Bacteria Penetrate the Inner Mucus Layer before Inflammation in the Dextran Sulfate Colitis Model

Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064-15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3-5 days, but early events explaining why DSS causes this has not been described.When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 microm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells.DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis

AGR2, an Endoplasmic Reticulum Protein, Is Secreted into the Gastrointestinal Mucus

The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2(-/-) mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine

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

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

Site-specific O-glycosylation on the MUC_{2} mucin protein inhibits cleavage by the porphyromonas gingivalis secreted cysteine protease (RgpB)

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation

Resource consumption and management associated with monitoring of warfarin treatment in primary health care in Sweden

BACKGROUND: Warfarin is used for the prevention and treatment of various thromboembolic complications. It is an efficacious anticoagulant, but it has a narrow therapeutic range, and regular monitoring is required to ensure therapeutic efficacy and at the same time avoid life-threatening adverse events. The objective was to assess management and resource consumption associated with patient monitoring episodes during warfarin treatment in primary health care in Sweden. METHODS: Delphi technique was used to systematically explore attitudes, demands and priorities, and to collect informed judgements related to monitoring of warfarin treatment. Two separate Delphi-panels were performed in three and two rounds, respectively, one concerning tests taken in primary health care centres, involving 34 GPs and 10 registered nurses, and one concerning tests taken in patients' homes, involving 49 district nurses. RESULTS: In the primary health care panel 10 of the 34 GPs regularly collaborated with a registered nurse. Average time for one monitoring episode was estimated to 10.1 minutes for a GP and 21.4 minutes for a nurse, when a nurse assisted a doctor. The average time for monitoring was 17.6 minutes for a GP when not assisted by a nurse. Considering all the monitoring episodes, 11.6% of patient blood samples were taken in the individual patient's home. Average time for such a monitoring episode was estimated to 88.2 minutes. Of all the visits, 8.2% were performed in vain and took on average 44.6 minutes. In both studies, approximately 20 different elements of work concerning management of patients during warfarin treatment were identified. CONCLUSION: Monitoring of patients during treatment with warfarin in primary health care in Sweden involves many elements of work, and demands large resources, especially when tests are taken in the patient's home

Mucus detachment by host metalloprotease meprin \beta requires shedding of its inactive pro-form, which is abrogated by the pathogenic protease RgpB

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota

International audienceSulfated glycans are ubiquitous nutrient sources for microbial communities that have co-evolved with eukaryotic hosts. Bacteria metabolise sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognise their glycan substrate remain poorly understood. Here, we utilise structural biology to determine how sulfatases from the human gut microbiota recognise sulfated glycans. We reveal 7 new carbohydrate sulfatase structures span four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent the first structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity towards related but distinct glycan targets. Collectively, these data reveal that Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: https://www.springernature.com/gp/open-research/policies/accepted-manuscript-term