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Objective: To decipher the mechanisms by which the major human milk oligosaccharide (HMO), 2'-fucosyllactose (2'FL), can affect body weight and fat mass gain on high-fat diet (HFD) feeding in mice. We wanted to elucidate whether 2'FL metabolic effects are linked with changes in intestinal mucus production and secretion, mucin glycosylation and degradation, as well as with the modulation of the gut microbiota, faecal proteome and endocannabinoid (eCB) system., Results: 2'FL supplementation reduced HFD-induced obesity and glucose intolerance. These effects were accompanied by several changes in the intestinal mucus layer, including mucus production and composition, and gene expression of secreted and transmembrane mucins, glycosyltransferases and genes involved in mucus secretion. In addition, 2'FL increased bacterial glycosyl hydrolases involved in mucin glycan degradation. These changes were linked to a significant increase and predominance of bacterial genera Akkermansia and Bacteroides , different faecal proteome profile (with an upregulation of proteins involved in carbon, amino acids and fat metabolism and a downregulation of proteins involved in protein digestion and absorption) and, finally, to changes in the eCB system. We also investigated faecal proteomes from lean and obese humans and found similar changes observed comparing lean and obese mice., Conclusion: Our results show that the HMO 2'FL influences host metabolism by modulating the mucus layer, gut microbiota and eCB system and propose the mucus layer as a new potential target for the prevention of obesity and related disorders., Competing Interests: Competing interests: PDC is an editor of the journal. PDC is inventor on patent applications dealing with the use bacteria on metabolic disorders. PDC was cofounders of The Akkermansia company SA and Enterosys., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

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., (© 2023. Springer Nature Limited.)

The human gastrointestinal (GI) tract harbors diverse microbial communities collectively known as the gut microbiota that exert a profound impact on human health and disease. The repartition and availability of sialic acid derivatives in the gut have a significant impact on the modulation of gut microbes and host susceptibility to infection and inflammation. Although N-acetylneuraminic acid (Neu5Ac) is the main form of sialic acids in humans, the sialic acid family regroups more than 50 structurally and chemically distinct modified derivatives. In the GI tract, sialic acids are found in the terminal location of mucin glycan chains constituting the mucus layer and also come from human milk oligosaccharides in the infant gut or from meat-based foods in adults. The repartition of sialic acid in the GI tract influences the gut microbiota composition and pathogen colonization. In this review, we provide an update on the mechanisms underpinning sialic acid utilization by gut microbes, focusing on sialidases, transporters, and metabolic enzymes., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced β-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial ( p -nitrophenyl β-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quantitative proteomics identified other enzymes and transporters involved in B. producta XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in L. reuteri and B. producta strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota.

Levan, a β-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography-mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 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., Competing Interests: The authors have declared that no competing interests exist.

Polysaccharides such as β-2,1-linked fructans including inulin or fructose oligosaccharides are well-known prebiotics with recognised immunomodulatory properties. In recent years, other fructan types covering β-2,6-linked fructans, particularly microbial levans, have gained increasing interest in the field. β-2,6-linked fructans of different degrees of polymerisation can be synthesised by plants or microbes including those that reside in the gastrointestinal tract. Accumulating evidence suggests a role for these β-2,6 fructans in modulating immune function. Here, we provide an overview of the sources and structures of β-2,6 fructans from plants and microbes and describe their ability to modulate immune function in vitro and in vivo along with the suggested mechanisms underpinning their immunomodulatory properties. Further, we discuss the limitations and perspectives pertinent to current studies and the potential applications of β-2,6 fructans including in gut health.

The human gut symbiont Ruminococcus gnavus scavenges host-derived N -acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase ( Rg NanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro- N -acetylneuraminic acid intermediate and NAD + regeneration. The crystal structure of Rg NanOx in complex with the NAD + cofactor showed a protein dimer with a Rossman fold. Guided by the Rg NanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of Rg NanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli ., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Bell et al.)

Serine-rich repeat proteins (SRRPs) have emerged as an important group of cell surface adhesins found in a growing number of Gram-positive bacteria. Studies focused on SRRPs from streptococci and staphylococci demonstrated that these proteins are O -glycosylated on serine or threonine residues and exported via an accessory secretion (aSec) system. In pathogens, these adhesins contribute to disease pathogenesis and represent therapeutic targets. Recently, the non-canonical aSec system has been identified in the genomes of gut microbes and characterization of their associated SRRPs is beginning to unfold, showing their role in mediating attachment and biofilm formation. Here we provide an update of the occurrence, structure, and function of SRRPs across bacteria, with emphasis on the molecular and biochemical properties of SRRPs from gut symbionts, particularly Lactobacilli. These emerging studies underscore the range of ligands recognized by these adhesins and the importance of SRRP glycosylation in the interaction of gut microbes with the host.

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.

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcβ(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein-carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly- N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule-grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.-Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Lactobacillus reuteri , a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP 100-23 ) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP 100-23 and SRRP 53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP 53608 -BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP 53608 -BR with PGA. Long molecular dynamics simulations showed that SRRP 53608 -BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Protein glycosylation is emerging as an important feature in bacteria. Protein glycosylation systems have been reported and studied in many pathogenic bacteria, revealing an important diversity of glycan structures and pathways within and between bacterial species. These systems play key roles in virulence and pathogenicity. More recently, a large number of bacterial proteins have been found to be glycosylated in gut commensal bacteria. We present an overview of bacterial protein glycosylation systems ( O - and N -glycosylation) in bacteria, with a focus on glycoproteins from gut commensal bacteria, particularly Lactobacilli. These emerging studies underscore the importance of bacterial protein glycosylation in the interaction of the gut microbiota with the host., Competing Interests: The authors declare no conflict of interest.

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)