Abstract Xylanases are central to lignocellulosic biomass degradation, yet current methods lack the specificity to resolve how enzymes distinguish complex xylan structures decorated with arabinofuranose (Ara f ) and 4- O -methyl-glucuronic acid (MeGlcA). Here, we report a suite of chemically-defined activity-based probes (ABPs) that enable the selective detection of arabinoxylan- and glucuronoxylan-specific xylanases (AXXs and GXXs). These cyclophellitol-derived ABPs covalently label retaining xylanases at their active sites, allowing precise mapping of substrate specificity across diverse glycoside hydrolase families. Crystallographic and mass spectrometric analyses reveal the molecular basis of probe selectivity, while in-gel and pull-down assays demonstrate their effectiveness in profiling xylanase activities in complex bacterial and fungal proteomes, including cellulosomes. By integrating activity-based protein profiling (ABPP) with sequence similarity networks (SSNs), we further show that xylanase specificity can be predicted from sequence alone, enabling rapid functional annotation of uncharacterized xylanases. This chemoproteomic strategy provides a powerful platform for discovering and engineering substrate-specific enzymes for biomass valorisation, microbial ecology, and biotechnological applications.

Exopolysaccharides are key matrix determinants that provide structural integrity and regulate biomechanical properties of microbial biofilms. Biofilm exopolysaccharides often undergo modifications that determine their functional properties and localization. In Bacillus cereus ATCC 10987, PelADA expressed from the pelDEADAFG operon is a putative deacetylase required for Pel-dependent biofilm formation. To understand the molecular basis of Pel deacetylation in B. cereus ATCC 10987, we determined the crystal structure of PelADA to 2.51 Å. PelADA adopts a distinct three-domain arrangement. We demonstrate in vitro that PelADA deacetylates α-1,4-linked GalNAc substrates in a length-dependent manner and that the N-terminal domain functions as a carbohydrate-binding module (CBM) capable of binding both GalNAc and partially deacetylated oligosaccharides. We found that the CBM domain together with the carbohydrate esterase (CE) domain forms an elongated carbohydrate binding cleft and that each domain is the founding member of two new CAZy families, CBM114 and CE26, respectively. Further, in vivo mutagenesis demonstrated that the catalytic activity of PelADA is required for Pel biosynthesis in B. cereus ATCC 10987. Employing AlphaFold, we propose a model wherein the N-terminal transmembrane helix of PelADA interacts with PelG. This interaction positions the protein to accept the polymer for deacetylation as it emerges from the cytoplasmic membrane. The work presented herein offers insight into the role of PelADA in Pel biosynthesis and modification in B. cereus ATCC 10987.

Staphylococcus aureus and Pseudomonas aeruginosa are major antimicrobial-resistant pathogens that often synergize in polymicrobial infections, such as chronic wound infections. These notorious and increasingly resistant bacteria contribute significantly to reduced antibiotic efficacy. Despite their substantial clinical burden, the urgent need to combat bacterial resistance and extensive research efforts, no vaccines currently exist for either bacterium. Glycoconjugate vaccines, which extend the range of suitable vaccine antigens to bacterial carbohydrates, could play a major role in this emergence. This study introduces a multiepitope vaccine conjugating S. aureus capsular polysaccharide serotype 8 to a chimeric protein fusing Hla and PcrV, two potent cytotoxins from S. aureus and P. aeruginosa, respectively. A conjugation strategy based on selective targeting of a purposefully introduced histidine tag was developed to preserve the structure and antigenicity of epitopes from the two proteins, leveraging their dual role as a carrier and antigen. This multivalent, multimeric and multipathogen construct successfully elicited antibodies against all three antigens as well as functional protection. This proof-of-concept highlights the potential for advanced vaccines targeting polymicrobial infections and bacteria with complex pathogenesis calling for multivalent formulations. It also points out the power of site-selective conjugation as a tool for vaccine manufacturing.

Bacterial infections represent a substantial global health challenge, impacting both human and veterinary health. The ongoing evolution of antibiotic-resistant pathogens, coupled with limited new antibiotic discoveries, urges the need for alternative strategies to treat and prevent these infections. Passive immunization with monoclonal antibodies (mAbs) is gaining interest as a promising alternative. Here, we report an experimental pipeline for generating human mAbs from healthy donor B cells using synthetic mimics of complex bacterial glycans. We identified functional mAbs recognizing discrete and unique epitopes on the surface glycans of two bacterial priority pathogens; Staphylococcus aureus and Streptococcus pyogenes. The use of chemically-defined synthetic glycans was critical for the discovery and systematic characterization of mAbs. From a heterogeneous mix of B cell specificities, antibody sequences were identified, leading to the production of mAbs with distinct reactivities against immunodominant but also to less common or even masked epitopes. The pipeline can be adapted to different glycan targets, donor material or specific antibody isotypes. This work thereby paves the way for the discovery of glycan-specific mAbs with clinical relevance to treat, prevent or diagnose infections with S. aureus, S. pyogenes or other bacterial pathogens.

Retaining glycosidases employ a two-step double displacement mechanism to hydrolyze their substrate glycosides. This mechanism involves a covalent enzyme-substrate adduct, and irreversible retaining glycosidase inhibitors have been designed based on this mechanism. Tagging such inhibitors with a reported moiety (biotin, fluorophore, bioorthogonal tag) provides activity-based retaining glycosidase probes. This chapter describes research on such activity-based probes that are inspired by the natural product retaining β-glucosidase inhibitor, cyclophellitol. Modulation of the configuration and substitution pattern yielded a suite of probes with which a host of retaining glycosidases are inhibited, and reported on, including enzymes involved in human pathologies (cancer, inherited lysosomal storage disorders). This chapter provides insights into their design and synthesis, their application in disease diagnosis, and their application in drug discovery, both as tools to uncover competitive inhibitors and as starting point for the design of covalent inhibitors.

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 currently 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 induced antibodies recognizing a wide range of antigenically-diverse pathogenic S. suis strains. This study provides new insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis . ### Competing Interest Statement A.A.C.J. and R.G. are employed by MSD Animal Health where a vaccine based on another antigen is currently under development. The other authors declare no competing interests. Netherlands Center for One Health, LSHM19137 Health~Holland, LSHM19137 Swedish Research Council, https://ror.org/03zttf063, 2022-03014 Knut and Alice Wallenberg Foundation, https://ror.org/004hzzk67 China Scholarship Council, https://ror.org/04atp4p48, CSC201909110078 Marie Sk łodowska-Curie grant, 861194 United States Department of Energy, DE-SC0015662

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), causes significant financial losses in the agricultural industry. Additionally, M. bovis transmission from animals to humans can result in zoonotic TB, especially in low- and middle-income countries (LMICs), highlighting the need to enhance One Health surveillance to mitigate this threat.Antibodies directed against a major mycobacterial cell wall component of M. leprae, phenolic glycolipid-I (PGL-I), have shown excellent performance in identifying M. leprae infection in humans and animals. In this study, we therefore investigated whether antibodies against M. bovis PGL similarly represent a useful biomarker for M. bovis infection in cattle.Comparing sera from naturally M. bovis-infected and the single intradermal comparative cervical tuberculin test (SICCT)-negative cattle, we assessed the potential of M. bovis PGL antibodies to identify this mycobacterial infection. Our results show that serum levels of anti-M. bovis PGL IgG and -IgM in M. bovis-infected cattle were significantly higher than in the SICCT-negative cattle. The sensitivity for anti-M. bovis PGL IgM in infected animals was, however, moderate (44.9 %) and the false-positive rate was 6.3 % in SICCT-negative cattle. Notably, vaccination with BCG- or heat-killed M. bovis did not affect serum levels of anti-M. bovis PGL IgM in cattle. Moreover, none of the 57 anti-M. bovis PGL-seropositive cattle tested positive in the anti-M. leprae PGL-I assay. This study shows for the first time that anti M. bovis PGL antibodies can be detected in infected cattle: anti-M. bovis PGL IgM is a highly specific, but moderately sensitive biomarker for M. bovis infection in cattle, showing potential for differentiate infected from vaccinated animals (DIVA). It could be a valuable component in a multi-biomarker approach for diagnosing bTB.

Glycosylation, the formation of glycosidic bonds, is a central yet challenging step in the chemical synthesis of complex carbohydrates due to its intricate regio- and stereochemical control. This study explores explicitly solvated, semi-empirical molecular dynamics (MD) simulations combined with multiple walker well-tempered metadynamics to investigate the mechanistic landscape of glycosylation involving a constrained glucose donor and a series of simple alcohol nucleophiles varying in nucleophilicity: ethanol, 2-monofluoroethanol, 2,2-difluoroethanol, and 2,2,2-trifluoroethanol. Our simulations reveal several mechanistic pathways depending on the nucleophile and substitution site. Stronger nucleophiles favor concerted SN2 displacement, while weaker nucleophiles increasingly promote dissociative SN1-like mechanisms and frontside attack pathways. This study demonstrates how semi-empirical MD simulations, combined with explicit solvation, can provide insights to understand the glycosylation reaction pathways.

Tetrahydropyran acetals bearing a fluorine atom adjacent to the acetal carbon atom can undergo highly stereoselective substitution reactions with nucleophilic alkenes to give the 1,2-cis products. By contrast, the chlorine- and bromine-substituted acetals give the 1,2-trans products. These results can be understood by considering oxocarbenium ion intermediates and their conformational preferences, which are dictated by hyperconjugative effects from axial substituents, with F ≪ H < Cl < Br. Reactions of the corresponding five-membered-ring acetals are also 1,2-cis selective in the case of fluorine and 1,2-trans selective with chlorine- and bromine-substituted acetals, but selectivities showed different trends of reactivity vs selectivity. The reactions with the five-membered-ring acetal were interpreted as requiring anomeric halides as reactive intermediates because of the conditions required to obtain substitution products.

Wall teichoic acids (WTAs) from the major Gram-positive foodborne pathogen Listeria monocytogenes are peptidoglycan-associated glycopolymers decorated by monosaccharides that, while not essential for bacterial growth, are required for bacterial virulence and resistance to antimicrobials. Here we report the structure and function of a bacterial WTAs rhamnosyltransferase, RmlT, strictly required for L. monocytogenes WTAs rhamnosylation. In particular, we demonstrated that RmlT transfers rhamnose from dTDP-L-rhamnose to naked WTAs, and that specificity towards TDP-rhamnose is not determined by its binding affinity. Structures of RmlT with and without its substrates showed that this enzyme is a dimer, revealed the residues responsible for interaction with the substrates and that the catalytic residue pre-orients the acceptor substrate towards the nucleophilic attack to the sugar. Additionally, the structures provided indications for two potential interaction pathways for the long WTAs on the surface of RmlT. Finally, we confirmed that WTAs glycosyltransferases are promising targets for next-generation strategies against Gram-positive pathogens by showing that inactivation of the RmlT catalytic activity results in a decreased infection in vivo.

The preparation of well-defined ADP-ribosylated peptides is essential for studying the functional implications of this post-translational modification. While methodologies exist for the chemical synthesis of short oligo-ADPr fragments and mono-ADP-ribosylated peptides separately, combining the two distinct chemistries, required to assemble them, has remained challenging. In this research, we employ a methodology for the regio- and chemoselective condensation of two phosphomonoesters to convergently install the pyrophosphate bond in ADP-ribosylated constructs. A diverse set of phosphoribosylated peptides, varying in the amino acid acceptor, was prepared and condensed with adenosine monophosphate to yield mono-ADP-ribosylated peptides. Furthermore, a solid-phase approach was developed to access a phosphoadenosyl-ADPr fragment, which was used to prepare a di-ADPr-peptide as the first example of a synthetic oligo-ADP-ribosylated peptide. Overall, the work presented here extends synthetic methodology for the preparation of well-defined ADP-ribosylated peptides.

ABSTRACT The World Health Organization (WHO) 2030 roadmap for schistosomiasis calls for development of highly sensitive and specific diagnostic tools to continue and sustain progress towards elimination. Serological assays are excellent for sensitive detection of primary schistosome infections and for schistosomiasis surveillance in near- and post-elimination settings. To develop accurate assay formats, it is necessary to identify defined antibody targets with low cross-reactivity and potential for standardized production. Here we aim to identify such target(s) with focus on defined schistosome glycan antigens. Target identification was performed by assessing antibody responses in well-characterized cross-sectional and cohort sample sets ( n = 366 individuals) on tailor-made antigen microarrays. IgM and IgG binding to candidate diagnostic targets was measured for serum/plasma samples from controlled human schistosome infection models, schistosome-infected travelers, soil-transmitted helminth-infected individuals, and non-infected individuals. We found that antibodies to a schistosome gut-associated glycan, the circulating anodic antigen (CAA), identify schistosome infection with high sensitivity (IgM ≥100%, IgG ≥97%) and specificity (IgM ≥93%, IgG ≥97%) in the test samples. Infection dose affected timing of anti-CAA antibody isotype switch. Furthermore, we demonstrate that other non-specific glycan epitopes in crude schistosome cercarial and egg antigen preparations can contribute to generation of false schistosomiasis positives, which is relevant for current serological assays based on these antigen mixtures. In conclusion, CAA is an excellent single glycan antigen target for development of highly sensitive and specific tools for schistosomiasis serology with use cases for travelers and surveillance in near- and post-elimination settings, as well as emerging transmission zones. IMPORTANCE The WHO 2030 roadmap deems diagnostics developments for schistosomiasis critically needed. Here we present identification of an antibody target with superior performance compared to traditionally used crude antigens in schistosomiasis serology. Access to unique controlled human infection model samples, traveler samples, and negative controls enabled this discovery, which forms the basis for development of new diagnostic tools urgently needed in travel medicine, surveillance in emerging transmission zones driven by climate change, and in pre- and post-elimination scenarios.

Cough drives respiratory pathogen transmission, yet how microbes directly engage host sensory neurons to trigger cough is largely unknown. We previously demonstrated that the Mycobacterium tuberculosis (Mtb) glycolipid sulfolipid-1 (SL-1) activates neurons and induces cough. Here, we reveal that phenolic glycolipid (PGL) produced by the hypertransmissible HN878 Mtb strain activates both mouse and human nociceptive neurons in vitro using calcium imaging and electrophysiology and is sufficient to induce cough using plethysmography. Combined with SL-1, PGL potently triggers neuronal activation. By synthesizing various PGL analogs, we show that neuroactivity is proportional to saccharide chain length and structure. Mechanistically, PGL stimulates rapid extracellular ATP release, which engages neuronal P2X3 purinergic receptors-an effect blocked by a P2X3 antagonist. These findings uncover a neuronal activation pathway co-opted by certain Mtb strains to enhance transmission via cough and suggest inhibition of purinergic signaling as a potential strategy to block airborne spread of Mtb.

During infection, the human opportunistic pathogen Pseudomonas aeruginosa forms protective biofilms, whose matrix consists of proteins, nucleic acids, and polysaccharides such as alginate, Psl, and Pel. Psl, a polymeric pentasaccharide composed of mannose, rhamnose, and glucose, is produced during the early stages of biofilm formation, serving as a protective barrier against antibiotics and the immune system. The Psl biosynthesis gene cluster, besides encoding various glycosyltransferases, also includes an endoglycosidase, PslG. Here, we show, by activity-based protein profiling, structural studies on enzyme-inhibitor complexes, and defined substrate processing, that PslG is not, as previously suggested, an endo-β-mannosidase but instead a retaining endo-β-glucosidase. This insight allows the design of both competitive and covalent PslG inhibitors, as we show for repeating pentasaccharide mimetics featuring either a reducing end deoxynojirimycin or cyclophellitol moiety. This work provides valuable tools to deepen the understanding of Psl biosynthesis, its function in biofilm formation, and its contribution to antibiotic resistance. We demonstrate the enzyme's actual endo-β-glucosidase activity, a means to monitor PslG activity in P. aeruginosa biofilms, and a blueprint for inhibitor design.

Staphylococcus aureus is a Gram-positive bacterium that is responsible for severe nosocomial infections. The protective capsular polysaccharides (CPs), which are key elements of the cell wall, have been proposed as promising candidate antigens. Several CP types have been identified including CP1, CP5, and CP8, and serotype 1 has been associated with increased resistance to phagocytosis and virulence. Here, the synthesis of a set of S. aureus CP 1 (strain M and D) trisaccharides, composed of an α-N-acetyl d-fucosamine and two α-N-acetyl d-galactosaminuronic acid residues, carrying taurine esters together with a nontaurinated hexasaccharide, is reported. To be able to tune the taurine substitution pattern, an orthogonal C-6-OH protecting group strategy for the galactosamine building blocks was developed, in conjunction with a postglycosylation oxidation protocol to site selectively introduce the taurine amide substitutions. The stereoselectivity in the glycosylations was secured using a silylene-protected 2-azido galactose synthon.

Staphylococcus aureus is a Gram-positive bacterium that is responsible for severe nosocomial infections. The rise of multidrug-resistant strains, which can pose significant health threats, prompts the development of new treatment interventions, and much attention has been directed at the development of prophylactic and therapeutic vaccination strategies. Capsular polysaccharides (CPs) are key protective elements of the S. aureus cell wall and have been proposed as promising candidate antigens. Thirteen different CP serotypes have been identified to date, of which types 5 and 8 are the most prominent. CP8 is composed of trisaccharide repeating units that are built up from an N-acetyl-4-O-acetyl-β-d-mannosaminuronic acid, that carries a C-4-O-acetyl, an N-acetyl-α-d-fucosamine, and an N-acetyl-α-l-fucosamine. Synthetic oligosaccharides are valuable tools to unravel the immunogenicity of bacterial oligosaccharides at the molecular level. However, the rare monosaccharides, cis-glycosidic linkages, and O-acetylation represent significant challenges for the synthesis of CP8 fragments. Here the stereoselective assembly of well-defined CP8 fragments, comprising a trimer, hexamer, nonamer, and dodecamer, is presented. This is the first time that fragments larger than a single repeating trisaccharide, which has been proven to be insufficient for antigenic activity, have been assembled. Structural studies have revealed a linear conformation for the oligosaccharides, with each trisaccharide repeat tilted ∼90° with respect to the flanking repeats, which is stabilized by the acetyl groups that prevent rotation around the glycosidic linkages. The N-acetyl groups in each repeating unit point in the same direction, generating a hydrophobic flank in the trisaccharide repeats. We applied the oligomers to generate model glycoconjugate vaccine modalities, which we then used to raise anti-CP8 antibodies. The antibody interaction and immunization studies have revealed a clear length dependent structure-activity relationship for the oligosaccharides, with an oligosaccharide of at least three repeating units required for an adequate immune response.

Aziridines are important structures in the contemporary organic synthesis and are used for several biological applications. Herein, we show that aziridines can be readily synthesized from alkenes by the reductive activation of sulfonyl azides, mediated by photoredox catalysis. Mechanistic studies indicate that the reaction proceeds through reactive nitrene radical anions instead of the more commonly encountered triplet nitrenes. A substrate scope is performed that showed good functional group compatibility.

Staphylococcus aureus is one of the most prominent pathogens responsible for life-threatening hospital acquired infections. Most clinical isolates belong to serotype 5 or 8, which express unique capsular polysaccharides (CP), composed of the rare N-acetyl-β-d-mannosaminuronic acid (β-d-ManNAcA), N-acetyl-α-l-fucosamine (α-l-FucNAc) and N-acetyl-β-d-fucosamine (β-d-FucNAc) that can be used for the development of conjugate vaccines. Different acetylation patterns of CP5 create microheterogeneous polymers, carrying partial zwitterionic character, which may be important for immunological activity. We here report on the assembly of a set of conjugation-ready CP5 oligosaccharides, ranging in length from trisaccharides to nonasaccharides. The developed protecting group strategy has allowed the incorporation of N-acetyl, -NH3 + and O-acetyl groups. The reported syntheses offer solutions for the construction of the challenging cis-glycosidic linkages, the incorporation of many different functional groups and the installation of an appropriate linker for future conjugation purposes. Conformational analysis of the O-acetylated oligomers has revealed a distinctive linear conformation with the repeating units (RUs) being flipped ∼180° with respect to the flanking RUs. Binding studies with CP5-antibodies revealed the trisaccharide to be too short for relevant binding, while the hexa- and nonasaccharides exhibited strong binding. The l-FucNAc acetyl esters and d-FucNAc acetamides were shown to be crucial for binding.

Nucleophilic substitution reactions of C-2-acyloxy furanosyl acetals can be highly diastereoselective. We here show that the presence of a less electron-donating p-nitrobenzoyloxy group at C-2 of a furanosyl acetal can be of use to control the 1,2-trans stereoselectivity of acetal substitution reactions with higher stereoselectivity than the analogue with the more electron-donating benzoyloxy group, just as what was observed in the pyranosyl system. Computational results support a reaction manifold involving both open oxocarbenium ions and cis-dioxolenium ions to provide the 1,2-cis and 1,2-trans products. Participation by the less electron-donating C-2-(p-nitrobenzoyloxy) group forms a less stabilized cis-dioxolenium ion that reacts with the incoming nucleophile more readily to provide 1,2-trans products. The relative stability of the furanosyl cis-dioxolenium ion versus the open oxocarbenium ion is much higher than the pyranosyl system as a result of the lower energy penalty for forming the cis-fused [5,5]-bicyclic dioxolenium ion.

Therapeutic antibodies are actively explored as alternative to treat or prevent bacterial infections. However, the narrow antigen specificity of IgG in combination with broad diversity in bacterial surface structures currently hampers the development of therapeutic antibodies against bacteria. Here we reveal that isotype conversion of three highly specific anti-staphylococcal antibodies from IgG into IgM does not only affect Fc effector functions but also modifies the interaction of Fab domains with bacterial surface antigens. These converted IgMs gain cross-reactivity for a broad range of bacterial species, including Gram-negatives such as Escherichia coli and Neisseria meningitidis and even protect against invasive infection with Streptococcus pyogenes in vivo. Mechanistic studies show that enhanced cross-specificity by IgM is conferred by changed ligand specificity and multivalent binding to high-density antigens. Altogether, these findings provide important insights for the development of antibody therapy for bacterial infections.