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ABSTRACT Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how Ruminococcus torques— a known mucin degrader that has been implicated in inflammatory bowel diseases (IBDs)—degrades mucin glycoproteins or their component O -linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that R. torques utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by R. torques revealed strong α-L-fucosidase, sialidase and β1,4-galactosidase activities. There was a lack of detectable sulfatase and weak β1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, Bacteroides thetaiotaomicron grows poorly on mucin glycoproteins, we demonstrate a clear ability of R. torques to liberate products from mucins, making them accessible to B. thetaiotaomicron . This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of R. torques to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which might contribute to its association with IBD. IMPORTANCE An important facet of maintaining healthy symbiosis between host and intestinal microbes is the mucus layer, the first defense protecting the epithelium from lumenal bacteria. Some gut bacteria degrade the various components of intestinal mucins, but detailed mechanisms used by different species are still emerging. It is imperative to understand these mechanisms as they likely dictate interspecies interactions and may illuminate species associated with bacterial mucus damage and subsequent disease susceptibility. Ruminococcus torques is positively associated with IBD in multiple studies. We identified mucin glycan-degrading enzymes in R. torques and found that it shares mucin degradation products with another species of gut bacteria, Bacteroides thetaiotaomicron . Our findings underscore the importance of understanding mucin degradation mechanisms in different gut bacteria and their consequences on interspecies interactions, which may identify keystone bacteria that disproportionately affect mucus damage and could therefore be key players in effects that result from reductions in mucus integrity.

Abstract Fiber free exclusive enteral nutrition (EEN) is an effective steroid-sparing treatment used to induce clinical remission in children with Crohn’s disease (CD). However, the mechanism underlying the beneficial effects of EEN remains obscure. We have generated a novel mouse strain that harbors mutations in two CD susceptibility genes (i.e, NOD2 and CYBB) and found that these mice spontaneously develop an early-onset (4 weeks of age), TH1-type gut inflammation, that closely recapitulates the human disease when exposed to a specific murine microbiota. Disease in Nod2/Cybb (DKO) mutants was triggered by a single mucus-dwelling anaerobe, Mucispirillum schaedleri, which markedly accumulates in the intestinal lumen and mucus layer prior to disease development. Given that our mouse model resembles early-onset CD, we have established a novel humanized mouse model of early-onset CD in which germ-free Nod2/Cybb mutants were colonized with microbiotas from early-onset-CD patients and their healthy first-degree relatives. We have found that the early-onset-CD-associated microbiota triggered intestinal inflammation in gnotobiotic DKO mice with all the hallmarks of the human disease. Using the murine and humanized models of early-onset CD we have found that administration of a fiber-free diet prevents the development of colitis and inhibits intestinal inflammation in colitic animals. Furthermore, identification of Mucispirillum as a trigger in our mouse model has allowed us to study the effects of dietary interventions on disease-causing microbes. Remarkably, we have found that a fiber-free diet alters the intestinal localization of Mucispirillum. Mechanistically, the absence of dietary fiber reduces the availability of nutrients and impairs Mucispirillum’s unique metabolic pathway in the intestinal mucus. Thus, appropriate localization of the pathobiont in the mucus layer is critical for the onset of disease, which can be disrupted by fiber exclusion. These results suggest that probing the intestinal niche and metabolism of disease-causing microbes will likely lead to the discovery of novel, more targeted therapies for the treatment of CD.

Mucus is a protective barrier secreted in the gastrointestinal tract to promote healthy separation between host tissues and the resident gut microbiota. Mucus forms two distinct layers covering the epithelium and is mainly composed of complex, highly glycosylated mucin monomers. Interestingly, some bacterial species occupy the outer mucus layer and utilize it as a nutrient source. While this is a feature of a healthy microbiota, mucin-degrading bacteria have been implicated in contributing to disease, such as inflammatory bowel disease, when present with other risk factors. Bacterial enzymes that target highly complex mucin glycoproteins have been identified, but a single enzymatic repertoire that enables a species to degrade specific components of mucins has not been identified. Thus, mucin-degrading mechanisms must be empirically characterized in individual species to understand how these bacteria access different components of mucin and how this activity influences the gut bacterial community.Here, I further characterize the mucin-degrading mechanisms of two gut bacteria: Ruminococcus torques and Bacteroides thetaiotaomicron. I found that R. torques degrades both intact mucin glycoproteins and free mucin glycans, predominantly using constitutively expressed, secreted enzymes. This mechanism allows R. torques to cross-feed degraded mucin products to B. thetaiotaomicron, which can only utilize free mucin glycans, as demonstrated in in vitro co-culture experiments and growth curves on R. torques pre-digested mucin. Thus, I have established that R. torques is a keystone mucin-degrader, which acts as a primary degrader of this complex substrate and releases simpler products that become available to species that cannot access mucin glycoprotein alone.While examining interspecies interactions is important to understand how mucin is degraded within the gut bacterial community, identifying and characterizing individual mucin-degrading enzymes is important to identify potential therapeutic targets to block bacterial mucin-degradation in vivo. To this end, we identified the activity of 36 putative mucin glycan-degrading glycoside hydrolase enzymes in B. thetaiotaomicron, including the discovery of novel endo-glycanase activity in three GH18 family enzymes. Interestingly, B. thetaiotaomicron encodes a redundant mucin-degrading enzyme repertoire, expressing multiple enzymes from the same glycoside hydrolase family or with the same substrate specificities. I assessed the contributions of a subset of these enzymes to the mucin-degrading ability and fitness of B. thetaiotaomicron by testing gene deletion mutant strains in in vitro growth assays on purified mucin substrates and in vivo competitions against the parent strain. Indeed, enzymatic redundancy did protect against loss of some enzymes, such as fucosidases. However, in other cases, such as with a mutant lacking three mucin-degrading loci, complementation with a single sulfatase enzyme was sufficient to rescue the loss of the other enzymes in these loci. Identification of these key enzymes critical to the ability of B. thetaiotaomicron to degrade mucin substrates will inform future experiments to develop approaches to blocking mucin-degradation.Together, these results underscore the importance of continued investigation and characterization of mucin-degrading mechanisms in additional species to understand both community interactions and individual enzyme contributions to this phenotype, which will facilitate the development of tools to block mucin-degradation in cases where it contributes to disease.

Secreted mucins are the major component of the mucus layer that protects intestinal epithelial surfaces by blocking excessive interactions with the microbiota. Mucins are complex glycoproteins decorated with over 100 different -glycans. Some bacteria can utilize mucins and excessive degradation has been associated with disruption of the mucus barrier and inflammation. Despite the importance of mucins, a detailed enzymatic pathway by which gut bacteria degrade colonic mucin -glycans and the impact of this process on gut colonization are unknown. Here, we identified >100 genes that are expressed by the symbiont during growth on different -glycan substrates, revealing effects of glycan structure on gene expression. The characterization of 33 glycoside hydrolase enzymes revealed the pathway for colonic -glycan degradation by this bacterium. competition experiments show that multiple exo-acting enzymes targeting mucin capping structures are central to gut colonization and may provide targets to inhibit bacterial mucin degradation.

Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how a known mucin-degrader that remains poorly studied despite its implication in inflammatory bowel diseases (IBDs)- degrades mucin glycoproteins or their component -linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by revealed strong fucosidase, sialidase and β1,4-galactosidase activities. There was a lack of detectable sulfatase and weak β1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, grows poorly on mucin glycoproteins, we demonstrate a clear ability of to liberate products from mucins, making them accessible to . This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which may contribute to its association with IBD. An important facet of maintaining healthy symbiosis between host and intestinal microbes is the mucus layer, the first defense protecting the epithelium from lumenal bacteria. Some gut bacteria degrade different components of intestinal mucins, but detailed mechanisms used by different species are still emerging. It is imperative to understand these mechanisms as they likely dictate interspecies interactions and may illuminate particular species associated with bacterial mucus destruction and subsequent disease susceptibility. is positively associated with IBD in multiple studies. We identified mucin glycan-degrading enzymes in and found that it shares mucin degradation products with another gut bacterium implicated in IBD, . Our findings underscore the importance of understanding the mucin degradation mechanisms of different gut bacteria and their consequences on interspecies interactions, which may identify keystone bacteria that disproportionately contribute to defects in mucus protection and could therefore be targets to prevent or treat IBD.

Akkermansia muciniphila, an obligate mucin degrader, is a major member of the human colonic microbiota and has been associated positive health outcomes. Mucins are complex glycoproteins that contain heavily sulfated O-glycans and form the protective colonic mucus layer. Bacterial carbohydrate sulfatases are required to metabolise these heavily sulfated mucin glycans and excessive bacterial foraging has been associated with several diseases. Sulfatases have been linked with inflammatory bowel disease, making these microbiota enzymes potential drug targets. A. muciniphila expresses carbohydrate sulfatases that can act on colonic mucins yet their roles in its metabolism remain opaque. Our data reveal that A. muciniphila requires glycopeptides/protein forms of colonic mucin for metabolism and its sulfatases have unique adaptations compared to Bacteroides species. Localisation studies reveal that desulfation of N-acetyl-D-glucosamine, but not D-galactose, is exclusively periplasmic. A cell surface sulfatase has a novel carbohydrate binding module that binds to colonic mucin. This paints a contrasting picture of sulfated mucin metabolism by Akkermansia muciniphila versus Bacteroides species. These data will be important for understanding the contexts for Akkermansia muciniphila’s positive health correlations.