Chemical Synthesis of a Complex-Type <i>N</i>‑Glycan Containing a Core Fucose

A chemical synthesis of a core fucose containing <i>N</i>-glycan was achieved. Asparagine was introduced at an early stage of the synthesis, and the sugar chain was convergently elongated. As for the fragment synthesis, we reinvestigated α-sialylation, β-mannosylation, and <i>N</i>-glycosylation to reveal that precise temperature control was essential for these glycosylations. Intermolecular hydrogen bonds involving acetamide groups were found to reduce the reactivity in glycosylations: the protection of NHAc as NAc₂ dramatically improved the reactivity. The dodecasaccharide–asparagine framework was constructed via the (4 + 4) glycosylation and the (4 + 8) glycosylation using the tetrasaccharide donor and the tetrasaccharide–asparagine acceptor. An ether-type solvent enhanced the yields of these key glycosylations between large substrates. After the whole deprotection of the dodecasaccharide, the target <i>N</i>-glycan was obtained

Identification of ORS285 mutants affected in the <i>O</i>-antigen synthesis.

<p>(<b>A</b>-<b>C)</b> Morphological aspect of the colonies of ORS285 (A), and two clones for which the Tn5 transposon is inserted in the CDS BRAO285v1_100023 (B) or BRAO285v1_100055 (C); scale bars, 5mm. (<b>D</b>) SDS-PAGE gel scan of the LPS of ORS285 (1), Tn5 mutant in BRAO285v1_100023 (2) and Tn5 mutant in BRAO285v1_100055 (3) mutants. The black box shows the canonical high molecular weight band of ORS285 LPS (with the <i>O</i>-antigen). All mutant strains displayed LPS lacking the complete <i>O</i>-antigen region with low molecular weight. (<b>E</b>) Genomic context of the two CDS, BRAO285v1_100023 and BRAO285v1_100055. The distance between the two CDS is about 44 kb. p., putative.</p

The <i>rfaL</i> and <i>gdh</i> mutants of ORS285 display a LPS lacking the <i>O</i>-antigen region.

<p>(<b>A</b>-<b>C</b>) GC-MS chromatogram of LPS from ORS285 (A), <i>rfal</i> mutant (B) and <i>gdh</i> mutant(C). (A) In the inset the compositional analysis is shown and at about 30 min retention time all the bradyrhizose peaks are also shown. In the section on the right the mass spectrum of ion peak 9 with fragmentation pattern of bradyrhizose is visible as well. Both mutant LPS do not display the typical ion peaks of bradyrhizose at retention time around 30 min. Pent, pentose; Man, mannose; Glc, glucose; Gal, galactose; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; C12:O, dodecanoic acid; C14:3OH, tetradecanoic acid; C16:O, hexadecanoic acid; C18:O, octadecanoic acid; C26:25OH, 25-hydroxy-hexacosanoid acid.</p

The lack of <i>O</i>-antigen has no impact on the free life of ORS285.

<p>(<b>A</b>-<b>C</b>) Colony morphotypes of ORS285 (A), <i>rfal</i> (B) and <i>gdh</i> (C) mutants; scale bars, 2mm. (<b>D</b>) SDS-PAGE gel scan of the LPS of ORS285 (1), <i>rfal</i> (2) and <i>gdh</i> (3) mutants. The black box shows the canonical high molecular weight band of ORS285 LPS (with the <i>O</i>-antigen) that is absent in the <i>rfal</i> and <i>gdh</i> mutants. (<b>E</b>) Growth of ORS285 (black), <i>rfal</i> (grey) and <i>gdh</i> (white) mutants in YM medium at 37°C. (<b>F</b>) Hydrogen peroxide (H₂O₂), hydrogen chloride (HCl) and sodium dodecyl sulfate (SDS) resistance of ORS285 (black bar), <i>rfal</i> (grey bar) and <i>gdh</i> (white bar) mutants, as determined by disk diffusion assays using 5 ml of 5.5M H₂O₂, 2N HCl or 10% of SDS. Error bars represent standard errors (n = 9); Tukey’s honestly significant difference test indicates no significant effect (<i>P</i> < 0.01). (<b>G</b>) Growth of ORS285 (black bar), <i>rfal</i> (grey bar) and <i>gdh</i> (white bar) mutants, in YM medium supplemented with various concentration of NaCl at 37°C. (<b>H</b>) Polymyxin B resistance of ORS285 (black bar), <i>rfal</i> (grey bar) and <i>gdh</i> (white bar) mutants, as determined by Etest (Etest®bioMérieux) on YM medium (performed and interpreted according to the manufacturer's procedures).</p

<i>O</i>-antigen minus mutants of ORS285 are not affected in their symbiotic properties with <i>Aeschynomene</i> legumes.

<p>(<b>A</b>, <b>B</b>) Comparison of the growth of <i>A</i>. <i>afraspera</i> (A) and <i>A</i>. <i>indica</i> (B) (aerial part), noninoculated (N.I.) or inoculated with ORS285, <i>rfal</i> or <i>gdh</i> mutants. (<b>C</b>, <b>D</b>) Quantification of acetylene reduction activity (ARA) and number of nodules per plant inoculated with ORS285 (black bars), <i>rfal</i> (grey bars) or <i>gdh</i> (white bars) mutants in <i>A</i>. <i>afraspera</i> (C) and <i>A</i>. <i>indica</i> (D). Error bars represent standard deviations (n = 10); Tukey’s honestly significant difference test indicates no significant effect(<i>P</i> < 0.01). (<b>E</b>-<b>J</b>) Whole roots of <i>A</i>. <i>afraspera</i> (E-G) and <i>A</i>. <i>indica</i> (H-J) inoculed with ORS285 (E, H), <i>rfaL</i> (F, I) or <i>gdh</i> (G, J) mutants; scale bars, 1 mm. (<b>K</b>-<b>P</b>) Nodule thin sections of <i>A</i>. <i>afraspera</i> (K-M) and <i>A</i>. <i>indica</i> (N-P), elicited by ORS285 (K, N), <i>rfaL</i> (L, O) or <i>gdh</i> (M, P) mutants and viewed by bright-field microscopy; scale bars, 400 μm. (<b>Q</b>-<b>V</b>) Confocal microscopy observations of nodules from <i>A</i>. <i>afraspera</i> (Q-S) and <i>A</i>. <i>indica</i> (T-V) elicited by ORS285 (Q, T), <i>rfal</i> (R, U) and <i>gdh</i> (S, V) mutants; scale bars, 20 μm.</p

DataSheet1_The chemistry of gut microbiome-derived lipopolysaccharides impacts on the occurrence of food allergy in the pediatric age.PDF

Introduction: Food allergy (FA) in children is a major health concern. A better definition of the pathogenesis of the disease could facilitate effective preventive and therapeutic measures. Gut microbiome alterations could modulate the occurrence of FA, although the mechanisms involved in this phenomenon are poorly characterized. Gut bacteria release signaling byproducts from their cell wall, such as lipopolysaccharides (LPSs), which can act locally and systemically, modulating the immune system function.Methods: In the current study gut microbiome-derived LPS isolated from fecal samples of FA and healthy children was chemically characterized providing insights into the carbohydrate and lipid composition as well as into the LPS macromolecular nature. In addition, by means of a chemical/MALDI-TOF MS and MS/MS approach we elucidated the gut microbiome-derived lipid A mass spectral profile directly on fecal samples. Finally, we evaluated the pro-allergic and pro-tolerogenic potential of these fecal LPS and lipid A by harnessing peripheral blood mononuclear cells from healthy donors.Results: By analyzing fecal samples, we have identified different gut microbiome-derived LPS chemical features comparing FA children and healthy controls. We also have provided evidence on a different immunoregulatory action elicited by LPS on peripheral blood mononuclear cells collected from healthy donors suggesting that LPS from healthy individuals could be able to protect against the occurrence of FA, while LPS from children affected by FA could promote the allergic response.Discussion: Altogether these data highlight the relevance of gut microbiome-derived LPSs as potential biomarkers for FA and as a target of intervention to limit the disease burden.</p

Additional file 1 of Microbial-derived imidazole propionate links the heart failure-associated microbiome alterations to disease severity

Additional file 1: Additional file 1. The document describes the primary/secondary outcomes of the GutHeart clincal trial

Molecular Insights into O‑Linked Sialoglycans Recognition by the Siglec-Like SLBR‑N (SLBR_UB10712) of Streptococcus gordonii

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3′SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections

Molecular Insights into O‑Linked Sialoglycans Recognition by the Siglec-Like SLBR‑N (SLBR_UB10712) of Streptococcus gordonii

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3′SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections

Molecular Insights into O‑Linked Sialoglycans Recognition by the Siglec-Like SLBR‑N (SLBR_UB10712) of Streptococcus gordonii

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3′SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections