Ruminococcus torques is a keystone degrader of intestinal mucin glycoprotein, releasing oligosaccharides used by Bacteroides thetaiotaomicron.

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 ß1,4-galactosidase activities. There was a lack of detectable sulfatase and weak ß1,3-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.IMPORTANCEAn 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.

Metaproteomics reveals parallel utilization of colonic mucin glycans and dietary fibers by the human gut microbiota.

A diet lacking dietary fibers promotes the expansion of gut microbiota members that can degrade host glycans, such as those on mucins. The microbial foraging on mucin has been associated with disruptions of the gut-protective mucus layer and colonic inflammation. Yet, it remains unclear how the co-utilization of mucin and dietary fibers affects the microbiota composition and metabolic activity. Here, we used 14 dietary fibers and porcine colonic and gastric mucins to study the dynamics of mucin and dietary fiber utilization by the human fecal microbiota in vitro. Combining metaproteome and metabolites analyses revealed the central role of the Bacteroides genus in the utilization of complex fibers together with mucin while Akkermansia muciniphila was the main utilizer of sole porcine colonic mucin but not gastric mucin. This study gives a broad overview of the colonic environment in response to dietary and host glycan availability.

The structure of the second CysD domain of MUC2 and role in mucin organization by transglutaminase-based cross-linking.

The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.

GLP-1R signaling modulates colonic energy metabolism, goblet cell number and survival in the absence of gut microbiota.

OBJECTIVES: Gut microbiota increases energy availability through fermentation of dietary fibers to short-chain fatty acids in conventionally raised mice. Energy deficiency in germ-free (GF) mice increases glucagon-like peptide-1 (GLP-1) levels, which slows intestinal transit. To further analyze the role of GLP-1-mediated signaling in this model of energy deficiency, we re-derived mice lacking GLP-1 receptor (GLP-1R KO) as GF. METHODS: GLP-1R KO mice were rederived as GF through hysterectomy and monitored for 30 weeks. Mice were subjected to rescue experiments either through feeding an energy-rich diet or colonization with a normal cecal microbiota. Histology and intestinal function were assessed at different ages. Intestinal organoids were assessed to investigate stemness. RESULTS: Unexpectedly, 25% of GF GLP-1R KO mice died before 20 weeks of age, associated with enlarged ceca, increased cecal water content, increased colonic expression of apical ion transporters, reduced number of goblet cells and loss of colonic epithelial integrity. Colonocytes from GLP-1R KO mice were energy-deprived and exhibited increased ER-stress; mitochondrial fragmentation, increased oxygen levels and loss of stemness. Restoring colonic energy levels either by feeding a Western-style diet or colonization with a normal gut microbiota normalized gut phenotypes and prevented lethality. CONCLUSIONS: Our findings reveal a heretofore unrecognized role for GLP-1R signaling in the maintenance of colonic physiology and survival during energy deprivation.

Ruminococcus torques is a keystone degrader of intestinal mucin glycoprotein, releasing oligosaccharides used by Bacteroides thetaiotaomicron.

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 remains poorly studied despite its implication 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 fucosidase, sialidase and ß1,4-galactosidase activities. There was a lack of detectable sulfatase and weak ß1,3-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 may 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 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. 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 gut bacterium implicated in IBD, Bacteroides thetaiotaomicron . 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.

Goblet cell interactions reorient bundled mucus strands for efficient airway clearance.

The respiratory tract of larger animals is cleared by sweeping bundled strands along the airway surface. These bundled strands can be millimetric in length and consist of MUC5B mucin. They are produced by submucosal glands, and upon emerging from these glands, the long axis of the bundled strands is oriented along the cilia-mediated flow toward the oral cavity. However, after release, the bundled strands are found to have turned orthogonal to the flow, which maximizes their clearance potential. How this unexpected reorientation is accomplished is presently not well understood. Recent experiments suggest that the reorientation process involves bundled strands sticking to MUC5AC mucus threads, which are tethered to the goblet cells. Such goblet cells are present in small numbers throughout the airway epithelium. Here, we develop a minimal model for reorientation of bundled mucus strands through adhesive interactions with surface goblet cells. Our simulations reveal that goblet cell interactions can reorient the bundled strands within 10 mm of release-making reorientation on the length scale of the tracheal tube feasible-and can stabilize the orthogonal orientation. Our model also reproduces other experimental observations such as strong velocity fluctuations and significant slow-down of the bundled strand with respect to the cilia-mediated flow. We further provide insight into the strand turning mechanism by examining the effect of strand shape on the impulse exerted by a single goblet cell. We conclude that goblet cell-mediated reorientation is a viable route for bundled strand reorientation, which should be further validated in future experiment.

Microbial experience through housing in a farmyard-type environment alters intestinal barrier properties in mouse colons.

To close the gap between ultra-hygienic research mouse models and the much more environmentally exposed conditions of humans, we have established a system where laboratory mice are raised under a full set of environmental factors present in a naturalistic, farmyard-type habitat-a process we have called feralization. In previous studies we have shown that feralized (Fer) mice were protected against colorectal cancer when compared to conventionally reared laboratory mice (Lab). However, the protective mechanisms remain to be elucidated. Disruption of the protective intestinal barrier is an acknowledged player in colorectal carcinogenesis, and in the current study we assessed colonic mucosal barrier properties in healthy, feralized C57BL/6JRj male mice. While we found no effect of feralization on mucus layer properties, higher expression of genes encoding the mucus components Fcgbp and Clca1 still suggested mucus enforcement due to feralization. Genes encoding other proteins known to be involved in bacterial defense (Itln1, Ang1, Retnlb) and inflammatory mechanisms (Zbp1, Gsdmc2) were also higher expressed in feralized mice, further suggesting that the Fer mice have an altered intestinal mucosal barrier. These findings demonstrate that microbial experience conferred by housing in a farmyard-type environment alters the intestinal barrier properties in mice possibly leading to a more robust protection against disease. Future studies to unravel regulatory roles of feralization on intestinal barrier should aim to conduct proteomic analyses and in vivo performance of the feralized mice intestinal barrier.

Intestinal mucus and their glycans: A habitat for thriving microbiota.

The colon mucus layer is organized with an inner colon mucus layer that is impenetrable to bacteria and an outer mucus layer that is expanded to allow microbiota colonization. A major component of mucus is MUC2, a glycoprotein that is extensively decorated, especially with O-glycans. In the intestine, goblet cells are specialized in controlling glycosylation and making mucus. Some microbiota members are known to encode multiple proteins that are predicted to bind and/or cleave mucin glycans. The interactions between commensal microbiota and host mucins drive intestinal colonization, while at the same time, the microbiota can utilize the glycans on mucins and affect the colonic mucus properties. This review will examine this interaction between commensal microbes and intestinal mucins and discuss how this interplay affects health and disease.

The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins.

The MUC2 mucin polymer is the main building unit of the intestinal mucus layers separating intestinal microbiota from the host epithelium. The MUC2 mucin is a large glycoprotein with a C-terminal domain similar to the MUC5AC and MUC5B mucins and the von Willebrand factor (VWF). A structural model of the C-terminal part of MUC2, MUC2-C, was generated by combining Cryo-electron microscopy, AlphaFold prediction, information of its glycosylation, and small angle X-ray scattering information. The globular VWD4 assembly in the N-terminal of MUC2-C is followed by 3.5 linear VWC domains that form an extended flexible structure before the C-terminal cystine-knot. All gel-forming mucins and VWF form tail-tail disulfide-bonded dimers in their C-terminal cystine-knot domain, but interestingly the MUC2 mucin has an extra stabilizing disulfide bond on the N-terminal side of the VWD4 domain, likely essential for a stable intestinal mucus barrier.

Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets.

BACKGROUND: The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000-5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. METHODS: We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. RESULTS: Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1-4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. CONCLUSIONS: These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1-4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.

Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis.

The metalloproteases meprin α and meprin ß are highly expressed in the healthy gut but significantly decreased in inflammatory bowel disease, implicating a protective role in mucosal homeostasis. In the colon, meprin α and meprin ß form covalently linked heterodimers tethering meprin α to the plasma membrane, therefore presenting dual proteolytic activity in a unique enzyme complex. To unravel its function, we applied N-terminomics and identified galectin-3 as the major intestinal substrate for meprin α/ß heterodimers. Galectin-3-deficient and meprin α/ß double knockout mice show similar alterations in their microbiome in comparison to wild-type mice. We further demonstrate that meprin α/ß heterodimers differentially process galectin-3 upon bacterial infection, in germ-free, conventionally housed (specific pathogen-free), or wildling mice, which in turn regulates the bacterial agglutination properties of galectin-3. Thus, the constitutive cleavage of galectin-3 by meprin α/ß heterodimers may play a key role in colon host-microbiome homeostasis.

Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection.

Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospecific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the regiospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colonization, but is highly sensitive to disruption by exposure to a WSD.

A Golgi oxygen sensor controls intestinal mucin glycosylation.

Intestinal mucin glycosylation is important for mucus-bacterial homeostasis and is altered in disease. In this issue of The EMBO Journal, Ilani et al (2022) identify the Golgi enzyme quiescin sulfhydryl oxidase 1 (QSOX1) as a novel mucus regulator by controlling mucin sialylation.

Mucus aberrant properties in CF: Insights from cells and animal models.

Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, is characterized by mucus accumulation in the lungs, the intestinal tract, and the pancreatic ducts. Mucins are high-molecular-weight glycoproteins that govern the biochemical and biophysical properties of mucus. In the CF lung, increased mucus viscoelasticity is associated with decreased mucociliary clearance and defects in host defense mechanisms. The link between defective ion channel and abnormal mucus properties has been investigated in studies involving cell and animal models. In this review article, we discuss recent progress toward understanding the different regions and cells that express CFTR in the airways and how mucus is produced and cleared from the lungs. In addition, we reflect on animal models that provided insights into the organization and the role of the mucin network and how mucus and antimicrobial activities act in concert to protect the lungs from invading pathogens.

Clearance of small intestinal crypts involves goblet cell mucus secretion by intracellular granule rupture and enterocyte ion transport.

Goblet cells in the small intestinal crypts contain large numbers of mucin granules that are rapidly discharged to clean bacteria from the crypt. Because acetylcholine released by neuronal and nonneuronal cells controls many aspects of intestinal epithelial function, we used tissue explants and organoids to investigate the response of the small intestinal crypt to cholinergic stimulation. The activation of muscarinic acetylcholine receptors initiated a coordinated and rapid emptying of crypt goblet cells that flushed the crypt contents into the intestinal lumen. Cholinergic stimulation induced an expansion of the granule contents followed by intracellular rupture of the mucin granules. The mucus expanded intracellularly before the rupture of the goblet cell apical membrane and continued to expand after its release into the crypt lumen. The goblet cells recovered from membrane rupture and replenished their stores of mucin granules. Mucus secretion from the goblet cells depended on Ca2+ signaling and the expansion of the mucus in the crypt depended on gap junctions and on ion and water transport by enterocytes adjacent to the goblet cells. This distinctive mode of mucus secretion, which we refer to as "expanding secretion," efficiently cleans the small intestine crypt through coordinated mucus, ion, and fluid secretion by goblet cells and enterocytes.

Goblet cells need some stress.

The intestinal tract is protected by epithelium-covering mucus, which is constantly renewed by goblet cells, a specialized type of epithelial cell. Mucus is largely composed of MUC2 mucin, an enormous molecule that poses a high demand on the endoplasmic reticulum (ER) for proper folding and protein assembly, creating a challenge for the secretory machinery in goblet cells. In this issue of the JCI, Grey et al. reveal that the ER resident protein and folding sensor ERN2 (also known as IRE1ß) was instrumental for goblet cells to produce sufficient amounts of mucus to form a protective mucus layer. In the absence of ERN2, mucus production was reduced, impairing the mucus barrier, which allowed bacteria to penetrate and cause an epithelial cell stress response. This study emphasizes the importance of a controlled unfolded protein response (UPR) for goblet cell secretion.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1ß and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

Transglutaminase 3 crosslinks the secreted gel-forming mucus component Mucin-2 and stabilizes the colonic mucus layer.

The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.