Dataset Information

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

ABSTRACT: 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.

SUBMITTER: Crouch LI

PROVIDER: S-EPMC7419316 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

Crouch Lucy I LI Liberato Marcelo V MV Urbanowicz Paulina A PA Baslé Arnaud A Lamb Christopher A CA Stewart Christopher J CJ Cooke Katie K Doona Mary M Needham Stephanie S Brady Richard R RR Berrington Janet E JE Madunic Katarina K Wuhrer Manfred M Chater Peter P Pearson Jeffery P JP Glowacki Robert R Martens Eric C EC Zhang Fuming F Linhardt Robert J RJ Spencer Daniel I R DIR Bolam David N DN

Nature communications 20200811 1

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 olig ...[more]

PMID: 32782292

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Project description:Glycans, the major source of energy for the human gut microbiota (HGM), are metabolised by numerous members of the Bacteroides genus. Arabinogalactan proteins (AGPs) are a ubiquitous and highly heterogenous group of plant glycans in which a B1,3-galactan backbone and B1,6-galactan side chains are conserved features. Diversity is provided by the extensive and highly variable nature of the sugars that decorate both the backbone and side chain galactans. The mechanisms by which nutritionally relevant AGPs are degraded at a cellular and biochemical level are poorly understood, as is the impact of this process on the ecology of the HGM. Here we have explored how the HGM organism Bacteroides thetaiotaomicron metabolises simple and highly complex AGPs. We propose a sequential degradative model in which a repertoire of exo-acting family GH43 B1,3-galactanases releases backbone galactose units that are attached to the side chains. The oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or glycoside hydrolase. We identified an a-L-arabinofuranosidase and B-D-glucuronidase that are the founders of two previously undiscovered glycoside hydrolase families. The crystal structures of the backbone exo-B1,3-galactanases identified a key specificity determinant and reveals significant departure from the canonical catalytic apparatus of other family GH43 enzymes. Growth studies of 20 HGM Bacteroidetes species on a complex AGP revealed three keystone organisms that facilitated utilisation of fragments of the glycan by the 17 other bacteria, which thus acted as recipients. The ability to function as a keystone organism was conferred by a surface endo-B1,3-galactanase, which, when engineered into a recipient enabled this bacterium to utilise complex AGPs and facilitate the growth of the other Bacteroidetes species. This study underpins future pre- and probiotic strategies to exploit AGPs to manipulate the function of the HGM.

Project description:BackgroundPost-weaning diarrhea in piglets reduces growth performance and increases mortality, thereby causing serious economic losses. The intestinal epithelial cells and microbiota reciprocally regulate each other in order to maintain intestinal homeostasis and control inflammation. However, a relative paucity of research has been focused on the host-derived regulatory network that controls mucin O-glycans and thereby changes gut microbiota during diarrhea in infancy. At the development stage just after birth, the ontogeny of intestinal epithelium, immune system, and gut microbiota appear similar in piglets and human infants. Here, we investigated the changes of mucin O-glycans associated with gut microbiota using a diarrheal post-weaned piglet model.ResultsWe found that diarrhea disrupted the colonic mucus layer and caused aberrant mucin O-glycans, including reduced acidic glycans and truncated glycans, leading to an impaired gut microenvironment. Subsequently, the onset of diarrhea, changes in microbiota and bacterial translocation, resulting in compromised epithelial barrier integrity, enhanced susceptibility to inflammation, and mild growth faltering. Furthermore, we found the activation of NLRP3 inflammasome complexes in the diarrheal piglets when compared to the healthy counterparts, triggered the release of proinflammatory cytokines IL-1β and IL-18, and diminished autophagosome formation, specifically the defective conversion of LC3A/B I into LC3A/B II and the accumulation of p62. Additionally, selective blocking of the autophagy pathway by 3-MA led to the reduction in goblet cell-specific gene transcript levels in vitro.ConclusionsWe observed that diarrheal piglets exhibited colonic microbiota dysbiosis and mucosal barrier dysfunction. Our data demonstrated that diarrhea resulted in the activation of inflammasomes and autophagy restriction along with aberrant mucin O-glycans including reduced acidic glycans and truncated glycans. The results suggested the mucin O-glycans-microbiota axis is likely associated with diarrheal pathogenesis. Our study provides novel insights into the pathophysiology of early-weaning-induced diarrheal disease in piglets and potentially understanding of disease mechanisms of diarrhea for human infants. Understanding the molecular pathology and pathogenesis of diarrhea is a prerequisite for the development of novel and effective therapies. Our data suggest that facilitating O-glycan elongation, modifying the microbiota, and developing specific inhibitors to some key inflammasomes could be the options for therapy of diarrhea including human infants. Video abstract.

Dataset Information

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

Publications

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

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