RESUMEN
Alterations in intestinal mucin glycosylation have been associated with increased intestinal permeability and sensitivity to inflammation and infection. Here, we used mice lacking core 3-derived O-glycans (C3GnT-/-) to investigate the effect of impaired mucin glycosylation in the gut-brain axis. C3GnT-/- mice showed altered microbial metabolites in the caecum associated with brain function such as dimethylglycine and N-acetyl-L-tyrosine profiles as compared to C3GnT+/+ littermates. In the brain, polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive granule cells showed an aberrant phenotype in the dentate gyrus of C3GnT-/- mice. This was accompanied by a trend towards decreased expression levels of PSA as well as ZO-1 and occludin as compared to C3GnT+/+. Behavioural studies showed a decrease in the recognition memory of C3GnT-/- mice as compared to C3GnT+/+ mice. Combined, these results support the role of mucin O-glycosylation in the gut in potentially influencing brain function which may be facilitated by the passage of microbial metabolites through an impaired gut barrier.
Asunto(s)
Microbioma Gastrointestinal , Mucinas , Animales , Ratones , Eje Cerebro-Intestino , Glicosilación , Encéfalo , PolisacáridosRESUMEN
Ruminococcus gnavus was first identified in 1974 as a strict anaerobe in the gut of healthy individuals, and for several decades, its study has been limited to specific enzymes or bacteriocins. With the advent of metagenomics, R. gnavus has been associated both positively and negatively with an increasing number of intestinal and extraintestinal diseases from inflammatory bowel diseases to neurological disorders. This prompted renewed interest in understanding the adaptation mechanisms of R. gnavus to the gut, and the molecular mediators affecting its association with health and disease. From ca. 250 publications citing R. gnavus since 1990, 94% were published in the last 10 years. In this review, we describe the biological characterization of R. gnavus, its occurrence in the infant and adult gut microbiota and the factors influencing its colonization of the gastrointestinal tract; we also discuss the current state of our knowledge on its role in host health and disease. We highlight gaps in knowledge and discuss the hypothesis that differential health outcomes associated with R. gnavus in the gut are strain and niche specific.
Asunto(s)
Microbioma Gastrointestinal , Ruminococcus , Adulto , Humanos , Tracto Gastrointestinal , Ruminococcus/fisiologíaRESUMEN
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.
Asunto(s)
Encéfalo , Clostridiales , Microbioma Gastrointestinal , Mucinas , Animales , Encéfalo/metabolismo , Ratones , Mucinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismoRESUMEN
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-ß-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.
Asunto(s)
Clostridiales/metabolismo , Mucina-1/metabolismo , Sistema del Grupo Sanguíneo ABO/inmunología , Antígenos de Grupos Sanguíneos/inmunología , Clostridiales/genética , Clostridiales/fisiología , Microbioma Gastrointestinal , Tracto Gastrointestinal , Glicósido Hidrolasas/metabolismo , Humanos , Mucinas/metabolismo , Oligosacáridos/metabolismo , Polisacáridos/metabolismo , Ruminococcus/genética , Ruminococcus/metabolismo , Especificidad por Sustrato , Espectrometría de Masas en Tándem/métodosRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridiales/metabolismo , Microbioma Gastrointestinal , alfa-L-Fucosidasa/metabolismo , Proteínas Bacterianas/química , Clostridiales/química , Clostridiales/enzimología , Tracto Gastrointestinal/microbiología , Glicoconjugados/metabolismo , Humanos , Oligosacáridos/metabolismo , Polisacáridos/metabolismo , Especificidad por Sustrato , alfa-L-Fucosidasa/químicaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glicoproteínas/metabolismo , Mucinas/metabolismo , Neuraminidasa/metabolismo , Ruminococcus/metabolismo , Proteínas Bacterianas/genética , Genoma Bacteriano , Glicoproteínas/genética , Neuraminidasa/genética , Ruminococcus/enzimología , Ruminococcus/genética , Ruminococcus/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Adaptación Fisiológica/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Glicoproteínas/metabolismo , Mucosa Intestinal/microbiología , Neuraminidasa/metabolismo , Ruminococcus/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Glicoproteínas/genética , Humanos , Mucinas/metabolismo , Neuraminidasa/genética , Ruminococcus/genética , Ruminococcus/metabolismoRESUMEN
The availability of host and dietary carbohydrates in the gastrointestinal (GI) tract plays a key role in shaping the structure-function of the microbiota. In particular, some gut bacteria have the ability to forage on glycans provided by the mucus layer covering the GI tract. The O-glycan structures present in mucin are diverse and complex, consisting predominantly of core 1-4 mucin-type O-glycans containing α- and ß- linked N-acetyl-galactosamine, galactose and N-acetyl-glucosamine. These core structures are further elongated and frequently modified by fucose and sialic acid sugar residues via α1,2/3/4 and α2,3/6 linkages, respectively. The ability to metabolize these mucin O-linked oligosaccharides is likely to be a key factor in determining which bacterial species colonize the mucosal surface. Due to their proximity to the immune system, mucin-degrading bacteria are in a prime location to influence the host response. However, despite the growing number of bacterial genome sequences available from mucin degraders, our knowledge on the structural requirements for mucin degradation by gut bacteria remains fragmented. This is largely due to the limited number of functionally characterized enzymes and the lack of studies correlating the specificity of these enzymes with the ability of the strain to degrade and utilize mucin and mucin glycans. This review focuses on recent findings unraveling the molecular strategies used by mucin-degrading bacteria to utilize host glycans, adapt to the mucosal environment, and influence human health.
RESUMEN
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.
Asunto(s)
Tracto Gastrointestinal/microbiología , Mucinas/metabolismo , Polisacáridos/metabolismo , Ruminococcus/metabolismo , Simbiosis , Secuencia de Bases , Metabolismo de los Hidratos de Carbono , Tracto Gastrointestinal/metabolismo , Orden Génico , Sitios Genéticos , Genoma Bacteriano , Humanos , Metaboloma , Datos de Secuencia Molecular , Familia de Multigenes , Resonancia Magnética Nuclear Biomolecular , Ruminococcus/genética , Ruminococcus/crecimiento & desarrollo , TranscriptomaRESUMEN
Ruminococcin C (RumC) is a trypsin-dependent bacteriocin produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from human feces. It consists of at least three similar peptides active against Clostridium perfringens. In this article, a 15-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumC was characterized. It harbored 17 open reading frames (called rum(c) genes) with predicted functions in bacteriocin biosynthesis and post-translational modification, signal transduction regulation, and immunity. An unusual feature of the locus is the presence of five genes encoding highly homologous, but nonidentical RumC precursors. The transcription levels of the rum(c) genes were quantified. The rumC genes were found to be highly expressed in vivo, when R. gnavus E1 colonized the digestive tract of mono-contaminated rats, whereas the amount of corresponding transcripts was below detection level when it grew in liquid culture medium. Moreover, the rumC-like genes were disseminated among 10 strains (R. gnavus or related species) previously isolated from human fecal samples and selected for their capability to produce a trypsin-dependant anti-C. perfringens compound. All harbored at least a rumC1-like copy, four exhibited rumC1-5 genes identical to those of strain E1.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriocinas/genética , Clostridium perfringens/efectos de los fármacos , Ruminococcus/genética , Animales , Proteínas Bacterianas/metabolismo , Bacteriocinas/metabolismo , Bacteriocinas/toxicidad , Secuencia de Bases , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Heces/microbiología , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Procesamiento Proteico-Postraduccional , Ratas , Ruminococcus/crecimiento & desarrollo , Ruminococcus/metabolismo , Tripsina/genética , Tripsina/metabolismoRESUMEN
The digestion of polysaccharides from the wheat cultivars Caphorn and Isengrain was investigated, and the efficiency of an enzyme preparation was tested using the TNO gastrointestinal model (TIM-1). The apparent digestibility (AD) of carbohydrates was determined based on the measurement of organic matter (OM), total monosaccharides, reducing ends (RE) and end products (EP: glucose, maltose and xylobiose). The AD of the OM from Caphorn and Isengrain measured using caecectomised cockerels did not differ from that measured using TIM-1: 72.0 (SD 2.6) v. 70.6 (SD 0.6) % for Caphorn (P = 0.580) and 73.0 (SD 2.3) v. 71.1 (SD 1.9) % for Isengrain (P = 0.252). After the 6 h TIM-1 digestion, 41.4-58.9 % of the OM, RE and EP were recovered from the jejunal compartment and 18.3-27.1 % from the ileal compartment, while ileal deliveries and digestive residues constituted the remainder. A commercial enzyme cocktail tested at 0.2 µl/g of wheat improved TIM-1 digestibility of Caphorn and Isengrain polysaccharides: 3.9 % (P = 0.0203) and 3.4 % (P = 0.0058) based on the OM; 9.7 % (P < 0.0001) and 3.1 % (P = 0.031) based on the total glucose; 47.2 % (P < 0.0001) and 14.2 % (P = 0.0004) based on the RE, respectively. The enzyme cocktail improved the release of the EP for Caphorn (3.8 %, P = 0.008) but not for Isengrain ( − 0.8 %, P = 0.561). The higher efficiency of the enzyme supplementation on the digestion of Caphorn polysaccharides compared with Isengrain seems to be linked to the higher soluble carbohydrate contents and/or less ramified arabinoxylan of Caphorn.