Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners.

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after ß-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

Structural characterization and metal biosorptive activity of the major polysaccharide produced by Pseudomonas veronii 2E.

Pseudomonas veronii 2E, an autochthonous bacterium isolated from sediments associated to a high-polluted watershed, produces a complex matrix of exopolymers with carbohydrates as main components. In this work, four polysaccharides were isolated from the extracellular material. The major acidic polysaccharide named EPO2, was purified and its structure was elucidated using Matrix-assisted laser desorption/ionization and Electrospray ionization mass spectrometry, Infrared spectroscopy, Nuclear magnetic resonance spectroscopy and chemical treatments. This heteropolysaccharide consists in an α(1-4) glucan substituted with N-Acetylglucosamine residues and with a branching α-D-GlcpA-(1-3)-L-Fucp disaccharide. The biosorption capacity of EPO2 and of the whole exopolysaccharide to Pb(II), Zn(II), Cu(II) and Fe(II) was evaluated. EPO2 showed a remarkable sorption capacity for Fe(II) with an efficiency of 70% and for Zn(II) 39%. When the whole exopolysaccharide fraction was tested it showed a significantly lower metal sorption ability than purified EPO2 suggesting the involvement of the distinct acidic branching disaccharide in this interaction.

Infrared spectroscopy with multivariate analysis to interrogate the interaction of whole cells and secreted soluble exopolimeric substances of Pseudomonas veronii 2E with Cd(II), Cu(II) and Zn(II).

Extracellular polymeric substances (EPS) are bacterial products associated to cell wall or secreted to the liquid media that form the framework of microbial mats. These EPS contain functional groups as carboxyl, amino, hydroxyl, phosphate and sulfhydryl, able to interact with cations. Thus, EPS may be considered natural detoxifying compounds of metal polluted waters and wastewaters. In this work Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) in combination with multivariate analysis (Principal Component Analysis-PCA-) were used to study the interaction of Cd(II), Cu(II) and Zn(II) and Pseudomonas veronii 2E cells, including bound EPS and cell wall, and its different soluble EPS fractions, previously characterized as Cd(II) ligands of moderate strength. Amino groups present in exopolysaccharide fraction were responsible for Zn(II) and Cu(II) complexation, while carboxylates chelated Cd(II). In lipopolysaccharide fraction, phosphoryl and carboxyl sites were involved in Cd(II) and Cu(II) binding, while Zn(II) interacted with amino groups. Similar results were obtained from cells. These studies confirmed that FTIR-PCA is a rapid analytical tool to provide valuable information regarding the functional groups in biomolecules related to metal interaction. Moreover, a discrimination and identification of functional groups present in both EPS and cells that interacted with Cd(II), Zn(II) and Cu(II) was demonstrated.

Bordetella bronchiseptica Glycosyltransferase Core Mutants Trigger Changes in Lipid A Structure.

Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-D-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2', O-acylated with C14:0(3-O-C10:0(3-OH) in C-3' and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4', both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane. Graphical Abstract.

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella.

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.

New Features in the Lipid A Structure of Brucella suis and Brucella abortus Lipopolysaccharide.

Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract ᅟ.

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated.

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive ß-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc5-8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. SIGNIFICANCE: A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp.

High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR.

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity.

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.

High molecular weight components containing N-linked oligosaccharides of ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.

Deletion of pilA, a Minor Pilin-Like Gene, from Xanthomonas citri subsp. citri Influences Bacterial Physiology and Pathogenesis.

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process.

A rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii.

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

Characterization of lipid A profiles from Shigella flexneri variant X lipopolysaccharide.

RATIONALE: In developing countries, Shigella flexneri (Sf) is the major causative agent of the endemic shigellosis (bacillary dysentery) responsible annually for one million fatalities mostly among infants. Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. Since lipid A is essential for the viability of the Gram-negative bacteria, it is subject to extensive chemical studies with new analytical techniques. METHODS: Lipid A was released by mild acid hydrolysis from the lipopolysaccharide which was obtained via the phenol/water extraction, purified and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and matrix-assisted laser desorption/ionization laser-induced dissociation tandem mass spectrometry (MALDI-LID-MS/MS). RESULTS: A detailed structural study of the whole lipid A obtained from S. flexneri variant X was carried out for the first time. Thus, we have shown that lipid A is a heterogeneous mixture having different numbers of acylated and phosphoethanolamine groups attached to the diglucosamine backbone. Furthermore, we found in the phenol phase an unusual hepta-acylated lipid A species, although the abundance was very low. CONCLUSIONS: MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.

A deep rough type structure in Bordetella bronchiseptica lipopolysaccharide modulates host immune responses.

The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains.

Structural analysis and involvement in plant innate immunity of Xanthomonas axonopodis pv. citri lipopolysaccharide.

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.

Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance.

We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.

Lack of galactose or galacturonic acid in Bradyrhizobium japonicum USDA 110 exopolysaccharide leads to different symbiotic responses in soybean.

Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.

Sialic acid residues are essential for the anaphylactic activity of murine IgG1 antibodies.

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to FcgammaRIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules.

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides.

Cyclic beta-1,2-glucans (CbetaG) are osmolyte homopolysaccharides with a cyclic beta-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CbetaG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked beta-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a beta-1,2-glucan phosphorylase present at the CbetaG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the beta-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this "length-controlling" phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots.

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant DeltaP22. This mutant has a deletion including the 3' region of exoP, exoT, and the 5' region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In DeltaP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in DeltaP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing DeltaP22 infectivity under N-starvation.