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Michel Fons - One of the best experts on this subject based on the ideXlab platform.
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Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli
Frontiers in Microbiology, 2018Co-Authors: Elvis Legala Ongey, Lorenz Adrian, Michel Fons, Robert Giessmann, Juri Rappsilber, Peter NeubauerAbstract:Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA∗). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA∗ was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA∗ per liter of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA∗, this amount would generate a final yield of approximately 1–2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogs with superior anti-infective properties.
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Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli.
Frontiers in microbiology, 2018Co-Authors: Elvis Legala Ongey, Lorenz Adrian, Michel Fons, Robert Giessmann, Juri Rappsilber, Peter NeubauerAbstract:Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalysed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA*). These modifications were achieved when the peptide was expressed as a fusion protein together with GFP, demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA* was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA* per litre of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA*, this amount would generate a final yield of approximately 1-2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several thousand-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogues with superior anti-infective properties.
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Data_Sheet_1_Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli.PDF
2018Co-Authors: Elvis Legala Ongey, Lorenz Adrian, Michel Fons, Robert Giessmann, Juri Rappsilber, Peter NeubauerAbstract:Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA∗). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA∗ was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA∗ per liter of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA∗, this amount would generate a final yield of approximately 1–2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogs with superior anti-infective properties.
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Ruminococcus gnavus e1 modulates mucin expression and intestinal glycosylation
Journal of Applied Microbiology, 2016Co-Authors: F. Graziani, Michel Fons, A. Pujol, C. Nicoletti, S. Dou, M. Maresca, T. Giardina, J. PerrierAbstract:Aims The molecular cross-talk between commensal bacteria and the gut play an important role in the maintenance of the intestinal homeostasis and general health. Here, we studied the impact of a major Gram-positive anaerobic bacterium of the human gut microbiota, that is, Ruminococcus gnavus on the glycosylation pattern and the production of intestinal mucus by the goblet cells. Methods and Results Our results showed that R. gnavus E1 specifically increases the expression and the glycosylation level of the intestinal glyco-conjugates by goblet cells in the colonic mucosa of mono-associated mice with R. gnavus E1 as well as in human HT29-MTX cells. Such an effect was mediated through induction of the level of mRNA encoding for the major intestinal gel-forming mucin such as MUC2 and various glycosyltransferase enzymes. Conclusions This study demonstrates for the first time that R. gnavus E1 possess the ability to modulate the glycosylation profile of the glyco-conjugate molecules and mucus in goblet cells. Significance and Impact of the Study Furthermore, we demonstrated that R. gnavus E1 modified specifically the glycosylation pattern and MUC2 expression by means of a small soluble factor of peptidic nature (<3 kDa) and heat stable in the HT29-MTX cell.
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ETUDE D’UNE ADHESINE IDENTIFIEE CHEZ Ruminococcus gnavus E1
European Scientific Journal ESJ, 2013Co-Authors: Radia Alatou, Gwenola Simon, Michel FonsAbstract:Ruminococcus gnavus E1 is a Gram positive strict anaerobic bacterium that was isolated from the dominant faecal microbiota of a healthy adult. A 6kb-long open reading fragment called radA was identified on the E1 chromosome, next to the genetic clusters involved in the biosynthesis of the RumA and RumC bacteriocins which are active against pathogenic Clostridium perfringens. radA shares a high sequence homology with genes of Staphylococcus aureus, Bacillus cereus and C. perfingens encoding adhesins of the MSCRAMMs family. RT-PCR experiments demonstrated that the radA gene was strongly expressed in vivo, when the E1 strain colonized the digestive tract of monoxenics animals, while little transcription occured in vitro. These results suggest that RadA could play an important role in the colonization of the digestive ecosystem.
C. David Owen - One of the best experts on this subject based on the ideXlab platform.
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Fucosidases from the human gut symbiont Ruminococcus gnavus
Cellular and Molecular Life Sciences, 2020Co-Authors: Haiyang Wu, Osmond Rebello, Emmanuelle H. Crost, C. David Owen, Samuel Walpole, Chloe Bennati-granier, Didier Ndeh, Serena Monaco, Thomas Hicks, Anna ColvileAbstract: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.
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Fucosidases from the human gut symbiont Ruminococcus gnavus
Cellular and Molecular Life Sciences, 2020Co-Authors: Osmond Rebello, Emmanuelle H. Crost, C. David Owen, Samuel Walpole, Chloe Bennati-granier, Didier Ndeh, Serena Monaco, Thomas Hicks, Anna Colvile, Paulina A. UrbanowiczAbstract: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.
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Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut
Nature Microbiology, 2019Co-Authors: Andrew Bell, C. David Owen, Laura Vaux, Jason Brunt, Emmanuelle Crost, Ridvan Nepravishta, Dimitrios Latousakis, An Xiao, Xi Chen, Martin A. WalshAbstract:Sialic acid ( N -acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus . R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans -sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan- cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that—in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains—the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease. Ruminococcus gnavus is a mucus-associated gut commensal that can release the sialic acid, 2,7-anhydro-Neu5Ac. Here, the authors identify the pathway for its transportation and metabolism in R. gnavus , and show that this pathway is essential for its spatial localization in vivo.
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Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus.
Nature communications, 2017Co-Authors: C. David Owen, Serena Monaco, Louise E. Tailford, Tanja Šuligoj, Laura Vaux, Romane Lallement, Zahra Khedri, Karine Lecointe, John WalshawAbstract:Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.
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Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus
Nature Communications, 2017Co-Authors: C. David Owen, Serena Monaco, Louise E. Tailford, Tanja Šuligoj, Laura Vaux, Romane Lallement, Zahra Khedri, Karine Lecointe, John Walshaw, Sandra TriboloAbstract:The mucus layer is an important physical niche within the gut which harbours a distinct microbial community. Here the authors show that specific carbohydrate-binding modules associated with bacterial carbohydrate-active enzymes are mucus adhesins that target regions of the distal colon rich in sialomucins. Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans -sialidase ( Rg NanH). Rg NanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of Rg NanH_CBM40 ( Rg CBM40). Rg CBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. Rg CBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that Rg CBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.
Louise E. Tailford - One of the best experts on this subject based on the ideXlab platform.
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Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus.
Nature communications, 2017Co-Authors: C. David Owen, Serena Monaco, Louise E. Tailford, Tanja Šuligoj, Laura Vaux, Romane Lallement, Zahra Khedri, Karine Lecointe, John WalshawAbstract:Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.
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Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus
Nature Communications, 2017Co-Authors: C. David Owen, Serena Monaco, Louise E. Tailford, Tanja Šuligoj, Laura Vaux, Romane Lallement, Zahra Khedri, Karine Lecointe, John Walshaw, Sandra TriboloAbstract:The mucus layer is an important physical niche within the gut which harbours a distinct microbial community. Here the authors show that specific carbohydrate-binding modules associated with bacterial carbohydrate-active enzymes are mucus adhesins that target regions of the distal colon rich in sialomucins. Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans -sialidase ( Rg NanH). Rg NanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of Rg NanH_CBM40 ( Rg CBM40). Rg CBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. Rg CBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that Rg CBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.
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The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular trans-sialidases.
Gut microbes, 2016Co-Authors: Emmanuelle H. Crost, Louise E. Tailford, Marie Monestier, David Swarbreck, Bernard Henrissat, Lisa C. Crossman, Nathalie JugeAbstract: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.
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Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation.
Nature communications, 2015Co-Authors: Louise E. Tailford, Emmanuelle H. Crost, C. David Owen, John Walshaw, Gwénaëlle Le Gall, Jemma Hardy-goddard, Willem M. De Vos, Garry L. Taylor, Nathalie JugeAbstract: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 α2-3-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.
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Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent
PloS one, 2013Co-Authors: Emmanuelle H. Crost, Michel Fons, Louise E. Tailford, Bernard Henrissat, Gwénaëlle Le Gall, Nathalie JugeAbstract: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.
Nathalie Juge - One of the best experts on this subject based on the ideXlab platform.
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Mechanistic Insights Into the Cross-Feeding of Ruminococcus gnavus and Ruminococcus bromii on Host and Dietary Carbohydrates.
Frontiers in microbiology, 2018Co-Authors: Emmanuelle H. Crost, Gwénaëlle Le Gall, Jenny A. Laverde-gomez, Indrani Mukhopadhya, Harry J. Flint, Nathalie JugeAbstract:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.
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Presentation_1_Mechanistic Insights Into the Cross-Feeding of Ruminococcus gnavus and Ruminococcus bromii on Host and Dietary Carbohydrates.pdf
2018Co-Authors: Emmanuelle H. Crost, Jenny A. Laverde-gomez, Indrani Mukhopadhya, Harry J. Flint, Gwenaelle Le Gall, Nathalie JugeAbstract:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.
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The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular trans-sialidases.
Gut microbes, 2016Co-Authors: Emmanuelle H. Crost, Louise E. Tailford, Marie Monestier, David Swarbreck, Bernard Henrissat, Lisa C. Crossman, Nathalie JugeAbstract: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.
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Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation.
Nature communications, 2015Co-Authors: Louise E. Tailford, Emmanuelle H. Crost, C. David Owen, John Walshaw, Gwénaëlle Le Gall, Jemma Hardy-goddard, Willem M. De Vos, Garry L. Taylor, Nathalie JugeAbstract: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 α2-3-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.
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Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent
PloS one, 2013Co-Authors: Emmanuelle H. Crost, Michel Fons, Louise E. Tailford, Bernard Henrissat, Gwénaëlle Le Gall, Nathalie JugeAbstract: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.
Emmanuelle H. Crost - One of the best experts on this subject based on the ideXlab platform.
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Fucosidases from the human gut symbiont Ruminococcus gnavus
Cellular and Molecular Life Sciences, 2020Co-Authors: Haiyang Wu, Osmond Rebello, Emmanuelle H. Crost, C. David Owen, Samuel Walpole, Chloe Bennati-granier, Didier Ndeh, Serena Monaco, Thomas Hicks, Anna ColvileAbstract: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.
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Fucosidases from the human gut symbiont Ruminococcus gnavus
Cellular and Molecular Life Sciences, 2020Co-Authors: Osmond Rebello, Emmanuelle H. Crost, C. David Owen, Samuel Walpole, Chloe Bennati-granier, Didier Ndeh, Serena Monaco, Thomas Hicks, Anna Colvile, Paulina A. UrbanowiczAbstract: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.
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Mechanistic Insights Into the Cross-Feeding of Ruminococcus gnavus and Ruminococcus bromii on Host and Dietary Carbohydrates.
Frontiers in microbiology, 2018Co-Authors: Emmanuelle H. Crost, Gwénaëlle Le Gall, Jenny A. Laverde-gomez, Indrani Mukhopadhya, Harry J. Flint, Nathalie JugeAbstract:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.
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Presentation_1_Mechanistic Insights Into the Cross-Feeding of Ruminococcus gnavus and Ruminococcus bromii on Host and Dietary Carbohydrates.pdf
2018Co-Authors: Emmanuelle H. Crost, Jenny A. Laverde-gomez, Indrani Mukhopadhya, Harry J. Flint, Gwenaelle Le Gall, Nathalie JugeAbstract:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.
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The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular trans-sialidases.
Gut microbes, 2016Co-Authors: Emmanuelle H. Crost, Louise E. Tailford, Marie Monestier, David Swarbreck, Bernard Henrissat, Lisa C. Crossman, Nathalie JugeAbstract: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.
