The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.

Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.

33The human gut microbiota (HGM) are closely associated with health, development and 34 disease. The thick intestinal mucus layer, especially in the colon, is the key barrier between 35 the contents of the lumen and the epithelial cells, providing protection against infiltration by 36 the microbiota as well potential pathogens. The upper layer of the colonic mucus is a niche for 37 a subset of the microbiota which utilise the mucin glycoproteins as a nutrient source and mucin 38 grazing by the microbiota appears to play a key role in maintaining barrier function as well as 39 community stability. Despite the importance of mucin breakdown for gut health, the 40 mechanisms by which gut bacteria access this complex glycoprotein are not well understood. 41The current model for mucin degradation involves exclusively exo-acting glycosidases that 42 sequentially trim monosaccharides from the termini of the glycan chains to eventually allow 43 access to the mucin peptide backbone by proteases. However, this model is in direct contrast 44 to the Sus paradigm of glycan breakdown used by the Bacteroidetes which involves 45 extracellular cleavage of glycans by surface located endo-acting enzymes prior to import of 46 the oligosaccharide products. Here we describe the discovery and characterisation of endo-47 acting family 16 glycoside hydrolases (GH16s) from prominent mucin degrading gut bacteria 48 that specifically target the oligosaccharide side chains of intestinal mucins from both animals 49 and humans. These endo-acting O-glycanases display β1,4-glactosidase activity and in 50 several cases are surface located indicating they are involved in the initial step in mucin 51 breakdown. The data suggest a new paradigm for mucin breakdown by the microbiota and 52 the endo-mucinases provide a potential tool to explore changes that occur in mucin structure 53 in intestinal disorders such as inflammatory bowel disease and colon cancer. 54 suggesting endo-like cleavage of the O-glycan chains. To investigate the identity of these 126 products in more detail, the products were labelled with the fluorophore procainamide and 127 analysed by liquid chromatography-fluorescence-detection-electrospray-mass spectrometry 128 (LC-FLD-ESI-MS) and the glycan structures determined by MS/MS (Fig. 2a). The data show 129 that oligosaccharides are produced by the GH16 enzymes with between 2 and 6 alternating 130 hexose and HexNAc sugars that are likely to be sections of the polyLacNAc repeats that 131 form the repeating unit of O-glycan chains (Fig. 1b). The reducing ends were all hexoses, 132 11 Desai, M. S. et al. A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus 595 Barrier and Enhances Pathogen Susceptibility. Egan, M. et al. Cross-feeding by Bifidobacterium breve UCC2003 during co-cultivation with 598Bifidobacterium bifidum PRL2010 in a mucin-based medium.

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFβ2 and IL1β. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

Background and objectives: Membranoproliferative Glomerulonephritis (MPGN) and C3 Glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific aetiological data for paediatric MPGN/C3 glomerulopathy are lacking, and outcome data are based upon retrospective studies without aetiological data. Design, setting, participants, and measurements: Eighty prevalent pediatric patients with MPGN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using COX proportional hazards model. Kidney and transplant graft survival was determined using Kaplan-Meier method. Results: Central histology review determined 39 C3 glomerulopathy, 31 immune-complex MPGN and 10 immune-complex glomerulonephritis (GN) cases. Patients were aged 2-15 (median 9 (IQR 7-11) years. Median complement C3 and C4 levels were 0.31g/L and 0.14g/L respectively; acquired (anti-complement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% patients respectively, across all groups including immune-complex GN. Median follow-up was 5.18 (IQR 2.13-8.08) years. Eleven patients (14%) progressed to kidney failure with 9 transplants performed in 8 patients, 2 of which failed due to recurrent disease. Presence of >50% crescents on initial biopsy was the sole variable associated with kidney failure in multivariable analysis (Hazard Ratio 6.2, p = 0.045; 95% CI 1.05 to 36.6). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% crescents on initial biopsy. Conclusions: Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric MPGN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.