Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development, and human health. No effective vaccines are now available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis, and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan “core” and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain elicited antibodies that recognized antigenically diverse pathogenic S. suis strains and induced complement deposition on encapsulated pathogenic S. suis strains. This study provides valuable insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

In mammals, glucose transporters (GLUTs) mediate organism-wide sugar distribution, yet the molecular basis of substrate specificity remains unclear. The bacterial xylose transporter XylE serves as a model for GLUTs. However, although xylose and glucose bind with a similar affinity, xylose is transported, but glucose acts as an inhibitor. Here, using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, we distinguished transported sugars from sugar inhibitors. Our findings revealed that only transported sugars generate STD NMR signals, which are abolished for xylose when XylE is trapped in either outward- or inward-facing conformations. Engineering the sugar-binding pocket and gating helix TM7b enabled glucose transport by XylE and corresponding STD signals. Using complementary molecular dynamics simulations, together with structural, biochemical and STD NMR analysis of related parasitic and mammalian GLUTs, we identified TM7b as a key determinant of occluded state formation. We conclude that, rather than the initial substrate-binding event observed in experimental structures, formation of a substrate-induced transition-state intermediate is the primary determinant of specificity in transporters.

The bacterium Yersinia enterocolitica serotype O:3 is targeted by two distinct agents, the bacteriophage φR1-37 and the bacteriocin-like enterocoliticin (a tailocin), which both utilize the lipopolysaccharide (LPS) outer core (OC) hexasaccharide as their primary host receptor. In order to understand this convergent recognition mechanism, we first characterized the enterocoliticin system, reporting the complete sequence of its large, biosynthetic gene cluster. Most of the 42 predicted gene products were functionally annotated by homology to known gene products. We then focused on identifying the receptor-binding proteins (RBPs) responsible for host attachment of both agents in order to elucidate a possible shared mechanism of binding. For phage φR1-37, the receptor binding complex was identified as the inseparable Gp298 tail fiber protein and its Gp297 trimerization chaperone, confirming its function as the RBP. Based on sequence identity with Gp298, the Orf39 gene product of the enterocoliticin cluster was predicted to be its corresponding RBP. An analytical comparison of the predicted RBPs revealed a highly conserved homologous region spanning 80–85 amino acid residues, which presents the only structural explanation for their identical receptor specificity. To resolve the binding mechanism, we generated high-confidence trimeric structural models for the Gp298 and Orf39 proteins using AlphaFold3-multimer. These models validated the high structural similarity of the RBP domains, despite global dissimilarity of the complete trimeric structures. Further docking simulations with a pentasaccharide ligand (generated by CarbBuilder) provided suggestive molecular models for the protein-carbohydrate interactions within the OC region. Intriguingly, a database search using the identified binding site motif revealed their wide and diverse presence in various phage tail proteins, suggesting that this motif is a specialized, common structure for carbohydrate recognition. This work identifies a conserved, novel sugar-binding motif as the molecular basis of host recognition for these key anti-Yersinia biologics.

Methyl 2,3-di-O-benzyl-α-d-(4-²H)-glucopyranoside, C₂₁H₂₅DO₆, is an intermediate used in synthesis of oligosaccharides. The hexopyranose ring has the ⁴C₁ chair conformation in the crystal structure. The exocyclic groups of the hexose sugar show for the glycosidic torsion angle ϕ =−52.8° and for the hydroxymethyl group the gauche-gauche conformation with ω = −64.7°, one of the two main orientations of the latter group in hexopyranose sugars that have the gluco-configuration, i.e., with an equatorial hydroxyl group at C4. The benzene rings of the benzyl groups are arranged with an angle of 56.9° to each other within the molecule and show intramolecular as well as intermolecular C-H···π interactions. A chain of intermolecular hydrogen bonds exists along the b-axis involving O4 and O6 atoms. The experimentally observed peak in the infrared spectrum at 2159 cm^− 1 was ascribed to the stretching of the C4–D4 bond based on DFT calculations.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of glycosaminoglycans (GAG) that transfers the first galactose unit to xylose in the linker region. Searching for new inhibitors of the GAG biosynthesis, we used saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy to evaluate the binding interactions between β4GalT7 and several pentosides in the presence of UDP donors. These investigations verified the glycosylation specificity of β4GalT7 and revealed that the naphthalene and the uridine moieties were significant contributors to the binding of the acceptor and the donor, respectively, while the galactose part was less important. Based on these findings, we set out to investigate conjugates of UDP and naphthoxylosides to function as transition state analogues. These compounds were synthesized using a one-pot procedure and tested as inhibitors in a β4GalT7 assay. Interestingly, one truncated analogue, a bisphosphonate-xyloside construct, showed a significant inhibition (IC₅₀: 188 μM). These findings open for the design of a new class of inhibitors of the GAG biosynthesis.

Streptococcus uberis is a causative pathogen of bovine mastitis with high genetic diversity. Rhamnose-rich polysaccharides (RPS) are abundant surface structures covalently anchored to peptidoglycan and represent promising vaccine candidates for several streptococcal pathogens. It was previously reported that the RPS of S. uberis strain 233 is composed of a repeating → 2)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → disaccharide backbone decorated with α-D-Glcp side-chains. In this study, we identified a hitherto unknown glycerol phosphate (GroP) modification at the 6-OH of the Glc residue in S. uberis 233 RPS using nuclear magnetic resonance analysis. Comparative genomic analysis of 592 S. uberis genomes revealed significant diversity in the RPS biosynthesis gene cluster with six major RPS genotypes. RPS genotypes 1-4, representing 97.5% of the analyzed strains, all contained the rhamnan backbone biosynthesis genes shared between several streptococcal species, as well as a putative GroP transferase gene. Using rhamnan-reactive immune serum, we further demonstrated that rhamnan is a conserved and accessible glycan motif in S. uberis RPS genotype 1 and 2 strains, but this motif is inferred to be shielded by side-chains in genotype 4 strains. Importantly, experiments with sera from cattle, challenged intramammarily with S. uberis, revealed that the rhamnan backbone of S. uberis RPS is an immunogenic glycan motif and remained accessible to bovine IgG antibodies in the presence of single residue RPS side-chains. Overall, this study suggests that S. uberis RPS are modified with GroP and reports that RPS in most strains contain a conserved, immunogenic and antibody accessible rhamnan glycan motif.

Outer membrane (OM) proteins play a vital role in the physiology of Gram-negative bacteria, and outer membrane protein F (OmpF) is one of the most studied porins in Escherichia coli. In this study, we have developed a comprehensive E. coli OM model with lipopolysaccharides (LPS), enterobacterial common antigen (ECA), and capsular polysaccharides (CPS) in the outer leaflet and with phospholipids in the inner leaflet. Using extensive all-atom molecular dynamics simulations of OmpF in this realistic asymmetric OM environment, we have investigated the structure and dynamics of OmpF within the OM and its interactions with the OM. The results demonstrate that the presence of ECA and CPS enhances the rigidity and stability of the OM while reducing the pore size of OmpF and increasing its cation selectivity. The complex and diverse interactions between OmpF and LPS/ECA/CPS contribute to these effects, resulting in a rigid and compact OmpF structure. These findings provide new insights into the complex interplay between bacterial OM components and OmpF porin, with potential implications for understanding bacterial resistance and developing novel antimicrobial strategies.

In posttranslational modifications of proteins and peptides by glycosylation, the two major classes are N-linked and O-linked glycans. The sugar residue proximal to the peptide chain is in N-glycans linked to L-asparagine, and in O-linked glycans, it is linked to either L-serine, L-threonine, or L-tyrosine, although other amino acids may be glycosylated. Identifying and assigning the ¹H and ¹³C nuclear magnetic resonance (NMR) chemical shifts of these glycoconjugates are a prerequisite for structural characterization as well as for subsequent conformational and interaction studies thereof. The web-based computer program CASPER (http://www.casper.organ.su.se/casper) is a tool that provides prediction of ¹H and ¹³C NMR chemical shift for glycans, as well as those linked to L-Asn, L-Ser, L-Thr, or L-Tyr, for which the predicted NMR chemical shifts of the glycan show good agreement to those from NMR experiments of glycopeptides and glycoproteins. This highlights that an approximation in which a single amino acid is present at the reducing end of the glycan structure is sufficient to predict NMR data well, as shown for different N-linked and O-linked glycans of various complexity.

Group A Streptococcus (Strep A) is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydrate biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within Escherichia coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.

This study introduces a novel enzymatic cascade featuring five recombinant enzymes for the efficient synthesis of fucosylated glycosides, using sucrose exclusively as the sugar donor substrate. In our approach, we employed a sucrose synthase sourced from tomato to generate GDP-glucose from sucrose and GDP. By repurposing CDP-tyvelose 2-epimerase from Salmonella enterica, chosen for its catalytic efficiency from a panel of 27 CDP-tyvelose 2-epimerase candidates, it was possible to epimerize GDP-glucose into GDP-mannose. The subsequent transformation of GDP-d-mannose to GDP-l-fucose was achieved by GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose epimerase/reductase, also derived from S. enterica. In the final stage, Helicobacter pylori α1,3-fucosyltransferase was employed to fucosylate para-nitrophenyl β-lactoside, resulting in the production of para-nitrophenyl 3-fucosyllactoside with a conversion of more than 40%. Analysis of the synthesized compound by LC-MS and NMR analyses substantiated its structure. This investigation not only highlights the utility of this five-enzyme fucosylation cascade but also establishes a novel methodological paradigm for the biocatalytic production of α-l-fucosides for biochemical research and for biotechnological applications.