The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.

n-Heptyl α-d-mannose (HM) is a nanomolar antagonist of FimH, a virulence factor of E. coli. Herein we report on the construction of multivalent HM-based glycopolymers as potent antiadhesives of type 1 piliated E. coli. We investigate glycopolymer/FimH and glycopolymer/bacteria interactions and show that HM-based glycopolymers efficiently inhibit bacterial adhesion and disrupt established cell-bacteria interactions in vitro at very low concentration (0.1 μM on a mannose unit basis). On a valency-corrected basis, HM-based glycopolymers are, respectively, 10(2) and 10(6) times more potent than HM and d-mannose for their capacity to disrupt the binding of adherent-invasive E. coli to T84 intestinal epithelial cells. Finally, we demonstrate that the antiadhesive capacities of HM-based glycopolymers are preserved ex vivo in the colonic loop of a transgenic mouse model of Crohn's disease. All together, these results underline the promising scope of HM-based macromolecular ligands for the antiadhesive treatment of E. coli induced inflammatory bowel diseases.

Antagonists of the Escherichia coli type-1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against acute and recurrent bacterial infections. In this study α-d-mannopyranosides O- or C-linked with an alkyl, alkene, alkyne, thioalkyl, amide, or sulfonamide were investigated to fit a hydrophobic substituent with up to two aryl groups within the tyrosine gate emerging from the mannose-binding pocket of FimH. The results were summarized into a set of structure-activity relationships to be used in FimH-targeted inhibitor design: alkene linkers gave an improved affinity and inhibitory potential, because of their relative flexibility combined with a favourable interaction with isoleucine-52 located in the middle of the tyrosine gate. Of particular interest is a C-linked mannoside, alkene-linked to an ortho-substituted biphenyl that has an affinity similar to its O-mannosidic analog but superior to its para-substituted analog. Docking of its high-resolution NMR solution structure to the FimH adhesin indicated that its ultimate, ortho-placed phenyl ring is able to interact with isoleucine-13, located in the clamp loop that undergoes conformational changes under shear force exerted on the bacteria. Molecular dynamics simulations confirmed that a subpopulation of the C-mannoside conformers is able to interact in this secondary binding site of FimH.

Fast, reliable and selective detection of microorganisms is of uttermost importance in clinical analysis, but also in food and water quality monitoring. In this study, we report on the construction of an immunosensor for sensitive and selective electrochemical detection of uropathogenic Escherichia coli (E. coli) UTI89 bacteria in aqueous and serum samples. We took benefit of electrophoretic deposition (EPD) to prepare, in a simple, controllable and cost effective way, gold electrodes modified with thin active layers of reduced graphene oxide/polyethylenimine (rGO/PEI). While rGO exhibits high surface area and favourable electrochemical properties, the presence of abundant-NH 2 groups on PEI offers a plethora of opportunities for the sensor's surface functionalization. To achieve selectivity of detection, the electrode surface was covalently modified with anti-fimbrial E. coli antibodies via amide bond formation. To minimize non-specific adsorption, the immunosensor was additionally modified with pyrene-polyethyleneglycol (pyrene-PEG) moieties prior to antibody immobilization. The detection of E. coli was based on the restriction of electron transfer of a redox mediator, in our case potassium ferrocyanide, to the rGO/PEI modified electrical transducer due to the formation of an immune complex. The developed immunosensor displayed a sigmoidal shape with a linear range from to 110 1-110 4 cfu mL-1 (R 2 =0.995) according to i(µA)=-16.66-20.5×log[E.coli] (cfu 3 mL-1) and a detection limit of 10 cfu mL-1. Additionally, the sensor performed well both in aqueous and urine media, which is essential for its potential use for clinical diagnosis of pathogenic diseases. Selectivity studies showed that the immunosensor was able to discriminate between E. coli UTI89 wild-type strain and UTI89 fim, without fim operon.

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Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio‐pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP‐sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human β‐d‐galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP‐sialic acids: CMP‐Neu5Ac, and CMP‐Neu5N‐(4pentynoyl)neuraminic acid (CMP‐SiaNAl), an unnatural CMP‐sialic acid donor with an extended and functionalized N‐acyl moiety.

A variety of physical and chemical parameters are of importance for adhesion of bacteria to surfaces. In the colonization of mammalian organisms for example, bacterial fimbriae and their adhesins not only seek particular glycan sequences exposed on diverse epithelial linings, they also enable the bacteria to overcome electrostatic repulsion exerted by their selected surfaces. In this work, we present a new technique based on simplified model systems for studying the adhesion strength of different Escherichia coli strains. For this purpose, gold-based surface plasmon resonance (SPR) interfaces were coated with thin films of reduced graphene oxide (rGO) through electrophoretic deposition. The rGO matrix was post-modified with polyethyleneimine (PEI), poly(sodium 4-styrenesulfonate) (PSS), mannose, and lactose through π-stacking and/or electrostatic interactions by simple immersion of the SPR interface into their respective aqueous solutions. The adhesion behaviors of one uropathogenic and two enterotoxigenic Escherichia coli clinical isolates, that each express structurally characterized fimbrial adhesins, were investigated. It was found that the UTI89 cystitis isolate that carries the mannose-binding FimH adhesin was most attracted to the PEI- and mannose-modified surfaces, whereas the att25 diarrhoeal strain with the N-acetylglucosamine-specific F17a-G adhesin disintegrated the lactose-modified rGO. The highly virulent 107/86 strain interacted strongly with the PSS-modified graphene oxide, in agreement with the polybasic surroundings of the ABH blood group-binding site of the FedF adhesin, and showed a linear SPR response in a concentration range between 1 × 10(2) and 1 × 10(9) cfu/mL.

Antagonists of the FimH adhesin, a protein almost universally present at the extremity of type-1 fimbriae expressed by Escherichia coli, have been abundantly in the spotlight as alternative treatments of urinary tract infections. The antagonists function as bacterial antiadhesives through highly specific α-d-mannose binding in a charged and polar pocket at the tip of the FimH lectin domain and by the stacking of alkyl or aromatic moieties substituted on the mannose with two tyrosine residues (Tyr48 and Tyr137) at the entrance of the mannose-binding pocket. Using high-resolution crystal data, interaction energies are calculated for the different observed aromatic stacking modes between the tyrosines and the antagonist. The dispersion component of the interaction energy correlates with the observed electron density. The quantum chemical reactivity descriptors local hardness and polarizability were successfully validated as prediction tools for ligand affinity in the tyrosine gate of FimH and therefore have potential for rapid drug screening.