acetate derived from <em>Bifidobacterium</em> <em>infantis</em>. <em>Bifidobacterium</em> <em>infantis</em> fully degraded lactose and the complete range of low molecular weight HMOS, pointing towards the key ecological role of bifidobacteria in providing substrates for other important emerging species in the infant gut. The gradual shift of the microbiota composition in the ecosystem contributing to the slow induction of butyrate could also be important for gut maturation. In chapter 3, the microbial network formation in the infant gut driven by another HMOS-degrading species, namely <em>Bacteroides thetaiotaomicron </em>was studied. We showed that <em>Bacteroides thetaiotaomicron </em>could drive the butyrogenic trophic chain with<em> Anaerostipes caccae</em>. <em>Bacteroides thetaiotaomicron </em>could metabolise lactose and HMOS. The bacterium showed different preference for specific HMOS structures when grown in co-culture. Subsequently, <em>Anaerostipes caccae </em>cross-fed on <em>Bacteroides thetaiotaomicron</em>-derived monosaccharides, lactate and acetate for growth and butyrate production. <em>Bacteroides thetaiotaomicron</em> might drive the establishment of the microbial network in the infant gut, leading to the sequential establishment of adult-like functional groups such as lactate-utilising and butyrate-producing bacteria. Furthermore, we observed stereospecific lactate isomer production in which <em>Bacteroides </em>spp. and <em>Bifidobacterium</em> spp. produced predominantly D- and L-lactate, respectively. The distinct lactate isomer production by these major glycans-degrading genera might affect the gut microbiota compositions by differential cross-feeding interaction with the lactate-utilisers. Chapter 4 and 5 studied the role of mucins in creating a micro-environment that leads to the formation of a microbial network at the intestinal mucosal layer. In chapter 4, we demonstrated that <em>Akkermansia muciniphila</em>, a gut symbiont specialised in mucin degradation, could support the growth of the butyrate-producing cross-feeders <em>Anaerostipes caccae</em>, <em>Eubacterium hallii</em>, and <em>Faecalibacterium prausnitzii</em>. <em>Akkermansia muciniphila</em> metabolised the complex mucin glycans into short chain fatty acids including acetate, propionate and 1,2-propanediol as well as the mucin-derived sugars. Subsequently, acetate and the liberated sugars could be used by the butyrate-producers for growth and concomitant butyrate production. Interestingly, a bidirectional cross-feeding was observed between <em>Akkermansia muciniphila </em>and <em>Eubacterium hallii.</em> Pseudo-vitamin B12 produced by <em>Eubacterium hallii </em>facilitated propionate production by <em>Akkermansia muciniphila</em> via the methylmalonyl-CoA pathway. Propionate could be beneficial to the human host by regulating satiety and lipid biosynthesis in the liver, indicative of a mutualistic host-microbial interaction driven by mucin glycans. In chapter 5, we studied the molecular mechanism of cross-feeding between <em>Akkermansia muciniphila</em> and <em>Anaerostipes caccae</em> by metatranscriptomics. We observed a differential transcriptional response of <em>Akkermansia muciniphila</em> grown in monoculture as compared to a co-culture together with <em>Anaerostipes caccae</em>. In particular, the expression of the extracellular mucin-degrading enzymes by <em>Akkermansia muciniphila</em> was heightened in co-cultures. As a result, the monosaccharides liberated from the breakdown of mucin oligosaccharides chain could support the central metabolism of both <em>Akkermansia</em> <em>muciniphila</em> and the butyrate-producer. This suggested that <em>Akkermansia muciniphila</em> plays a key role in supporting the microbial community at the mucosal environment of the intestine by increasing the availability of substrates. In summary, this thesis demonstrated the key functional role of milk- and mucin-degrading symbionts in fostering a butyrogenic microbial network via cross-feeding. Ecologically, HMOS-degraders are critical to drive the establishment of a healthy infant gut microbiota, whilst mucin-degraders are vital to maintain a beneficial mucosal community. A better understanding of the complex nature of both the microbial network and the host-secreted glycans could aid in the design of nutritional intervention for health improvement. To this end, innovative avenues using novel probiotic strains (key species including <em>Akkermansia muciniphila</em>, <em>Bacteroides </em>spp. and butyrate-producing <em>Clostridium</em>), prebiotics (HMOS in early nutrition) and microbiota-targeted nutrients (iron and vitamin B12) deem promising.