A very rapid, versatile and high yield synthesis of Manα1,2Man derivatives using a common orthogonal benzoyl/acetyl protection strategy compatible with the presence of azido groups and the use of CuAAC for conjugating the α(1–2)mannobiosides to different scaffolds, reducing the cost and time of the synthesis and improving the overall yield.

Lactobacillus displacement from the vaginal microbiome associates with adverse health outcomes and is linked to increased risk of preterm birth. Glycans mediate bacterial adhesion events involved in colonisation and infection. Using customised glycan microarrays, we establish glycan interaction profiles of vaginal bacteria implicated in reproductive health. Glycan binding signatures of the opportunistic pathogens Escherichia coli, Fusobacterium nucleatum and Streptococcus agalactiae to oligomannose N-glycans, galactose-terminating glycans and hyaluronic acid, respectively are highly distinct from Lactobacillus commensals. Binding to sulphated glycosaminoglycans by vaginal bacteria is pH dependent, as is binding to neutral and sialic acid-terminating glycans by F. nucleatum. Adhesion of Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis, S. agalactiae and F. nucleatum to vaginal epithelial cells is partially mediated by chondroitin sulphate. S. agalactiae binding to chondroitin sulphate C oligosaccharides is inhibited by L. crispatus. This study highlights glycans as mediators of vaginal bacterial binding events involved in reproductive health and disease.

Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewis^x analogues (‘glycofluoroforms’) using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewis^x. Notably, we show that for two proteins with unique binding sites for Lewis^x, glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow.

Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover. Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. We tested HA binding in solution and using a glycan microarray. Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. We did not observe HA binding in two assay formats, suggesting a modest affinity at best. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for k cat of approximately 10-5 min-1. Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.

Chimeric antigen receptors (CARs) offer promising prospects for innovative cell-based therapies against invasive fungal infections such as invasive candidiasis. Here, we have developed 4 CARs targeting Candida albicans with distinct single-chain variable fragments (scFvs): scFv3-CAR, scFv5-CAR, scFv12-CAR, and scFvκ3-1-CAR. In T cells, scFv5-CAR induced IL-2 expression in response to C. albicans hyphae, while scFv3-CAR and scFv12-CAR did not mediate cell activation against C. albicans. Notably, scFvκ3-1-CAR mediated the strongest cell activation against C. albicans yeast, hyphae, and other clinically relevant Candida species. scFvκ3-1-CAR-NK-92 cells exhibited elevated IFN-γ and CD107a expression, reducing C. albicans viability. NOD scid gamma (NSG) mice treated with scFvκ3-1-CAR-NK-92 cells had reduced C. albicans burden in the kidneys 24 hours postinfection. We showed that scFvκ3-1-CAR targets C. albicans mannan but no other glycans in glycan microarray screening analyses. These findings reveal the scFvκ3-1-CAR potential as a therapeutic strategy for treating Candida spp. by modifying peripheral blood mononuclear cells.

Density depended binding and selectivity is studied on glycodendron microarray with defined valency, which were prepared by on-chip synthesis and analysed by in situ MALDI-TOF MS.