Search

Sialyltransferases (ST) are key enzymes found in, among others, mammals and bacteria that are responsible for producing sialylated glycans, which play critical roles in human health and disease. However, chemical tools to study sialyltransferases have been limited to non-covalent inhibitors and probes that do not allow isolation and profiling of these important enzymes. Here we report a new class of covalent affinity-based probes (AfBP) for ST by using ligand-directed chemistry (LDchem). Our affinity-based probes are armed with a simple to synthesise but robust O -nitrobenzoxadiazole ( O -NBD) warhead, which is a lysine-specific S N Ar electrophilic warhead with an advantageous turn-on fluorescence property. We chemoenzymatically synthesised a series of CMP-Neu5Ac based probes and demonstrated their high specificity in labelling a range of recombinant STs with submicromolar sensitivity. Importantly, with our LDchem ST probe, we successfully labelled the endogenous lipooligosaccharide ST (Lst) in live Neisseria gonorrhoeae , a clinically relevant human pathogen. Our results demonstrated that this new class of covalent ST probes offer a robust platform for ST profiling and future studies of STs in their native environments., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Carrot rhamnogalacturonan-I (cRG-I) is a polydisperse polysaccharide with molecular weights of 7-250 kDa. Using size exclusion chromatography cRG-I was fractionated and pooled in fractions (PF1-6). All fractions contained the same RG-I monosaccharides and similar glycosidic linkages although in varying relative amounts. The main differences were in rhamnose substitution, arabinan- and galactan side chain length and in levels of acetylation and methyl esterification. Atomic force microscopy showed either spheric or elongated structures for cRG-I and its derived fractions. To gain insight in the structure-function relationship of cRG-I, the immunomodulatory effect of the six fractions and their saponified derivatives was assessed in vitro. All fractions, except PF2, dose-dependently stimulated TNFα, IL-6, IL-1β, IL-8 and IL-10 production in peripheral blood mononuclear cells (PBMCs) of three healthy donors. Cytokine levels were largely influenced by the Mw and degree of esterification of the individual fractions. Notably, the highest Mw fraction (100 kDa) displayed the most potent activity, which was strongly reduced after the removal of ester residues by saponification. In contrast, the 75 kDa Mw population (PF2) proved inactive while its saponified counterpart exhibited substantial immunomodulatory activity. This confirmed the role of ester residues on the immune profile of RG-I subpopulations., Competing Interests: Declaration of competing interest The authors declare the following financial interests which may be considered as potential competing interests. NutriLeads holds intellectual property rights for the use cRG-I. Annick Mercenier and Ruud Albers were employed at NutriLeads and remain shareholders affiliated to NutriLeads for scientific matters., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Lipooligosaccharides are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their lipooligosaccharides with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their lipooligosaccharides. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the lipooligosaccharides of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering and Selective Exo-Enzymatic Labeling, and that together they provide tools to modify, label, detect and visualize sialylation of bacterial lipooligosaccharides., (© The Author(s) 2024. Published by Oxford University Press.)

Sialoglycan-binding enveloped viruses often possess receptor-destroying activity to avoid being immobilized by non-functional decoy receptors. Sialic acid (Sia)-binding paramyxoviruses contain a hemagglutinin-neuraminidase (HN) protein that possesses both Sia-binding and -cleavage activities. The multivalent, dynamic receptor interactions of paramyxovirus particles provide virion motility and are a key determinant of host tropism. However, such multivalent interactions have not been exhaustively analyzed, because such studies are complicated by the low affinity of the individual interactions and the requirement of high titer virus stocks. Moreover, the dynamics of multivalent particle-receptor interactions are difficult to predict from Michaelis-Menten enzyme kinetics. Therefore, we here developed Ni-NTA nanoparticles that multivalently display recombinant soluble HN tetramers via their His tags (HN-NPs). Applying this HN-NP platform to Newcastle disease virus (NDV), we investigated using biolayer interferometry (BLI) the role of important HN residues in receptor-interactions and analyzed long-range effects between the catalytic site and the second Sia binding site (2SBS). The HN-NP system was also applicable to other paramyxoviruses. Comparative analysis of HN-NPs revealed and confirmed differences in dynamic receptor-interactions between type 1 human and murine parainfluenza viruses as well as of lab-adapted and clinical isolates of human parainfluenza virus type 3, which are likely to contribute to differences in tropism of these viruses. We propose this novel platform to be applicable to elucidate the dynamics of multivalent-receptor interactions important for host tropism and pathogenesis, particularly for difficult to grow sialoglycan-binding (paramyxo)viruses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.

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., (© The Author(s) 2021. Published by Oxford University Press.)

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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The influenza A virus (IAV) neuraminidase (NA) is essential for virion release from cells and decoy receptors and an important target of antiviral drugs and antibodies. Adaptation to a new host sialome and escape from the host immune system are forces driving the selection of mutations in the NA gene. Phylogenetic analysis shows that until 2015, 16 amino acid substitutions in NA became fixed in the virus population after introduction in the human population of the pandemic IAV H1N1 (H1N1pdm09) in 2009. The accumulative effect of these substitutions, in the order in which they appeared, was analyzed using recombinant proteins and viruses in combination with different functional assays. The results indicate that NA activity did not evolve to a single optimum but rather fluctuated within a certain bandwidth. Furthermore, antigenic and enzymatic properties of NA were intertwined, with several residues affecting multiple properties. For example, the substitution K432E in the second sialic acid binding site, next to the catalytic site, was shown to affect catalytic activity, substrate specificity, and the pH optimum for maximum activity. This substitution also altered antigenicity of NA, which may explain its selection. We propose that the entanglement of NA phenotypes may be an important determining factor in the evolution of NA. IMPORTANCE Since its emergence in 2009, the pandemic H1N1 influenza A virus (IAV) has caused significant disease and mortality in humans. IAVs contain two envelope glycoproteins, the receptor-binding hemagglutinin (HA) and the receptor-destroying neuraminidase (NA). NA is essential for virion release from cells and decoy receptors, is an important target of antiviral drugs, and is increasingly being recognized as an important vaccine antigen. Not much is known, however, about the evolution of this protein upon the emergence of the novel pandemic H1N1 virus, with respect to its enzymatic activity and antigenicity. By reconstructing the evolutionary path of NA, we show that antigenic and enzymatic properties of NA are intertwined, with several residues affecting multiple properties. Understanding the entanglement of NA phenotypes will lead to better comprehension of IAV evolution and may help the development of NA-based vaccines., (Copyright © 2021 Dai et al.)

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.

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., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)