Kinetic analysis of paramyxovirus-sialoglycan receptor interactions reveals virion motility.

Many viruses initiate infection by binding to sialoglycan receptors at the cell surface. Binding to such receptors comes at a cost, however, as the sheer abundance of sialoglycans e.g. in mucus, may immobilize virions to non-functional decoy receptors. As a solution, sialoglycan-binding as well as sialoglycan-cleavage activities are often present in these viruses, which for paramyxoviruses are combined in the hemagglutinin-neuraminidase (HN) protein. The dynamic interactions of sialoglycan-binding paramyxoviruses with their receptors are thought to be key determinants of species tropism, replication and pathogenesis. Here we used biolayer interferometry to perform kinetic analyses of receptor interactions of animal and human paramyxoviruses (Newcastle disease virus, Sendai virus, and human parainfluenza virus 3). We show that these viruses display strikingly different receptor interaction dynamics, which correlated with their receptor-binding and -cleavage activities and the presence of a second sialic acid binding site. Virion binding was followed by sialidase-driven release, during which virions cleaved sialoglycans until a virus-specific density was reached, which was largely independent of virion concentration. Sialidase-driven virion release was furthermore shown to be a cooperative process and to be affected by pH. We propose that paramyxoviruses display sialidase-driven virion motility on a receptor-coated surface, until a threshold receptor density is reached at which virions start to dissociate. Similar motility has previously been observed for influenza viruses and is likely to also apply to sialoglycan-interacting embecoviruses. Analysis of the balance between receptor-binding and -cleavage increases our understanding of host species tropism determinants and zoonotic potential of viruses.

Structure-activity relationship of 2,4-D correlates auxinic activity with the induction of somatic embryogenesis in Arabidopsis thaliana.

2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic analogue of the plant hormone auxin that is commonly used in many in vitro plant regeneration systems, such as somatic embryogenesis (SE). Its effectiveness in inducing SE, compared to the natural auxin indole-3-acetic acid (IAA), has been attributed to the stress triggered by this compound rather than its auxinic activity. However, this hypothesis has never been thoroughly tested. Here we used a library of forty 2,4-D analogues to test the structure-activity relationship with respect to the capacity to induce SE and auxinic activity in Arabidopsis thaliana. Four analogues induced SE as effectively as 2,4-D and 13 analogues induced SE but were less effective. Based on root growth inhibition and auxin response reporter expression, the 2,4-D analogues were classified into different groups, ranging from very active to not active auxin analogues. A halogen at the 4-position of the aromatic ring was important for auxinic activity, whereas a halogen at the 3-position resulted in reduced activity. Moreover, a small substitution at the carboxylate chain was tolerated, as was extending the carboxylate chain with an even number of carbons. The auxinic activity of most 2,4-D analogues was consistent with their simulated TIR1-Aux/IAA coreceptor binding characteristics. A strong correlation was observed between SE induction efficiency and auxinic activity, which is in line with our observation that 2,4-D-induced SE and stress both require TIR1/AFB auxin co-receptor function. Our data indicate that the stress-related effects triggered by 2,4-D and considered important for SE induction are downstream of auxin signalling.

Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia muciniphila with Bioorthogonal Chemical Reporters.

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.

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes.

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

From the freezer to the clinic: Antifreeze proteins in the preservation of cells, tissues, and organs.

Understanding the mechanisms by which natural anti-freeze proteins protect cells and tissues from cold could help to improve the availability of donor organs for transplantation.

Sweet impersonators: Molecular mimicry of host glycans by bacteria.

All bacteria display surface-exposed glycans that can play an important role in their interaction with the host and in select cases mimic the glycans found on host cells, an event called molecular or glycan mimicry. In this review, we highlight the key bacteria that display human glycan mimicry and provide an overview of the involved glycan structures. We also discuss the general trends and outstanding questions associated with human glycan mimicry by bacteria. Finally, we provide an overview of several techniques that have emerged from the discipline of chemical glycobiology, which can aid in the study of the composition, variability, interaction and functional role of these mimicking glycans.

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues.

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.

Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Bacteroides fragilis fucosidases facilitate growth and invasion of Campylobacter jejuni in the presence of mucins.

The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilisation. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion.

Development of a 1,2-difluorofucoside activity-based probe for profiling GH29 fucosidases.

GH29 α-l-fucosidases catalyze hydrolysis of terminal α-l-fucosyl linkages with varying specificity and are expressed by prominent members of the human gut microbiota. Both homeostasis and dysbiosis at the human intestinal microbiota interface have been correlated with altered fucosidase activity. Herein we describe the development of a 2-deoxy-2-fluoro fucosyl fluoride derivative with an azide mini-tag as an activity-based probe (ABP) for selective in vitro labelling of GH29 α-l-fucosidases. Only catalytically active fucosidases are inactivated by this ABP, allowing their functionalization with a biotin reporter group via the CuAAC reaction and subsequent in-gel detection at nanogram levels. The ABP we present here is shown to be active against a GH29 α-l-fucosidase from Bacteroides fragilis and capable of labeling two other GH29 α-l-fucosidases with different linkage specificity, illustrating its broader utility. This novel ABP is a valuable addition to the toolbox of fucosidase probes by allowing identification and functional studies of the wide variety of GH29 fucosidases, including those in the gut microbiota.

Metabolic Labeling of Legionaminic Acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase.

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.

A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase.

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining ß-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.

Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

BACKGROUND: Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. RESULTS: Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. CONCLUSIONS: Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.

Exploring the Chemistry of Bicyclic Isoxazolidines for the Multicomponent Synthesis of Glycomimetic Building Blocks.

Starting from a chiral furanone, the nitrone-olefin [3 + 2] cycloaddition can be used to obtain bicyclic isoxazolidines for which we report a set of reactions to selectively modify each functional position. These synthetically versatile bicyclic isoxazolidines allowed us to obtain complex glycomimetic building blocks, like iminosugars, via multicomponent chemistry. For example, a library of 20 pipecolic acid derivatives, a recurring motif in various prescription drugs, could be obtained via a one-pot Staudinger/aza-Wittig/Ugi three-component reaction of a bicyclic isoxazolidine-derived azido-hemiacetal. Notably, specific pipecolic acids in this library were obtained via hydrolysis of an unique tricyclic imidate side product of the Ugi reaction. The azido-hemiacetal was also converted into an aza-C-glycoside iminosugar via an unprecendented one-pot Staudinger/aza-Wittig/Mannich reaction.

Versatile (bio)functionalization of bromo-terminated phosphonate-modified porous aluminum oxide.

Porous aluminum oxide (PAO) is a nanoporous material used for various (bio)technological applications, and tailoring its surface properties via covalent modification is a way to expand and refine its application. Specific and complex chemical modification of the PAO surface requires a stepwise approach in which a secondary reaction on a stable initial modification is necessary to achieve the desired terminal molecular architecture and reactivity. We here show that the straightforward initial modification of the bare PAO surface with bromo-terminated phosphonic acid allows for the subsequent preparation of PAO with a wide scope of terminal reactive groups, making it suitable for (bio)functionalization. Starting from the initial bromo-terminated PAO, we prepared PAO surfaces presenting various terminal functional groups, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS). We also show that this wide scope of easily accessible tailored reactive PAO surfaces can be used for subsequent modification with (bio)molecules, including carbohydrate derivatives and fluorescently labeled proteins.

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase.

N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (ß/α)(8) barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

Ambient surface analysis of organic monolayers using direct analysis in real time Orbitrap mass spectrometry.

A better characterization of nanometer-thick organic layers (monolayers) as used for engineering surface properties, biosensing, nanomedicine, and smart materials will widen their application. The aim of this study was to develop direct analysis in real time high-resolution mass spectrometry (DART-HRMS) into a new and complementary analytical tool for characterizing organic monolayers. To assess the scope and formulate general interpretation rules, DART-HRMS was used to analyze a diverse set of monolayers having different chemistries (amides, esters, amines, acids, alcohols, alkanes, ethers, thioethers, polymers, sugars) on five different substrates (Si, Si3N4, glass, Al2O3, Au). The substrate did not play a major role except in the case of gold, for which breaking of the weak Au-S bond that tethers the monolayer to the surface, was observed. For monolayers with stronger covalent interfacial bonds, fragmentation around terminal groups was found. For ester and amide-terminated monolayers, in situ hydrolysis during DART resulted in the detection of ions characteristic of the terminal groups (alcohol, amine, carboxylic acid). For ether and thioether-terminated layers, scission of C-O or C-S bonds also led to the release of the terminal part of the monolayer in a predictable manner. Only the spectra of alkane monolayers could not be interpreted. DART-HRMS allowed for the analysis of and distinction between monolayers containing biologically relevant mono or disaccharides. Overall, DART-HRMS is a promising surface analysis technique that combines detailed structural information on nanomaterials and ultrathin films with fast analyses under ambient conditions.

Stability of (bio)functionalized porous aluminum oxide.

Porous aluminum oxide (PAO), a nanostructured support for, among others, culturing microorganisms, was chemically modified in order to attach biomolecules that can selectively interact with target bacteria. We present the first comprehensive study of monolayer-modified PAO using conditions that are relevant to microbial growth with a range of functional groups (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane). Their stability was initially assessed in phosphate-buffered saline (pH 7.0) at room temperature. The most stable combination (PAO with phosphonic acids) was further studied over a range of physiological pHs (4-8) and temperatures (up to 80 °C). Varying the pH had no significant effect on the stability, but it gradually decreased with increasing temperature. The stability of phosphonic acid-modified PAO surfaces was shown to depend strongly on the other terminal group of the monolayer structure: in general, hydrophilic monolayers were less stable than hydrophobic monolayers. Finally, an alkyne-terminated PAO surface was reacted with an azide-linked mannose derivative. The resulting mannose-presenting PAO surface showed the clearly increased adherence of a mannose-binding bacterium, Lactobacillus plantarum, and also allowed for bacterial outgrowth.

Microwave-assisted formation of organic monolayers from 1-alkenes on silicon carbide.

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.