Mucin and human milk oligosaccharides utilization: a strategy of Akkermansia muciniphila to ensure survival in the human gut

The research in this thesis aims to understand the mechanism used by Akkermansia muciniphila to survive and thrive in the complex and competitive ecosystems of the human gut. We evaluated the ability of A. muciniphila to utilize and degrade HMOs and mucin glycans as a carbohydrate specialist and the advantage of this phenotype in this highly competitive environment. As it has been demonstrated, A. muciniphila can have an important role in human (metabolic) health and it is essential to understand the functions and resilience of A. muciniphila in such communities. This can further expand our knowledge on the benefits that A. muciniphila can bring for human health, but potentially also help to design (nutritional) therapies to support A. muciniphila’s activity in the gut to even further support human health.

In Chapter 1, a brief overview of the human intestinal microbiota is provided, focusing on the host-derived glycans that affect the physiology and composition of the gut microbiota. Furthermore, A. muciniphila, a resident of the mucosal layer in the gut, is introduced. The capacity of A. muciniphila to use glycoside hydrolases (GHs) to degrade glycosidic linkages found in mucin and human milk oligosaccharides (HMOs) constituted the motivation for the research described in this thesis.

Chapter 2 provides an overview on the presence and functionality of A. muciniphila in different parts of the gastrointestinal tract. Akkermansia muciniphila has been found in human milk, the oral cavity, the pancreas, the biliary system, the small intestine, and the appendix. Hypothetical functions of A. muciniphila in these different niches are proposed, highlighting also that further research is needed to fully understand the versatile roles that A. muciniphila may have in the digestive tract.

In Chapter 3 we hypothesized that the presence of A. muciniphila in infant’s intestine is the result of its ability to use its glycan degrading enzymes to break down HMOs. After growth of A. muciniphila in human milk, we identified HMOs being utilized by A. muciniphila due to the expression of GHs such as α-l-fucosidases, exo-α-sialidases, β-galactosidases, and β-hexosaminidases. We confirmed our hypothesis by testing heterologously expressed and purified GHs against the degradation of pure HMOs (2’-FL and LNT) and human milk derivatives (lactose, LNT2). Based on the experimental data, we proposed a model for the utilization of 2’-FL, 3’-SL, LNT, LNT2, and lactose by A. muciniphila. The ability of A. muciniphila to degrade HMOs leads to the release of metabolites, like mono- and disaccharides, which contribute to cross-feeding pathways in the gut and providing nutrients to the resident bacteria. These findings explain how A. muciniphila is able to colonize the gut in early life.

Chapter 4 describes the microbe-to-microbe interaction of A. muciniphila with another mucin-degrading bacterium, Bacteroides thetaiotaomicron, a more generalist micro-organism, under continuous influx of mucin glycans in vitro. A. muciniphila exhibited little or no alterations in its gene expression profiles for exploitation of mucin glycans, while B. thetaiotaomicron showed a trend for increased expression of its GHs coding gene to utilize the available nutrients in the co-culture. We also observed that B. thetaiotaomicron tried to gain competitive advantage by expressing genes coding for antimicrobial proteins/peptides. We hypothesize that A. muciniphila is able to counteract this by expressing LPS associated biosynthesis genes and ABC transporters that make the organism resistant against antimicrobial proteins. Finally colonizing germ-free mice with the A. muciniphila and B. thetaiotaomicron showed no significant differences in the gene expression of both species between mono- and co-colonization mice. We explain this by the fact that the high fiber diet used in mice, might sustain two different ecological niches in the rodent gut, and that the micro-organisms do not compete for substrate nor space.

In Chapter 5, we describe the assembly of a synthetic community in controlled bioreactors of 16 different species including A. muciniphila in order to monitor the trophic and metabolic interactions between mucin degrading species and species that were part of the core microbiota. The members of the minimal microbiome were continuously fed with mucin, while three times per day, dietary fibers (pectin, starch, inulin, and xylan) were fed into the bioreactors. We identified the establishment of four different trophic guilds in the minimal microbiome driven by the available nutrients in the community.

In Chapter 6 a comparison of the transcriptional response of A. muciniphila between different experiments of chapter 4 and 5 is described. We studied the transcriptional landscape of A. muciniphila under varying conditions such as complexity of the community, introduction of carbohydrates, media composition, and experimental design (in vitro vs. in vivo). Special focus was given to key functions of A. muciniphila, e.g. mucin degradation, EPS production and pili associated genes expression. We found that A. muciniphila was able to perform these key functions both in vitro and in vivo, independently of the environmental changes, the complexity of culture and media composition. The consistency in expression of key functions from A. muciniphila might indicate how robust and resilient A. muciniphila is in an environment rich in mucin glycans.

Finally, in Chapter 7, all the research findings both in vitro and in vivo of this thesis are discussed, in detail highlighting the ability of A. muciniphila to thrive in complex environments as the human intestine via degradation of host-secreted glycans. With the research described in this thesis, we have attempted to unravel the metabolic capabilities of A. muciniphila and important microbe-microbe interactions of this unique species in complex environments. We described the capability of A. muciniphila to utilize and degrade HMOs via glycan-utilizing pathways that are also essential for growth in the mucus environment in the gut. The trait of being able to utilize HMO’s also indicates the important role that A. muciniphila may play in early life colonization. Finally, potential applications are suggested for the future application of A. muciniphila as a probiotic strain either alone or in a combination with HMOs or other prebiotics (synbiotic mixture) to support human health.