The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Structural and genetic basis for the binding of a mouse monoclonal antibody to Flavobacterium psychrophilum lipopolysaccharide.

A mouse monoclonal antibody (mAb FL100A) previously prepared against Flavobacterium psychrophilum (Fp) CSF259-93 has now been examined for binding to lipopolysaccharides (LPS) of this strain and Fp 950106-1/1. The corresponding O-polysaccharides (O-PS) of these strains are formed by identical trisaccharide repeats composed of l-Rhamnose (l-Rha), 2-acetamido-2-deoxy-l-fucose (l-FucNAc) and 2-acetamido-4-R1-2,4-dideoxy-d-quinovose (d-Qui2NAc4NR1) where R1 represents a dihydroxyhexanamido moiety. The O-PS loci of these strains are also identical except for the gene (wzy1 or wzy2) that encodes the polysaccharide polymerase. Accordingly, adjacent O-PS repeats are joined through d-Qui2NAc4NR1 and l-Rha by wzy2-dependent α(1-2) linkages in Fp CSF259-93 versus wzy1-dependent ß(1-3) linkages in Fp 950106-1/1. mAb FL100A reacted strongly with Fp CSF259-93 O-PS and LPS but weakly or not at all with Fp 950106-1/1 LPS and O-PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5-times stronger to homologous Fp CSF259-93 LPS than to LPS from a strain with a different R-group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O-PS. Thus, Fp CSF259-93 O-PS formed a stable well-defined compact helix in which the R1 groups were displayed in a regular pattern on the helix exterior while unreactive Fp 950106-1/1 O-PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2-linked F. psychrophilum O-PS and LPS.

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-ß-d-Fucf-(1→3)-ß-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-ß-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

Antigenic and Structural Properties of the Lipopolysaccharide of the Uropathogenic Proteus mirabilis Dm55 Strain Classified to a New O85 Proteus Serogroup.

The aim of the study was the serological and structural characterization of the lipopolysaccharide (LPS) O antigen from P. mirabilis Dm55 coming from the urine of a patient from Lodz. The Dm55 LPS was recognized in ELISA only by the O54 antiserum, suggesting a serological distinction of the Dm55 O antigen from all the 84 Proteus LPS serotypes described. The obtained polyclonal rabbit serum against P. mirabilis Dm55 reacted in ELISA and Western blotting with a few LPSs (including O54), but the reactions were weaker than those observed in the homologous system. The LPS of P. mirabilis Dm55 was subjected to mild acid hydrolysis, and the obtained high-molecular-mass O polysaccharide was chemically studied using sugar and methylation analyses, mass spectrometry, and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The Dm55 O unit is a branched three-saccharide, and its linear fragment contains α-GalpNAc and ß-Galp, whereas α-GlcpNAc occupies a terminal position. The Dm55 OPS shares a disaccharide epitope with the Proteus O54 antigen. Due to the structural differences of the studied O antigen from the other described Proteus O polysaccharides, we propose to classify the P. mirabilis Dm55 strain to a new Proteus O85 serogroup.

Functional Activities of O-Polysaccharide and Hemolysin Coregulated Protein 1 Specific Antibodies Isolated from Melioidosis Patients.

Melioidosis is a fatal tropical disease caused by the environmental Gram-negative bacterium, Burkholderia pseudomallei. This bacterium is intrinsically resistant to several antibiotics and treatment of melioidosis requires prolonged antibiotic administration. To date, there are no vaccines available for melioidosis. Previous studies have shown that humoral immunity is critical for surviving melioidosis and that O-polysaccharide (OPS) and hemolysin coregulated protein 1 (Hcp1) are important protective antigens in animal models of melioidosis. Our previous studies revealed that melioidosis patients had high levels of OPS- and Hcp1-specific antibodies and that IgG against OPS (IgG-OPS) and Hcp1 (IgG-Hcp1) were associated with patient survival. In this study, we characterized the potential function(s) of IgG-OPS and IgG-Hcp1 from melioidosis patients. IgG-OPS and IgG-Hcp1 were purified from pooled serum obtained from melioidosis patients using immuno-affinity chromatography. Antibody-dependent cellular phagocytosis assays were performed with pooled serum from melioidosis patients and compared with serum obtained from healthy controls. Serum from melioidosis patients significantly enhanced B. pseudomallei uptake into the human monocytic cell line THP-1 compared with pooled serum from healthy donors. Enhanced opsonization was observed with IgG-OPS and IgG-Hcp1 in a dose-dependent manner. Antibody-dependent complement deposition assays were performed with IgG-OPS and IgG-Hcp1 using flow cytometry and showed that there was enhanced C3b deposition on the surface of B. pseudomallei treated with IgG-OPS but to a lesser degree with IgG-Hcp1. This study provides insight into the function of IgG-OPS and IgG-Hcp1 in human melioidosis and supports that OPS and Hcp1 are potential vaccine antigens for immunization against melioidosis.

Outer Membrane Vesicles Derived from Salmonella enterica Serotype Typhimurium Can Deliver Shigella flexneri 2a O-Polysaccharide Antigen To Prevent Shigella flexneri 2a Infection in Mice.

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has been developed yet. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella, due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively biosynthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in the serum, with similar trends in IgA levels from vaginal secretions and fluid from bronchopulmonary lavage, both intranasally and intraperitoneally. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay, opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. IMPORTANCEShigella, the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. In addition, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella, from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms.

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

Synthesis of the tetrasaccharide repeating unit of the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T using linear and one-pot iterative glycosylations.

A straightforward synthetic strategy was developed for the synthesis of the tetrasaccharide repeating unit corresponding to the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T in a very good yield adopting sequential glycosylation followed by removal of the p-methoxybenzyl (PMB) group in the same pot. Further, the synthetic strategy was modified by carrying out three stereoselective iterative glycosylations followed by in situ removal of the PMB group in one pot. The stereochemical outcome of the newly formed glycosidic linkages was excellent using thioglycoside derivatives as glycosyl donors and a combination of N-iodosuccinimide (NIS) and perchloric acid supported on silica (HClO4-SiO2) as the glycosyl activator.

Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera.

Lipopolysaccharides (LPSs) of Gram-negative bacteria comprise lipid A, core, and O-polysaccharide (OPS) components. Studies have demonstrated that LPSs isolated from the pathogenic species Burkholderia pseudomallei and Burkholderia mallei and from less-pathogenic species, such as Burkholderia thailandensis, are potent immune stimulators. The LPS structure of B. pseudomallei, the causative agent of melioidosis, is highly conserved in isolates from Thailand; however, the LPSs isolated from other, related species have not been characterized to enable understanding of their immune recognition and antigenicities. Here, we describe the structural and immunological characteristics of the LPSs isolated from eight Burkholderia species and compare those for B. pseudomallei to those for the other seven species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gas chromatography (GC), SDS-PAGE, Toll-like receptor 4 (TLR4) stimulation, and immunoblot analysis were performed on these Burkholderia species. MALDI-TOF profiles demonstrated that Burkholderia lipid A contains predominantly penta-acylated species modified with 4-amino-4-deoxy-arabinose residues at both terminal phosphate groups. The lipid A could be differentiated based on mass differences at m/z 1,511, 1,642, 1,773, and 1,926 and on fatty acid composition. LPSs of all species induced TLR4-dependent NF-κB responses; however, while SDS-PAGE analysis showed similar LPS ladder patterns for B. pseudomallei, B. thailandensis, and B. mallei, these patterns differed from those of other Burkholderia species. Interestingly, immunoblot analysis demonstrated that melioidosis patient sera cross-reacted with OPSs of other Burkholderia species. These findings can be used to better understand the characteristics of LPS in Burkholderia species, and they have implications for serological diagnostics based on the detection of antibodies to OPS.

Salmonella enterica Serovar Typhimurium Uses PbgA/YejM To Regulate Lipopolysaccharide Assembly during Bacteremia.

Salmonella enterica serovar Typhimurium (S Typhimurium) relies upon the inner membrane protein PbgA to enhance outer membrane (OM) integrity and promote virulence in mice. The PbgA transmembrane domain (residues 1 to 190) is essential for viability, while the periplasmic domain (residues 191 to 586) is dispensable. Residues within the basic region (residues 191 to 245) bind acidic phosphates on polar phospholipids, like for cardiolipins, and are necessary for salmonella OM integrity. S Typhimurium bacteria increase their OM cardiolipin concentrations during activation of the PhoPQ regulators. The mechanism involves PbgA's periplasmic globular region (residues 245 to 586), but the biological role of increasing cardiolipins on the surface is not understood. Nonsynonymous polymorphisms in three essential lipopolysaccharide (LPS) synthesis regulators, lapB (also known as yciM), ftsH, and lpxC, variably suppressed the defects in OM integrity, rifampin resistance, survival in macrophages, and systemic colonization of mice in the pbgAΔ191-586 mutant (in which the PbgA periplasmic domain from residues 191 to 586 is deleted). Compared to the OMs of the wild-type salmonellae, the OMs of the pbgA mutants had increased levels of lipid A-core molecules, cardiolipins, and phosphatidylethanolamines and decreased levels of specific phospholipids with cyclopropanated fatty acids. Complementation and substitution mutations in LapB and LpxC generally restored the phospholipid and LPS assembly defects for the pbgA mutants. During bacteremia, mice infected with the pbgA mutants survived and cleared the bacteria, while animals infected with wild-type salmonellae succumbed within 1 week. Remarkably, wild-type mice survived asymptomatically with pbgA-lpxC salmonellae in their livers and spleens for months, but Toll-like receptor 4-deficient animals succumbed to these infections within roughly 1 week. In summary, S Typhimurium uses PbgA to influence LPS assembly during stress in order to survive, adapt, and proliferate within the host environment.

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→ O-PS2 →2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. METHOD AND RESULTS: The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. CONCLUSIONS: Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development.

Heterologous expression of Shigella dysenteriae serotype 1 O-antigen analog in Escherichia coli K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis.

OBJECTIVE: To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster. RESULTS: The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached. CONCLUSIONS: Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

The O-antigen structure of bacterium Comamonas aquatica CJG.

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1H and 13C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

O-polysaccharide is important for Salmonella Pullorum survival in egg albumen, and virulence and colonization in chicken embryos.

The pathogen Salmonella Pullorum is the causative agent of persistent systemic infection of poultry, leading to economic losses in developing countries due to morbidity, mortality and reduction in egg production. These infections may result in vertical transmission to eggs or progeny. Limited information is available regarding the mechanisms involved in the survival of Salmonella Pullorum in egg albumen and developing chicken embryos. Hence, we investigated the role of O-polysaccharide in the contamination of eggs and the colonization of chicken embryos. Compared with the wild-type strain, the isogenic waaL mutant exhibited an O-antigen-deficient rough phenotype, and increased sensitivity to egg albumen and chicken serum, as well as reduced adherence to DF-1 cells. Infection with Salmonella Pullorum lacking O-polysaccharide resulted in significantly reduced embryo lethality and bacterial colonization. These results suggest that O-polysaccharide is essential for Salmonella Pullorum colonization in eggs, both post-lay and developing embryos. The chicken embryo infection model could be used to characterize the interaction between Salmonella Pullorum and developing embryos, and it will also contribute to the development of more rational vaccines to protect laying hens and embryos.

Partial structure and immunological properties of lipopolysaccharide from marine-derived Pseudomonas stutzeri KMM 226.

The partial structure and immunology of the lipopolysaccharide (LPS) of Pseudomonas stutzeri KMM 226, a bacterium isolated from a seawater sample collected at a depth of 2000 m, was characterised. The O-polysaccharide was built up of disaccharide repeating units constituted by L-Rhap and D-GlcpNAc: →2)-α-L-Rhap-(1→3)-α-D-GlcpNAc-(1→. The structural analysis of the lipid A showed a mixture of different species. The major species were hexa-acylated and penta-acylated lipids A, bearing the 12:0(3-OH) in amide linkage and 10:0(3-OH) in ester linkage, while the secondary fatty acids were present only as 12:0. The presence of 12:0(2-OH) was not detected. The immunology experiments demonstrated that P. stutzeri KMM 226 LPS displayed a low ability to induce TNF-α, IL-1ß, IL-6, IL-8 and IL-10 cytokine production and acted as an antagonist of hexa-acylated Escherichia coli LPS in human blood in vitro.

Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PglB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.

Influence of lipopolysaccharide outer-core in the intrinsic resistance to antimicrobial peptides and virulence in Edwardsiella ictaluri.

The genus Edwardsiella consists of bacteria with an intrinsic resistance to cyclic cationic antimicrobial peptides (CAMPs). Edwardsiella ictaluri, a pathogen of the catfish (Ictalurus punctatus) and the causative agent of a systemic infection, is highly resistant to CAMPs. Previously, we determined that the oligo-polysaccharide (O-PS) of the lipopolysaccharide (LPS) does not play a role in the E. ictaluri CAMP resistance and an intact core-lipid A structure is necessary for CAMPs resistance. Here, we evaluated the influence of the outer-core in the CAMPs resistance and fish virulence. E. ictaluri wabG, a gene that encodes for the UDP-glucuronic acid transferase that links the lipid A-inner-core to the outer-core-oligopolysaccharides, was deleted. Deletion of ΔwabG caused a pleiotropic effect, influencing LPS synthesis, CAMPs resistance, growth, and biofilm formation. E. ictaluri ΔwabG was attenuated in zebrafish indicating the important role of LPS during fish pathogenesis. Also, we evaluated the inflammatory effects of wabG LPS in catfish ligated loop model, showing a decreased inflammatory effect at the gut level respects to the E. ictaluri wild type. We conclude that E. ictaluri CAMPs resistance is related to the molecules present in the LPS outer-core and that fish gut inflammation triggered by E. ictaluri is LPS dependent, reinforcing the hypothesis that fish gut recognizes LPS in an O-PS dependent fashion.

Structural studies of the O polysaccharides from the lipopolysaccharides of Azospirillum thiophilum BV-ST and Azospirillum griseum L-25-5w-1T.

Diazotrophic bacteria of the genus Azospirillum are known widely, because they are ubiquitous in the rhizosphere and can promote the growth and performance of nonlegume plants. Recently, more Azospirillum species have been isolated from sources other than plants or soil. We report the structures of the O polysaccharides (OPSs) from the lipopolysaccharides of the type strains A. thiophilum BV-ST (1) and A. griseum L-25-5w-1T (2), isolated from aquatic environments. Both structures have a common tetrarhamnan in the repeating-unit, which is decorated with a side xylose in the OPS of A. thiophilum BV-ST.

New synthesis of oligosaccharides modelling the M epitope of the Brucella O-polysaccharide.

Brucellosis is a dangerous zoonotic disease caused by bacteria of the genus Brucella. Diagnosis of brucellosis is based on the detection in animal and human sera of antibodies to the O-polysaccharide of Brucella lipopolysaccharide. The currently employed serodiagnosis of brucellosis relies on the use of the Brucella O-polysaccharide as a diagnostic antigen. However, the existence of bacterial species, which also express O-polysaccharides structurally similar to that of Brucella, may decrease the specificity of the brucellosis detection due to false-positive test results. It has been shown that the efficiency of the test can be significantly improved by using synthetic oligosaccharides that correspond to the so-called M epitope of the Brucella O-antigen. This epitope is characterized by an α-(1→3)-linkage between d-perosamine units and is unique to Brucella. Here we report on an efficient approach to the synthesis of oligosaccharides that model the M epitope of the Brucella O-polysaccharide. The approach is based on the use of the α-(1→3)-linked disaccharide thioglycoside as the key donor block. Its application allowed the straightforward assembly of a set of four protected oligosaccharides, which includes a disaccharide, two trisaccharides, and a tetrasaccharide, in five glycosylation steps. The synthesized oligosaccharides are planned to be used in the development of diagnostic tools for identifying brucellosis in humans and domestic animals, as well as a potential vaccine against it.