The mucin-degradation strategy of Ruminococcus gnavus:The importance of intramolecular trans-sialidases

We previously identified and characterized an intramolecular trans-sialidase (IT-sialidase) in the gut symbiont Ruminococcus gnavus ATCC 29149, which is associated to the ability of the strain to grow on mucins. In this work we have obtained and analyzed the draft genome sequence of another R. gnavus mucin-degrader, ATCC 35913, isolated from a healthy individual. Transcriptomics analyses of both ATCC 29149 and ATCC 35913 strains confirmed that the strategy utilized by R. gnavus for mucin-degradation is focused on the utilization of terminal mucin glycans. R. gnavus ATCC 35913 also encodes a predicted IT-sialidase and harbors a Nan cluster dedicated to sialic acid utilization. We showed that the Nan cluster was upregulated when the strains were grown in presence of mucin. In addition we demonstrated that both R. gnavus strains were able to grow on 2,7-anyhydro-Neu5Ac, the IT-sialidase transglycosylation product, as a sole carbon source. Taken together these data further support the hypothesis that IT-sialidase expressing gut microbes, provide commensal bacteria such as R. gnavus with a nutritional competitive advantage, by accessing and transforming a source of nutrient to their own benefit

Formate cross-feeding and cooperative metabolic interactions revealed by transcriptomics in co-cultures of acetogenic and amylolytic human colonic bacteria

© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.Peer reviewedPublisher PD

Fucosidases from the human gut symbiont Ruminococcus gnavus

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers

Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation

The gastrointestinal mucus layer is colonized by a dense community of microbes catabolizing dietary and host carbohydrates during their expansion in the gut. Alterations in mucosal carbohydrate availability impact on the composition of microbial species. Ruminococcus gnavus is a commensal anaerobe present in the gastrointestinal tract of > 90% of humans and overrepresented in inflammatory bowel diseases (IBD). Using a combination of genomics, enzymology and crystallography, we show that the mucin-degrader R. gnavus ATCC 29149 strain produces an intramolecular trans-sialidase (IT-sialidase) that cleaves off terminal alpha 2-3-linked sialic acid from glycoproteins, releasing 2,7-anhydro-Neu5Ac instead of sialic acid. Evidence of IT-sialidases in human metagenomes indicates that this enzyme occurs in healthy subjects but is more prevalent in IBD metagenomes. Our results uncover a previously unrecognized enzymatic activity in the gut microbiota, which may contribute to the adaptation of intestinal bacteria to the mucosal environment in health and disease.Peer reviewe

The human gut symbiont <i>Ruminococcus gnavus</i> shows specificity to blood group A antigen during mucin glycan foraging:Implication for niche colonisation in the gastrointestinal tract

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection

The human gut symbiont <i>Ruminococcus gnavus</i> shows specificity to blood group A antigen during mucin glycan foraging:Implication for niche colonisation in the gastrointestinal tract

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection

The role of the mucin-glycan foraging Ruminococcus gnavus in the communication between the gut and the brain

Ruminococcus gnavus is a prevalent member of the human gut microbiota, which is over-represented in inflammatory bowel disease and neurological disorders. We previously showed that the ability of R. gnavus to forage on mucins is strain-dependent and associated with sialic acid metabolism. Here, we showed that mice monocolonized with R. gnavus ATCC 29149 (Rg-mice) display changes in major sialic acid derivatives in their cecum content, blood, and brain, which is accompanied by a significant decrease in the percentage of sialylated residues in intestinal mucins relative to germ-free (GF) mice. Changes in metabolites associated with brain function such as tryptamine, indolacetate, and trimethylamine N-oxide were also detected in the cecal content of Rg-mice when compared to GF mice. Next, we investigated the effect of R. gnavus monocolonization on hippocampus cell proliferation and behavior. We observed a significant decrease of PSA-NCAM immunoreactive granule cells in the dentate gyrus (DG) of Rg-mice as compared to GF mice and recruitment of phagocytic microglia in the vicinity. Behavioral assessments suggested an improvement of the spatial working memory in Rg-mice but no change in other cognitive functions. These results were also supported by a significant upregulation of genes involved in proliferation and neuroplasticity. Collectively, these data provide first insights into how R. gnavus metabolites may influence brain regulation and function through modulation of granule cell development and synaptic plasticity in the adult hippocampus. This work has implications for further understanding the mechanisms underpinning the role of R. gnavus in neurological disorders

The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract

AU The:human Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly gut symbiont Ruminococcus gnavus displays strain-specific : repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection

Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent

Commensal bacteria often have an especially rich source of glycan-degrading enzymes which allow them to utilize undigested carbohydrates from the food or the host. The species Ruminococcus gnavus is present in the digestive tract of ≥90% of humans and has been implicated in gut-related diseases such as inflammatory bowel diseases (IBD). Here we analysed the ability of two R. gnavus human strains, E1 and ATCC 29149, to utilize host glycans. We showed that although both strains could assimilate mucin monosaccharides, only R. gnavus ATCC 29149 was able to grow on mucin as a sole carbon source. Comparative genomic analysis of the two R. gnavus strains highlighted potential clusters and glycoside hydrolases (GHs) responsible for the breakdown and utilization of mucin-derived glycans. Transcriptomic and functional activity assays confirmed the importance of specific GH33 sialidase, and GH29 and GH95 fucosidases in the mucin utilisation pathway. Notably, we uncovered a novel pathway by which R. gnavus ATCC 29149 utilises sialic acid from sialylated substrates. Our results also demonstrated the ability of R. gnavus ATCC 29149 to produce propanol and propionate as the end products of metabolism when grown on mucin and fucosylated glycans. These new findings provide molecular insights into the strain-specificity of R. gnavus adaptation to the gut environment advancing our understanding of the role of gut commensals in health and disease