Abstract The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae , using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP‐sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6‐linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6‐sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans.
Abstract Campylobacter jejuni is the major human food‐borne pathogen. Its bipolar flagella are heavily O ‐glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide‐modified Leg precursors. The hereby azido‐Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC–MS detection of labeled CMP‐Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.
The rate of formation of covalently linked organic monolayers on HF-etched silicon carbide (SiC) is greatly increased by microwave irradiation. Upon microwave treatment for 60 min at 100 °C (60 W), 1-alkenes yield densely packed, covalently attached monolayers on flat SiC surfaces, a process that typically takes 16 h at 130 °C under thermal conditions. This approach was extended to SiC microparticles. The monolayers were characterized by X-ray photoelectron spectroscopy and static water contact angle measurements. The microwave-assisted reaction is compatible with terminal functionalities such as alkenes that enable subsequent versatile "click" chemistry reactions, further broadening the range and applicability of chemically modified SiC surfaces.
The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and β-glucosidase 2 that are enzymes involved in glucosylceramide metabolism. The study has resulted in many novel inhibitors of these three enzymes among which several that improve upon the inhibition profile of the lead
compound in this study. The successful use of lipophilic iminosugars in type 2 diabetes models and the partial elucidation of their mechanism of action therein provide prospects for their development towards therapeutics for diabetes type 2.
Abstract Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota‐host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin‐degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila . Two novel non‐toxic tetrazine click‐compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species.
Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100 000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors.
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