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.

The serological properties of the O-antigen polysaccharide region of the lipopolysaccharides are used to differentiate E. coli strains into serogroups. In this study, we report the structure elucidation of the O-specific chain of E. coli O179 using NMR data, the program CASPER and analysis of biosynthetic information available in the E. coli O-antigen Database (ECODAB). The presence of genes that encode enzymes involved in the biosynthesis of the GDP-Man and UDP-GlcA within the O-antigen gene cluster of the bacteria indicates that the corresponding residues could be present in the polysaccharide. Furthermore, the occurrence of four genes that encode for glycosyltransferases indicates that the polysaccharide is composed of pentasaccharide repeating units; a bioinformatics approach based on predictive glycosyltransferase functions present in ECODAB revealed that the β-d-Manp-(1→4)-β-d-Manp-(1→3)-d-GlcpNAc structural element could be present in the O-specific chain. NMR spectroscopy data obtained from homonuclear and heteronuclear 2D NMR spectra (¹H,¹H-TOCSY, ¹H,¹³C-HSQC, ¹H,¹³C-H2BC and ¹H,¹³C-HMBC) were analyzed using the CASPER program, revealing the following arrangement of monosaccharide residues as the most probable structure: →4)-α-d-GlcpA-(1→3)-[β-d-Glcp-(1→2)]β-d-Manp-(1→4)-β-d-Manp-(1→3)-β-d-GlcpNAc-(1→, which was further confirmed using 2D homonuclear ¹H,¹H-COSY and ¹H,¹H-NOESY spectra. The functions of the α-gluconosyltransferase and the β-glucosyltransferase were predicted using structural alignment of AlphaFold-predicted 3D structures. This O-antigen polysaccharide shares structural similarities with those of E. coli O6 and O188, S. boydii type 16, and the capsular polysaccharide of E. coli K43, explaining the serological cross-reactivities observed with strains belonging these O- and K-antigen groups.

Streptococcus mutans, the causative agent of human dental caries, expresses a cell wall attached Serotype c-specific Carbohydrate (SCC) that is critical for cell viability. SCC consists of a polyrhamnose backbone of →3)α-Rha(1 → 2)α-Rha(1→ repeats with glucose (Glc) side-chains and glycerol phosphate (GroP) decorations. This study reveals that SCC has one predominant and two more minor Glc modifications. The predominant Glc modification, α-Glc, attached to position 2 of 3-rhamnose, is installed by SccN and SccM glycosyltransferases and is the site of the GroP addition. The minor Glc modifications are β-Glc linked to position 4 of 3-rhamnose installed by SccP and SccQ glycosyltransferases, and α-Glc attached to position 4 of 2-rhamnose installed by SccN working in tandem with an unknown enzyme. Both the major and the minor β-Glc modifications control bacterial morphology, but only the GroP and major Glc modifications are critical for biofilm formation.

Carbohydrates, vital components of biological systems, are well-known for their structural diversity. Nuclear Magnetic Resonance (NMR) spectroscopy plays a crucial role in understanding their intricate molecular arrangements and is essential in assessing and verifying the molecular structure of organic molecules. An important part of this process is to predict the NMR chemical shift from the molecular structure. This work introduces a novel approach that leverages E(3) equivariant graph neural networks to predict carbohydrate NMR spectral data. Notably, our model achieves a substantial reduction in mean absolute error, up to threefold, compared to traditional models that rely solely on two-dimensional molecular structure. Even with limited data, the model excels, highlighting its robustness and generalization capabilities. The model is dubbed GeqShift (geometric equivariant shift) and uses equivariant graph self-attention layers to learn about NMR chemical shifts, in particular since stereochemical arrangements in carbohydrate molecules are characteristics of their structures.

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles phi and psi have been investigated for O-methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in alpha-l-Fucp-(1 -> 3)-beta-d-Glcp-OMe, alpha-l-Fucp-(1 -> 3)-alpha-d-Galp-OMe and alpha-d-Glcp-(1 -> 4)-alpha-d-Galp-OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one-dimensional 1H,1H-NOESY NMR experiments to obtain transglycosidic proton-proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3JCH transglycosidic coupling constants related to torsion angles phi and psi. Computed effective proton-proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the alpha-(1 -> 3)-linked disaccharides and revealed that for the bimodal distribution at the psi torsion angle for the alpha-(1 -> 4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter-residue hydrogen bonding pattern for the alpha-(1 -> 4)-linked disaccharide, including a nonconventional hydrogen bond between H5 ' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1H NMR chemical shift displacement compared to alpha-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles phi and psi in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation. The importance of solvation on the preferred conformation of saccharides in solution and in crystals is unraveled by solution-state NMR and computational MD studies of solvated disaccharides. Crystal structures containing solvated glycan structures have glycosidic linkage conformations similar to those of the carbohydrate molecules in solution. 

Here we report an enzymatic approach to synthesize N-formylneuraminic acid (Neu5Fo) containing sialosides, through a five-enzyme cascade. This method stands as an alternative to traditional chemical syntheses, aiming for precision and efficiency in generating sialosides with a tailored N-formyl group generated directly from formic acid. The newly synthesized Neu5Fo was characterized using various NMR techniques revealing a conformational equilibrium at the amide bond of the formyl group in slow exchange on the NMR time scale with a trans : cis ratio of ∼2 : 1. This work not only suggests potential for exploring the biological roles of sialosides but also points to the possibility of developing novel therapeutic agents.

Glycans in the form of oligosaccharides, polysaccharides, and glycoconjugates are ubiquitous in nature, and their structures range from linear assemblies to highly branched and decorated constructs. Solution state NMR spectroscopy facilitates elucidation of preferred conformations and shapes of the saccharides, motions, and dynamic aspects related to processes over time as well as the study of transient interactions with proteins. Identification of intermolecular networks at the atomic level of detail in recognition events by carbohydrate-binding proteins known as lectins, unraveling interactions with antibodies, and revealing substrate scope and action of glycosyl transferases employed for synthesis of oligo- and polysaccharides may efficiently be analyzed by NMR spectroscopy. By utilizing NMR active nuclei present in glycans and derivatives thereof, including isotopically enriched compounds, highly detailed information can be obtained by the experiments. Subsequent analysis may be aided by quantum chemical calculations of NMR parameters, machine learning-based methodologies and artificial intelligence. Interpretation of the results from NMR experiments can be complemented by extensive molecular dynamics simulations to obtain three-dimensional dynamic models, thereby clarifying molecular recognition processes involving the glycans.

Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)-mediated inflammation, including upregulation of various transcription factors and the activation of pro-inflammatory pathways important for immune surveillance. Dysfunction of PRRs-mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs-driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure-based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, Toll-like Receptor 4 (TLR4). Taking advantage of an exo-anomeric conformation and specific molecular shape of synthetic nonreducing β,β-diglucosamine, which was investigated by NMR, we developed two sets of lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro-inflammatory signaling. Stereoselective 1,1′-glycosylation towards fully orthogonally protected nonreducing GlcNβ(1↔1′)βGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4-specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant- and immunotherapy candidates, while anionic glycolipids with TLR4-inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.

Methodology development in carbohydrate chemistry entails the stereoselective formation of C-O bonds as a key step in the synthesis of oligo- and polysaccharides. The anomeric selectivity of a glycosylation reaction is affected by a multitude of parameters, such as the nature of the donor and acceptor, activator/promotor system, temperature and solvent. The influence of different solvents on the stereoselective outcome of glycosylation reactions employing thioglucopyranosides as glycosyl donors with a non-participating protecting group at position 2 has been studied. A large change in selectivity as a function of solvent was observed and a correlation between selectivity and the Kamlet-Taft solvent parameter pi* was found. Furthermore, molecular modeling using density functional theory methodology was conducted to decipher the role of the solvent and possible reaction pathways were investigated.

Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often polymerized by the inner membrane bound Wzy-dependent pathway. By employing cryo-electron microscopy we show that WzzE, the co-polymerase component of this pathway that is responsible for the length modulation of the enterobacterial common antigen, is octameric with alternating up-down conformations of its L4 loops. The alternating up-down nature of these essential loops, located at the top of the periplasmic bell, are modulated by clashing helical faces between adjacent protomers that flank the L4 loops around the octameric periplasmic bell. This alternating arrangement and a highly negatively charged binding face create a dynamic environment in which the polysaccharide chain is extended, and suggest a ratchet-type mechanism for polysaccharide elongation. Cryo-EM structure of bacterial polysaccharide co-polymerase WzzE provides insight into possible mechanisms of lipopolysaccharide elongation and length regulation.