Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide.

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

Quantification of Protein Glycosylation Using Nanopores.

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis.

More than sugar in the milk: human milk oligosaccharides as essential bioactive molecules in breast milk and current insight in beneficial effects.

Human milk is the gold standard for newborn infants. Breast milk not only provides nutrients, it also contains bioactive components that guide the development of the infant's intestinal immune system, which can have a lifelong effect. The bioactive molecules in breast milk regulate microbiota development, immune maturation and gut barrier function. Human milk oligosaccharides (hMOs) are the most abundant bioactive molecules in human milk and have multiple beneficial functions such as support of growth of beneficial bacteria, anti-pathogenic effects, immune modulating effects, and stimulation of intestine barrier functions. Here we critically review the current insight into the benefits of bioactive molecules in mother milk that contribute to neonatal development and focus on current knowledge of hMO-functions on microbiota and the gastrointestinal immune barrier. hMOs produced via genetically engineered microorganisms are now applied in infant formulas to mimic the nutritional composition of breast milk as closely as possible, and their prospects and scientific challenges are discussed in depth.

Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus PlantarumWCFS1in an In Vitro Model with Intestinal Peristaltic Shear Force.

BACKGROUND: The intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells. OBJECTIVES: We investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium. METHODS: We applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2'-fucosyllactose (2'-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin ß-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified. RESULTS: Shear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0-94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). CONCLUSIONS: 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.

Direct and Regioselective Di-α-fucosylation on the Secondary Rim of ß-Cyclodextrin.

A straightforward glycosylation method is described to regio- and stereoselectively introduce two α-l-fucose moieties directly to the secondary rim of ß-cyclodextrin. Using NMR and MS fragmentation studies, the nonasaccharide structure was determined, which was also visualized using molecular dynamics simulations. The reported glycosylation method proved to be robust on gram-scale, and may be generally applied to directly glycosylate ß-cyclodextrins to make well-defined multivalent glycoclusters.

Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface.

Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes.

P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.

A Modular Approach to Phosphoglycosyltransferase Inhibitors Inspired by Nucleoside Antibiotics.

Phosphoglycosyltransferases (PGTs) represent "gatekeeper" enzymes in complex glycan assembly pathways by catalyzing transfer of a phosphosugar from an activated nucleotide diphosphosugar to a membrane-resident polyprenol phosphate. The unique structures of selected nucleoside antibiotics, such as tunicamycin and mureidomycin A, which are known to inhibit comparable biochemical transformations, are exploited as the foundation for the development of modular synthetic inhibitors of PGTs. Herein we present the design, synthesis, and biochemical evaluation of two readily manipulatable modular scaffolds as inhibitors of monotopic bacterial PGTs. Selected compounds show IC50 values down to the 40 µm range, thereby serving as lead compounds for future development of selective and effective inhibitors of diverse PGTs of biological and medicinal interest.

Catalytic mechanism and mode of action of the periplasmic alginate epimerase AlgG.

Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1-4-linked ß-D-mannuronate. As the polymer passages through the periplasm, 22-44% of the mannuronate residues are converted to α-L-guluronate by the C5-epimerase AlgG to produce a polymer of alternating ß-D-mannuronate and α-L-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel ß-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His(319) acts as the catalytic base and that Arg(345) neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca(2+) dependence.

A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining ß-glucosidases.

Retaining ß-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining ß-glucosidases. Whereas ß-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining ß-glucosidases, ß-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining ß-glucosidases by the combined use of cyclophellitol ß-epoxide- and ß-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent ß-aziridine but not ß-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the ß-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining ß-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining ß-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.

Acceptor reactivity in the total synthesis of alginate fragments containing α-L-guluronic acid and ß-D-mannuronic acid.

The total synthesis of mixed-sequence alginate oligosaccharides, featuring both ß-D-mannuronic acid (M) and α-L-guluronic acid (G), is reported for the first time. A set of GM, GMG, GMGM, GMGMG, GMGMGM, GMGMGMG, and GMGGMG alginates was assembled using GM building blocks, having a guluronic acid acceptor part and a mannuronic acid donor side to allow the fully stereoselective construction of the cis-glycosidic linkages. It was found that the nature of the reducing-end anomeric center, which is ten atoms away from the reacting alcohol group in the key disaccharide acceptor, had a tremendous effect on the efficiency with which the building blocks were united. This chiral center determines the overall shape of the acceptor and it is revealed that the conformational flexibility of the acceptor is an all-important factor in determining the outcome of a glycosylation reaction.

Gut Epithelial Barrier Function is Impacted by Hyperglycemia and Secondary Bile Acids in Vitro: Possible Rescuing Effects of Specific Pectins.

Gut epithelial barrier disruption is commonly observed in Western diseases like diabetes and inflammatory bowel disease (IBD). Enhanced epithelial permeability triggers inflammatory responses and gut microbiota dysbiosis. Reduced bacterial diversity in IBD affects gut microbiota metabolism, altering microbial products such as secondary bile acids (BAs), which potentially play a role in gut barrier regulation and immunity. Dietary fibers such as pectin may substitute effects of these BAs. The study examines transepithelial electrical resistance of gut epithelial T84 cells and the gene expression of tight junctions after exposure to (un)sulfated secondary BAs. This is compared to the impact of the dietary fiber pectin with different degrees of methylation (DM) and blockiness (DB), with disruption induced by calcium ionophore A23187 under both normal and hyperglycemic conditions. Unsulfated lithocholic acid (LCA) and deoxycholic acid (DCA) show a stronger rescuing effect, particularly evident under 20 mM glucose levels. DM19 with high DB (HB) and DM43HB pectin exhibit rescuing effects under both glucose conditions. Notably, DM19HB and DM43HB display higher rescue effects under 20 mM glucose compared to 5 mM glucose. The study demonstrates that specific pectins such as DM19HB and DM43HB may serve as alternatives for preventing barrier disruption in the case of disturbed DCA metabolism.

Exopolysaccharide ß-(2,6)-levan-type fructans have a molecular-weight-dependent modulatory effect on Toll-like receptor signalling.

SCOPE: Fructans are a group of dietary fibers which are known to have many beneficial effects including immune-modulating effects. A family of fructans are ß-(2,6)-linked levan-type fructans that are known to serve as exopolysaccharides in the cell wall of many species of bacteria including commensal bacteria and probiotics. It is still largely unknown whether and how they can serve as immunomodulating molecules. RESULTS: Microbial ß-(2,6)-fructans were found to induce TLR-dependent activation of THP-1 cells, in a dose-dependent fashion. Low molecular weight (Mw), medium Mw and high Mw ß-(2,6)-fructans activated both TLR2 and 4 in a dose- and molecular weight-dependent fashion. In addition, it was found that ß-(2,6)-fructans were able to inhibit signalling of various TLRs with the strongest effect on TLR5 and 8, which were inhibited by all the ß-(2,6)-fructans in a dose- and molecular weight-dependent fashion. The final effect of this activation and inhibition of TLRs on cytokine responses in human dendritic cells (DCs) was minor which may be explained by the counter-activating effects of the different ß-(2,6)-linked levan-type fructans on inhibition of TLR signalling in the DCs. CONCLUSION: A mechanism by which exopolysaccharide levan ß-(2,6)-fructans can be immune-modulating is by impacting TLR signalling. This knowledge could lead to food in which exopolysaccharide levan ß-(2,6)-fructans are added for preventing disorders where TLR-signalling is modulated.

Influence of O6 in mannosylations using benzylidene protected donors: stereoelectronic or conformational effects?

The stereoselective synthesis of ß-mannosides and the underlying reaction mechanism have been thoroughly studied, and especially the benzylidene-protected mannosides have gained a lot of attention since the corresponding mannosyl triflates often give excellent selectivity. The hypothesis for the enhanced stereoselectivity has been that the benzylidene locks the molecule in a less reactive conformation with the O6 trans to the ring oxygen (O5), which would stabilize the formed α-triflate and subsequent give ß-selectivity. In this work, the hypothesis is challenged by using the carbon analogue (C7) of the benzylidene-protected mannosyl donor, which is investigated in terms of diastereoselectivity and reactivity and by low-temperature NMR. In terms of diastereoselectivity, the C-7-analogue behaves similarly to the benzylidene-protected donor, but its low-temperature NMR reveals the formation of several reactive intermediate. One of the intermediates was found to be the ß-oxosulfonium ion. The reactivity of the donor was found to be in between that of the "torsional" disarmed and an armed donor.

Chair interconversion and reactivity of mannuronic acid esters.

Mannopyranosyluronic acids display a very unusual conformation behavior in that they often prefer to adopt a (1)C4 chair conformation. They are endowed with a strikingly high reactivity when used in a glycosylation reaction as a glycosyl donor. To investigate the unusual conformational behavior a series of mannuronic acid ester derivatives, comprising anomeric triflate species and O-methyl glycosides, was examined by dynamic NMR experiments, through lineshape analysis of (1)H and (19)F NMR spectra at various temperatures from -80 °C to 0 °C. Exchange rates between (4)C1 and (1)C4 chair conformations were found to depend on the electronic properties and the size of the C2 substituent (F, N3 or OBn) and the aglycon, with higher exchange rates for the glycosyl triflates and smaller C2 substituents. Low temperature (19)F exchange spectroscopy experiments showed that the covalently bound anomeric triflates did not exchange with free triflate species present in the reaction mixture. To relate the conformational behavior of the intermediate triflates to their reactivity in a glycosylation reaction, their relative reactivity was determined via competition reactions monitored by (1)H NMR spectroscopy at low temperature. The 2-O-benzyl ether compound was found to be most reactive whereas the 2-fluoro compound - the most flexible of the studied compounds - was least reactive. Whereas the ring-flip of the mannuronic acids is important for the enhanced reactivity of the donors, the rate of the ring-flip has little influence on the relative reactivity.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR.

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Efficacy of pectins with different degrees of methyl-esterification and of blockiness in preventing gut epithelial cell barrier disruption and the impact on sodium-glucose co-transporter expression under low and high glucose conditions.

Pectins support intestinal barrier function and have anti-diabetic effects, and can differ in the degree of methyl-esterification (DM) and the distribution of non-esterified galacturonic acid residues (DB). The mechanisms and effects of pectin type at different glucose levels are unknown. Pectins with different DM/DB on T84 cells were tested in the presence and absence of the barrier disruptor A23187 at 5 mM and 20 mM glucose. DM19 and DM43 pectins with high DB do rescue the intestinal barrier from disruption. Their effects were as strong as those of the barrier-rescuing anti-diabetic drug metformin, but effects with metformin were restricted to high glucose levels while pectins had effects at both low and high glucose levels. At high glucose levels, DM43HB pectin, which enhanced trans-epithelial electrical resistance, also increased the expressions of claudin1, occludin, and ZO-1. Low and high DM pectins decrease the apical expression of the sodium-glucose co-transporter (SGLT-1) and thereby influence glucose transport, explaining the anti-diabetogenic effect of pectin. Higher DB pectins had the strongest effect. Their impact on SGLT-1 was stronger than that of metformin. Pectin's rescuing effect on barrier disruption and its impact on glucose transportation and anti-diabetogenic effects depend on both the DB and the DM of pectins.

Evaluation of Kdo-8-N3 incorporation into lipopolysaccharides of various Escherichia coli strains.

8-Azido-3,8-dideoxy-α/ß-d-manno-oct-2-ulosonic acid (Kdo-8-N3) is a Kdo derivative used in metabolic labeling of lipopolysaccharide (LPS) structures found on the cell membrane of Gram-negative bacteria. Several studies have reported successful labeling of LPS using Kdo-8-N3 and visualization of LPS by a fluorescent reagent through click chemistry on a selection of Gram-negative bacteria such as Escherichia coli strains, Salmonella typhimurium, and Myxococcus xanthus. Motivated by the promise of Kdo-8-N3 to be useful in the investigation of LPS biosynthesis and cell surface labeling across different strains, we set out to explore the variability in nature and efficiency of LPS labeling using Kdo-8-N3 in a variety of E. coli strains and serotypes. We optimized the chemical synthesis of Kdo-8-N3 and subsequently used Kdo-8-N3 to metabolically label pathogenic E. coli strains from commercial and clinical origin. Interestingly, different extents of labeling were observed in different E. coli strains, which seemed to be dependent also on growth media, and the majority of labeled LPS appears to be of the 'rough' LPS variant, as visualized using SDS-PAGE and fluorescence microscopy. This knowledge is important for future application of Kdo-8-N3 in the study of LPS biosynthesis and dynamics, especially when working with clinical isolates.

Novel activity-based probes for broad-spectrum profiling of retaining ß-exoglucosidases in†situ and in†vivo.

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian ß-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian ß-glucosidases (see picture). These probes offer new ways to study ß-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Efficient isolation of membrane-associated exopolysaccharides of four commercial bifidobacterial strains.

Bifidobacteria confer many health effects, such as fiber digestion, pathogen inhibition and immune system maturation, especially in the newborn infant. The bifidobacterial exopolysaccharides (EPS) are often associated with important health effects, but their thorough investigation is hampered by lack of knowledge of the EPS localization, which is important for efficient EPS isolation. Here we present a straightforward isolation procedure to obtain EPS of four commercial bifidobacterial strains (B. adolescentis, B. bifidum, B. breve, and B. infantis), that are localized at the cell membrane (evidenced using cryo-EM). This procedure can be applied to other bifidobacterial strains, to facilitate the easy isolation and purification for biological experiments and future application in nutraceuticals. In addition, we demonstrate structural differences in the EPS of the four bifidobacterial strains, in terms of monosaccharide composition and size, highlighting the potential of the isolated EPS for determining specific structure-activity effects of bifidobacteria.