Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila

<strong>Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila</strong> <strong>Noora A. Ottman</strong> The human gastrointestinal tract is colonized by a complex community of micro-organisms, the gut microbiota. The majority of these are bacteria, which perform various functions involved in host energy metabolism and immune system stimulation. The field of gut microbiology is continuously expanding as novel species are isolated and high-throughput techniques are developed. The research focus is shifting from DNA-based techniques, looking at microbial community composition, to techniques relying on analysis of RNA and proteins, which reveal more about the activity and functionality of the microbiota. The mucosa-associated microbiota forms a distinct population in the gut, and is influenced by the close proximity of the epithelial layer and nutrients present in the mucus layer. One of the key players in this community is the mucus degrader Akkermansia muciniphila. This Gram-negative, anaerobic bacterium can use mucin, the main component of mucus, as the sole carbon and nitrogen source for growth. A. muciniphila belongs to the phylum Verrucomicrobia and is present in the majority of humans, starting from early life. Interestingly, the levels of A. muciniphila are negatively correlated with several disorders, including inflammatory bowel diseases and diabetes. A. muciniphila lives in a symbiosis with its host, harvesting energy from mucin; whether the relationship is mutualistic, and thereby also beneficial to the host, remains to be discovered. In this thesis, the ability of A. muciniphila to utilize the host-derived glycans mucin and human milk oligosaccharides was studied in detail. In addition, the host-bacterial interactions were examined by immunological assays, focusing especially on the effect of A. muciniphila outer membrane proteins on host immune response. The genome of A. muciniphila encodes numerous enzymes involved in mucin degradation. Transcriptome analysis comparing the gene expression of A. muciniphila grown on mucin or the non-mucin sugar glucose confirmed the activity of these genes and revealed most of them to be upregulated in the presence of mucin. This was also confirmed by a proteome analysis, reinforcing the adaptation of A. muciniphila to the mucosal environment. A genome-based metabolic model was constructed to test amino acid auxotrophy, vitamin biosynthesis, and sugar-degrading capacities of A. muciniphila. The model predicted A. muciniphila to be able to synthesize all the essential amino acids, with the exception of threonine, which was added to the mucin-free medium designed to test A. muciniphila growth on single sugars. A. muciniphila was able to individually metabolize all the main monomeric sugars present in mucin, albeit with limited efficiency in comparison to mucin. As mucin shares structural similarities with human milk oligosaccharides (HMOs), which stimulate the bacterial community colonizing the gut in early life, growth of A. muciniphila on human milk and its components was tested. A. muciniphila showed metabolic activity on human milk and one of the HMOs, 2&rsquo;-fucosyllactose. Comparison of A. muciniphila activity during growth on human milk or mucin revealed that the expression of genes involved in mucin degradation was similar for both experimental conditions, suggesting that A. muciniphila might be capable of also using the corresponding gene products for utilization of human milk glycans. The capacity to survive in the early life environment by degrading and consuming human milk components would be beneficial for A. muciniphila during initial colonization before reaching the mucosal layer in the intestine. Several mouse studies have indicated that A. muciniphila is able to modulate the host immune system, possibly to the benefit of the host, but not much is known about its immunological mechanism of action. The cell envelope structures of bacteria can have a big influence on their immunostimulatory capacities, and therefore the outer membrane (OM) proteome of A. muciniphila was characterized. The membrane structure of A. muciniphila is also of interest because it belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum, which contains bacteria with features that differentiate them from classical Gram-negative bacteria, including a complex endomembrane system. Mass spectrometry data, coupled with bioinformatics analysis, revealed the presence of highly abundant OM proteins involved in secretion, transport and biogenesis of the Gram-negative membranes, as well as proteins predicted to take part in formation of the fimbriae-like structures observed in A. muciniphila by electron microscopy. Live A. muciniphila and the identified OM proteins induced production of a wide range of cytokines and activated the intestinal Toll-like receptors 2 and 4. Moreover, a 30 kDa protein that was predicted to form a part of the fimbriae, increased transepithelial resistance, indicating it may be involved in improving gut barrier function. Based on the evidence from in vitro and in vivo studies, A. muciniphila is a promising candidate for a next-generation probiotic. However, further confirmation of causal relationships between disease development and presence of this species in the gut is required. The findings of this thesis provide valuable insight into the bacterial lifestyle and host interactions of the gut symbiont A. muciniphila