Total synthesis of the Bacteroides fragilis zwitterionic polysaccharide A1 repeating unit

Nearly all bacteria capsular polysaccharides are T-cell-independent antigens that do not promote immunoglobulin class switching from IgM to IgG nor memory responses. In contrast, zwitterionic polysaccharides activate T-cell-dependent immune responses by major histocompatability complex class II presentation, a mechanism previously believed to be reserved for peptidic antigens. The best studied zwitterionic polysaccharide, polysaccharide A1 (PS A1) is found on the capsule of the commensal bacteria Bacteroides fragilis . Its potent immunomodulatory properties have been linked to postoperative intra-abdominal abscess formation. Here, we report the synthesis of the PS A1 tetrasaccharide repeating unit (2) as a tool to investigate the biological role of this polysaccharide. A modular synthetic strategy originating from the reducing end of the PS A1 repeating unit was unsuccessful and illustrated the limitations of glycosylation reactions between highly armed glycosylating agents and poor nucleophiles. Thus, a [3 + 1] glycosylation relying on trisaccharide 5 and pyruvalated galactose 6 was used to complete the first total synthesis of the PS A1 repeating unit (2).     

Tuning chemotactic responses with synthetic multivalent ligands

BACKGROUND: Multivalent ligands have been used previously to investigate the role of ligand valency and receptor clustering in eliciting biological responses. Studies of multivalent ligand function, however, typically have employed divalent ligands or ligands of undefined valency. How cells respond to multivalent ligands of distinct valencies, which can cluster a signaling receptor to different extents, has never been examined. The chemoreceptors, which mediate chemotactic responses in bacteria, are localized, and clustering has been proposed to play a role in their function. Using multivalent ligands directed at the chemoreceptors, we hypothesized that we could exploit ligand valency to control receptor occupation and clustering and, ultimately, the cellular response. RESULTS: To investigate the effects of ligand valency on the bacterial chemotactic response, we generated a series of linear multivalent arrays with distinct valencies by ring-opening metathesis polymerization. We report that these synthetic ligands elicit bacterial chemotaxis in both Escherichia coli and Bacillus subtilis. The chemotactic response depended on the valency of the ligand; the response of the bacteria can be altered by varying chemoattractant ligand valency. Significantly, these differences in chemotactic responses were related to the ability of the multivalent ligands to cluster chemoreceptors at the plasma membrane. CONCLUSIONS: Our results demonstrate that ligand valency can be used to tune the chemotactic responses of bacteria. This mode of regulation may arise from changes in receptor occupation or changes in receptor clustering or both. Our data implicate changes in receptor clustering as one important mechanism for altering cellular responses. Given the diverse events modulated by changes in the spatial proximity of cell surface receptors, our results suggest a general strategy for tuning biological responses.     

Mechanism regulating cell surface expression and activation of Toll-like receptor 4

The Toll family of receptors senses microbial invasion and activates defense responses. Toll-like receptor 4 (TLR4) is a member of the Toll family that senses lipopolysaccharide (LPS), a principal membrane component from Gram-negative bacteria. LPS is known as an endotoxin that strongly activates immune cells such as macrophages and dendritic cells. LPS recognition by TLR4 requires an additional accessory molecule, MD-2. MD-2 is associated with the extracellular portion of TLR4, directly binds to LPS, and regulates subsequent LPS-induced TLR4 clustering. LPS recognition occurs on the cell surface. The subcellular distribution of TLR was shown to influence TLR responses. An endoplasmic reticulum (ER) chaperone, glycoprotein 96, is required for the stability of TLR4 and the formation of a TLR4/MD-2 complex in ER. MD-2 facilitates TLR4 glycosylation and its trafficking to the cell surface. Recently, another molecule, a protein associated with Toll-like receptor 4 (PRAT4A), was shown to play a critical role in cell surface expression of TLR4. These molecules control LPS responsiveness by regulating the subcellular distribution of TLR4.     

Combining Spectroscopy and Theory to Evaluate Structural Models of Metalloenzymes: A Case Study on the Soluble [NiFe] Hydrogenase from Ralstonia eutropha

Hydrogenases catalyse the reversible cleavage of molecular hydrogen into protons and electrons. While most of these enzymes are inhibited under aerobic conditions, some hydrogenases are catalytically active even at ambient oxygen levels. In particular, the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 couples reversible hydrogen cycling to the redox conversion of NAD(H). Its insensitivity towards oxygen has been formerly ascribed to the putative presence of additional cyanide ligands at the active site, which has been, however, discussed controversially. Based on quantum chemical calculations of model compounds, we demonstrate that spectroscopic consequences of the proposed non‐standard set of inorganic ligands are in contradiction to the underlying experimental findings. In this way, the previous model for structure and function of this soluble hydrogenase is disproved on a fundamental level, thereby highlighting the efficiency of computational methods for the evaluation of experimentally derived mechanistic proposals.     

Application of Polysaccharide Microarray Technology for the Serodiagnosis of Burkholderia pseudomallei Infection (Melioidosis) in Humans

Burkholderia pseudomallei is the causative agent of melioidosis, a bacterial infection endemic in tropical regions including southeast Asia and northern Australia. B. pseudomallei contains structurally unique polysaccharides (capsular polysaccharide and O?antigen saccharides of lipopolysaccharide). A polysaccharide microarray platform was developed by immobilizing these polysaccharides onto glass slides. Employing this microarray, we were able to demonstrate the presence of antibodies to these polysaccharide antigens in the sera of melioidosis patients, but not in serum from nonmelioidosis human subjects. The advantages of this polysaccharide microarray technology over the conventional tests for the serodiagnosis of melioidosis are discussed.     

Polysaccharide conjugate vaccine:A kind of vaccine with great development potential

Diseases caused by microbial bacteria such as Haemophilus influenzae type b(Hib),Streptococcus pneumoniae and Neisseria meningitidis are still very serious disease,which has brought a lot of burden to many countries.Development of vaccine has brought hope for the prevention of such diseases.Polysaccharide conjugate vaccines have been shown to have very good effects in preventing such diseases.The polysaccharide co njugate vaccine adds the positive characteristics of protein antigens to the polysaccharide antigen,thereby improving the immunogenicity of the polysaccharide antigen,solving the problem that the polysaccharide vaccine cannot be effectively applicated in toddler or children,which greatly promoting the development of this vaccine.This review introduces the progress of polysaccharide conjugate vaccines.We introduce the typical polysaccharide conjugate vaccines currently on the market firstly,and then elucidate the protein carriers,the coupling chemistry methods and quality control that required in the preparation of polysaccharide conjugate vaccines.We can see that polysaccharide conjugate vaccine is a kind of vaccine with great development potential,which can be a sharp edge for us to prevent diseases.     

Recognition of lipopolysaccharide pattern by TLR4 complexes

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. Minute amounts of LPS released from infecting pathogens can initiate potent innate immune responses that prime the immune system against further infection. However, when the LPS response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of LPS in diverse bacterial species is recognized by a cascade of LPS receptors and accessory proteins, LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)–MD-2 complex. The structures of these proteins account for how our immune system differentiates LPS molecules from structurally similar host molecules. They also provide insights useful for discovery of anti-sepsis drugs. In this review, we summarize these structures and describe the structural basis of LPS recognition by LPS receptors and accessory proteins.     

Modulating surface rheology by electrostatic protein/polysaccharide interactions

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/polysaccharide complexes or by sequential adsorption of complexes or polysaccharides to a previously formed protein layer. Even though the final protein and polysaccharide bulk concentrations are the same, the behavior of the adsorbed layers can be very different, depending on the method of preparation. The surface shear modulus of a sequentially formed beta-lactoglobulin/pectin layer can be up to a factor of 6 higher than that of a layer made by simultaneous adsorption. Furthermore, the surface dilatational modulus and surface shear modulus strongly (up to factors of 2 and 7, respectively) depend on the bulk -lactoglobulin/pectin mixing ratio. On the basis of the surface rheological behavior, a mechanistic understanding of how the structure of the adsorbed layers depends on the protein/polysaccharide interaction in bulk solution, mixing ratio, ionic strength, and order of adsorption to the interface (simultaneous or sequential) is derived. Insight into the effect of protein/polysaccharide interactions on the properties of adsorbed layers provides a solid basis to modulate surface rheological behavior.     

Mast cells in UV-B-induced immunosuppression

Degranulating dermal mast cells in UV-B-irradiated skin have been implicated for many years in the mechanisms of irradiation erythema. There is now considerable evidence that dermal mast cells are important to the processes by which both UV-B radiation and cis-urocanic acid (cis-UCA) suppress immune responses to sensitizing antigens applied to non-irradiated/non-cis-UCA-exposed sites. Mast-cell-depleted mice are resistant to the immunosuppressive effects of UV-B radiation and cis-UCA for 'systemic' immunomodulation. However, these mice gain responsiveness if the dorsal skin is reconstituted six weeks prior to irradiation or cis-UCA administration at that site with cultured bone-marrow-derived mast cells from +/+ mice. The molecular triggers for initiating mast-cell degranulation are being actively sought. Evidence suggests that histamine, and not tumour necrosis factor alpha, is the major mast-cell product that signals altered immune responses to sensitizing antigens applied to non-irradiated, non-cis-UCA-exposed sites. Histamine may have multiple roles, but experiments with indomethacin administered to mice have shown that one process involves induction of prostanoid production.     

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.     

Immune diversity and genomic stability: opposite goals but similar paths.

DNA damage response mechanisms serve to protect cells from exogenous and endogenous DNA damaging agents with the aim of maintaining genomic stability. In contrast, the generation of an efficient immune response requires the creation of a repertoire of distinct immunoglobulin and T cell receptor genes able to recognise the huge array of antigens that may be encountered in a lifetime. Surprisingly, cells have exploited the same mechanisms used to maintain genomic integrity to create genetic diversity during immune development. Here, we review the damage response mechanisms operating on DNA double strand breaks and their function during development of the immune response. We discuss disorders that are associated with immunodeficiency and defective responses to the presence of DNA double strand breaks.     

Total chemical synthesis by native chemical ligation of the all-D immunoglobulin-like domain 2 of Axl

Axl is a tyrosine kinases receptor playing crucial role in several cellular responses. The deregulation of Axl signaling has been associated to many high impact diseases ranging from cancer to multiple sclerosis. We report the successful procedure for the chemical synthesis of the Ig2 domain of Axl, one of the key extracellular regions of the receptor involved in ligand binding. The protein was synthesized in its d-enantiomeric form (D-Axl-2), opening the way to the selection of D-peptides selectively targeting Axl receptor through the mirror-image phage display peptide library screening approach.     

Activation of T lymphocytes by the Fc portion of immunoglobulin

T lymphocytes are stimulated to release T-cell-replacing factors in response to Fc fragments of human IgG, Lyt 1+23- T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage-dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc-mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti-SRBC responses.     

Surface display on gram positive bacteria

Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, "second generation" vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Gram-positive bacterial surface display, and discusses current uses and possible future trends of this emerging technology.     

Innate Immunity Modulating Impurities and the Immunotoxicity of Nanobiotechnology-Based Drug Products

Innate immunity can be triggered by the presence of microbial antigens and other contaminants inadvertently introduced during the manufacture and purification of bionanopharmaceutical products. Activation of these innate immune responses, including cytokine secretion, complement, and immune cell activation, can result in unexpected and undesirable host immune responses. These innate modulators can also potentially stimulate the activation of adaptive immune responses, including the formation of anti-drug antibodies which can impact drug effectiveness. To prevent induction of these adverse responses, it is important to detect and quantify levels of these innate immunity modulating impurities (IIMIs) that may be present in drug products. However, while it is universally agreed that removal of IIMIs from drug products is crucial for patient safety and to prevent long-term immunogenicity, there is no single assay capable of directly detecting all potential IIMIs or indirectly quantifying downstream biomarkers. Additionally, there is a lack of agreement as to which of the many analytical assays currently employed should be standardized for general IIMI screening. Herein, we review the available literature to highlight cellular and molecular mechanisms underlying IIMI-mediated inflammation and its relevance to the safety and efficacy of pharmaceutical products. We further discuss methodologies used for direct and indirect IIMI identification and quantification.     

Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.     

Purification of the Escherichia coli K5 capsular polysaccharide and use of high-performance capillary electrophoresis to qualitative and quantitative monitor the process

A rapid, highly sensitive, and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K5 bacteria Bi8337/41 010:K5:H4. This natural polysaccharide having the structure of a desulfo-heparin composed of -4)-alphaGlcUA-(1,4)-alpha-GlcNAc-(1- is separated (GlcUA = D-glucuronic acid; GlcNAc = D-glucosamine) and qualitatively and quantitatively determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient > approximately 0.99) was found for the polymer over a wide range of concentrations, from approx. 60 to 1500 ng, with a detection sensitivity of < approximately 60 ng. Furthermore, this qualitative and quantitative HPCE approach was applied to the K5 extraction and purification process from cultured bacteria in several stages. HPCE was also able to separate several molecular species mainly due to the presence of polysaccharides of distinct and increasing mean chain lengths. A linear relationship was found for migration time and log molecular mass of different K5 polysaccharide species, and this model was used to calculate the molecular mass of the main K5 species producing a result of approx. 17,000, also confirmed by high-performance size-exclusion chromatography analysis, yielding approx. 17 200.     

Trivalent antigens for degranulation of mast cells

Degranulation of basophils and mast cells plays a central role in allergic reactions. Degranulation is a response to cell surface receptor aggregation caused by association of receptors with antibodies bound to multivalent antigens. Tools used in studying this process have included small-molecule divalent antigens, but they suffer from weak signaling apparently due to small aggregate size. We have prepared trivalent antigens that allow formation of larger aggregates and potent responses from mast cells.