Synthetic Teichoic Acid Conjugate Vaccine against Nosocomial Gram-Positive Bacteria

Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria

Synthetic Teichoic Acid Conjugate Vaccine against Nosocomial Gram-Positive Bacteria

Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria

On the reactivity and selectivity of donor glycosides in glycochemistry and glycobiology:trapped covalent intermediates

The reactivity of sugar donors and the stability of covalent intermediates formed in both chemical and biological systems is an active subject of study in both glycochemistry and glycobiology. Knowledge of the structure of these intermediates is vital for understanding reactivity and stereoselectivity in glycosidic bond formation, and in glycosidic bond destruction in the case of enzymatic hydrolysis. For chemical reactions, tuning of the electron-withdrawing power of the carbohydrate side chains allows for stabilization of covalent anomeric triflates thereby enabling chemo-, regio- and stereoselective glycosylations. Retaining glycosidase-mediated hydrolysis reactions in turn often involve a covalent intermediate. The existence of such covalent intermediates was convincingly demonstrated at the beginning of this century by making use of modified glycosyl substrates tuned such that stable adducts are formed efficiently but the ensuing hydrolysis is slowed down. Recently this concept has also been used in the design of glycosidase activity-based probes. This review describes recent investigations on different carbohydrate decoration patterns to influence both chemical and biological reactivity and selectivity

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

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.NWO15.002.Metals in Catalysis, Biomimetics & Inorganic MaterialsBio-organic Synthesi

Synthetic zwitterionic polysaccharides

Bio-organic Synthesi

Chair interconversion and reactivity of mannuronic acid esters

Mannopyranosyluronic acids display a very unusual conformation behavior in that they often prefer to adopt a C-1(4) 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 H-1 and F-19 NMR spectra at various temperatures from -80 degrees C to 0 degrees C. Exchange rates between C-4(1) and C-1(4) chair conformations were found to depend on the electronic properties and the size of the C2 substituent (F, N-3 or OBn) and the aglycon, with higher exchange rates for the glycosyl triflates and smaller C2 substituents. Low temperature F-19 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 H-1 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

Mannosazide Methyl Uronate Donors. Glycosylating Properties and Use in the Construction of beta-ManNAcA-Containing Oligosaccharides

Mannosazide methyl uronate donors equipped with a variety of anomeric leaving groups (beta- and a-S-phenyl, beta- and alpha-N-phenyltrifluoroacetimidates, hydroxyl, P-sulfoxide, and (R-s)- and (S-s)-alpha-sulfoxides) were subjected to activating conditions, and the results were monitored by H-1 NMR. While the S-phenyl and imidate donors all gave a conformational mixture of anomeric alpha-triflates, the hemiacetal and beta- and alpha-sulfoxides produced an oxosulfonium triflate and beta- and alpha-sulfonium bistriflates, respectively. The beta-S-phenyl mannosazide methyl uronate performed best in both activation experiments and glycosylation studies and provided the 1,2-cis mannosidic linkage with excellent selectivity. Consequently, an alpha-Glc-(l -> 4)-,beta-ManN(3)A-SPh disaccharide, constructed by the stereo-selective glycosylation of a 6-O-Fmoc-protected glucoside and beta-S-phenyl mannosazide methyl uronate, was used as the repetitive donor building block in the synthesis of tri-, penta-, and heptasaccharide fragments corresponding to the Micrococcus luteus teichuronic acid

Uronic Acids in Oligosaccharide and Glycoconjugate Synthesis

This chapter describes the assembly of uronic acid containing oligosaccharides and glycoconjugates. Two strategies are available to access these target molecules, namely a pre-glycosylation oxidation approach, in which uronic acid building blocks are used, and a post-glycosylation oxidation strategy, which employs an oxidation step after the assembly of the oligosaccharide chain. Because uronic acid building blocks are generally considered to be less reactive than their non-oxidized counterparts, the latter approach has found most application in carbohydrate synthesis. With the aid of selected examples of recent syntheses of biologically relevant oligosaccharides and glycoconjugates, the reactivity of different uronic acid building blocks is evaluated. From these examples it is apparent that the generally assumed low reactivity of uronic acids does not a priori rule out an efficient assembly of these target compounds. Besides influencing the reactivity of a given pyranoside, the C-5 carboxylic acid function can also have a profound effect on the stereochemical course of a glycosylation reaction, which can be exploited in the stereoselective formation of glycosidic bonds.</p

A multiplexing activity-based protein profiling platform for dissection of a native bacterial xyloglucan-degrading system

Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) is complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a “low-tech”, easy-to-use and sensitive, multiplexing activity-based protein profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide degrading systems, by modelling probe structures to those of desired substrates