L-Rhamnose Is Often an Important Part of Immunodominant Epitope for Pneumococcal Serotype 23F Polysaccharide Antibodies in Human Sera Immunized with PPV23

Streptococcus pneumoniae is a major human pathogen which expresses more than 90 serologically distinct capsular polysaccharides (PS) on the surface. Since pneumococcal PSs elicit protective antibodies against pneumococcal diseases, it is important to identify the immunological epitope eliciting anti-pneumococcal PS antibodies. L-rhamnose is a part of the 23F PS repeating unit and is known to be a critical part of immunodominant epitope which elicits antibodies against pneumococcal serotype 23F PS. In order to determine if L-rhamnose is a part of epitope recognized by functional antibodies specific for serotype 23F PS in human serum samples, we evaluated the opsonophagocytic killing of serotype 23F pneumococci by serum antibodies specific for L-rhamnose. Using 10 mM L-rhamnose, opsonic capacities (opsonic indices) of serum antibodies were inhibited by 60% in 19 sera (36%) and 30–60% in 16 sera (30%) out of 53 sera from young and old adults immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). Interestingly, when IgM antibodies were depleted from immune sera in order to preferentially study IgG antibodies, the proportion of young adult sera showing more than 60% inhibition in opsonic capacity by 10 mM of L-rhamnose increased from 33% (11/31) to 68% (21/31). On the other hand, IgM depletion did not alter the proportion for old adult sera. Therefore, young and old adults may produce different antigen binding profiles of IgG antibodies against serotype 23F PS.     

A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the β-mannosamine glycosylation step

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343, 2545–2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl α-l-rhamnopyranoside acceptors 7 and 8. Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity (α/β = 3.4) leading to the protected trisaccharide α-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide α-15, a constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (α/β = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4″, the trisaccharide α-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers.     

Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-[alpha-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->. The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-[-alpha-Manp-(1-->2)]-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-[beta-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->.     

The complete structure of the core of the LPS from Plesiomonas shigelloides 302-73 and the identification of its O-antigen biological repeating unit

Plesiomonas shigelloides is a Gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal infections, which especially invades immunocompromised patients and neonates. The lipopolysaccharides are one of the major virulence determinants in Gram-negative bacteria and are structurally composed of three different domains: the lipid A, the core oligosaccharide and the O-antigen polysaccharide.In the last few years we elucidated the structures of the O-chain and the core oligosaccharide from the P. shigelloides strain 302-73. In this paper we now report the characterization of the linkage between the core and the O-chain. The LPS obtained after PCP extraction contained a small number of O-chain repeating units. The product obtained by hydrazinolysis was analysed by FTICR-ESIMS and suggested the presence of an additional Kdo in the core oligosaccharide. Furthermore, the LPS was hydrolysed under mild acid conditions and a fraction that contained one O-chain repeating unit linked to a Kdo residue was isolated and characterized by FTICR-ESIMS and NMR spectroscopy. Moreover, after an alkaline reductive hydrolysis, a disaccharide α-Kdo-(2→6)-GlcNol was isolated and characterized. The data obtained proved the presence of an α-Kdo in the outer core and allowed the identification of the O-antigen biological repeating unit as well as its linkage with the core oligosaccharide.     

Synthesis of the 6-deoxytalose-containing tri- and hexasaccharides of the O-antigen polysaccharide from Mesorhizobium huakuii IFO15243T

The synthesis of a trisaccharide and a hexasaccharide, the monomer and dimer of the repeating unit of O-antigen polysaccharide from Mesorhizobium huakuii IFO15243, has been accomplished through suitable protecting group manipulations and stereoselective glycosylation reactions starting from commercially available l-rhamnose. The target oligosaccharides in the form of their p-methoxyphenyl glycosides are suitable for further glycoconjugate formation via selective cleavage of this group.     

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel l-FucNAc and d-FucNAc synthons and immunochemical evaluation

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-β-d-ManNAcA-(1→4)-α-l-FucNAc(3OAc)-(1→3)-β-d-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile l- and d-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM₁₉₇. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide–CRM₁₉₇ conjugate.     

Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans

Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single beta(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions.     

Engineering Escherichia coli to produce Bordetella pertussis oligosaccharide with multiple trisaccharide units

The immunogenicity of the pertussis vaccine can be significantly improved by adding Bordetella pertussis oligosaccharide with multiple trisaccharide units. The more trisaccharide units there are, the better the efficiency of the immune response induction. However, natural B. pertussis oligosaccharides usually contain only a single terminal trisaccharide unit. In addition, B. pertussis is pathogenic, and there are potential safety hazards when preparing oligosaccharides from B. pertussis. In this study, Escherichia coli MG1655 was engineered to produce B. pertussis oligosaccharides containing multiple trisaccharide units. Fifty-nine genes relevant to the biosynthesis of the O-antigen and core oligosaccharide of lipopolysaccharide, enterobacterial common antigen, and colanic acid were deleted in MG1655, resulting in strain MDCO020. Then, 25 genes relevant to the biosynthesis of the oligosaccharide antigen in B. pertussis and 3 genes relevant to the repeating trisaccharide unit in Pseudomonas aeruginosa PAO1 were overexpressed in MDCO020, resulting in the recombinant E. coli MDCO020/pWpBpD5. The production of B. pertussis oligosaccharide with multiple trisaccharide units by MDCO020/pWpBpD5 was confirmed by SDS-PAGE and ¹H NMR analyses, and its immune response-stimulating activity was confirmed by using rabbit anti-pertussis serum.     

Structural elucidation of the O-chain of the lipopolysaccharide from Xanthomonas campestris strain 8004

A novel O-specific polysaccharide containing 3-acetamido-3-deoxy-alpha-D-fucose (Fuc3NAc) and D-rhamnose was isolated from the phenol-soluble lipopolysaccharide fraction of the plant associated bacterium Xanthomonas campestris strain 8004. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a branched trisaccharide repeating unit, as shown below: [formula: see text].     

Concise synthesis of the pentasaccharide O-antigen of Escherichia coli O83:K24:H31 present in the Colinfant vaccine

A block synthetic approach is presented for the synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O83:K24:H31 strain, present in the “Colifant” vaccine. The target pentasaccharide has been synthesized by coupling a disaccharide with a trisaccharide in excellent yield. Yields are quite satisfactory in all intermediate steps. A concise synthesis of the pentasaccharide repeating unit of the O-antigen of E. coli O83:K24:H31 strain, present in the COLINFANT vaccine is presented. The target pentasaccharide has been synthesized following a block synthetic strategy by coupling a disaccharide with a trisaccharide in excellent yield.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

Identification of the Immunodominant Regions of Staphylococcus aureus Fibronectin-Binding Protein A

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional ‘microbial surface components recognizing adhesive matrix molecules'' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F1_30-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F1_30-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B_110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B_110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F1_30-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.     

The structure of O-specific polysaccharide of Yersinia frederiksenii serotype O:16,29 lipopolysaccharide

A branched chain octose, 3,6-dideoxy-4-C-(L-glycero-1'-hydroxyethyl)-D-xylo- hexose, was isolated from the lipopolysaccharide of Yersinia frederiksenii, serovar O: 16.29 and identified as yersiniose A from Y. pseudotuberculosis, serovar VI. Mild hydrolysis of the lipopolysaccharide with acetic acid afforded a rhamnan. Structural features of the trisaccharide repeating unit were elucidated on the basic of 13C NMR spectral data, methylation studies and periodate oxidation. Using these data as well as data on sugar composition and methylation studies of the lipopolysaccharide, the following structural pattern of the repeating unit of O-specific polysaccharide was proposed: (formula; see text)     

Chemical structure of a polysaccharide from Campylobacter jejuni 176.83 (serotype O:41) containing only furanose sugars.

A neutral polysaccharide was obtained by hot phenol-water extraction of biomass from Campylobacter jejuni 176.83 and subsequently separated from acid-liberated core oligosaccharide of lipopolysaccharide by sequential GPC on Bio-Gel P6 and TSK-40 columns. All sugar components of the trisaccharide repeating unit of the polysaccharide were found to be of the furanose ring form. The major trisaccharide contained beta-L-arabinose, 6-deoxy-beta-D-altro-heptose (beta-D-6d-altHep) and 6-deoxy-beta-L-altrose (beta-L-6d-Alt), whereas in the minor trisaccharide the beta-L-6d-Alt is replaced by its C-5 epimer alpha-D-Fuc. On the basis of 1H and 13C NMR spectroscopic studies, including 2D ROESY, HMQC and HMQC-TOCSY experiments, the following structures of the repeating units were established: [formula: see text]     

Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide.

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 degrees C or 0.1 M NaOH-AcOH, pH 4.5, 5 h, 100 degrees C), the O-specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O-specific polysaccharide was shown to be the following trisaccharide: -->4)-alpha-Neup5Ac(2-->3)-L-alpha-FucpNAc(1-->3)-D-beta-Glc pNAc(1--> The polysaccharide O-chain was substituted with a single molar equivalent of O-acetyl group, distributed between the Neu5Ac O-9 and O-7 positions, in an approximate ratio of 7 : 3.     

The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13

An acidic exopolysaccharide was isolated from P. fluorescens strain H13. The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques. The repeating unit was characterized as a trisaccharide composed of -glucose, 2-acetamido-2-deoxy- -glucose and

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acid.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.     

Synthesis of the pentasaccharide repeating unit of the major O-antigen component from Pseudomonas syringae pv. ribicola NVPPB 1010

The synthesis of the repeating unit of the major O-antigen component from Pseudomonas syringae pv. ribicola NVPPB 1010 is reported. The strategy used was based on the successive coupling of a trisaccharide rhamnosyl trichloroacetimidate with a rhamnosyl acceptor with a free hydroxyl group on C-2. The pentasaccharide was then obtained by coupling with a N-Troc-tri-O-acetyl-glucosamine trichloroacetimidate. The synthesis allowed the oligomerisation of the repeating unit.     

Characterization of an enterobacterial common antigen (ECA) epitope recognized by anti-ECA-tetanus toxoid conjugate serum

The antigenic reactivity of both native and chemically modified enterobacterial common antigen (ECA) with anti-ECA-tetanus toxoid (TT) conjugate serum was investigated. The results obtained suggest that reduction of the carboxyl group of the mannosaminuronic acid component of ECA diminishes, but does not destroy its antigenic reactivity. Each of the sugar components were found to contribute to its reactivity with anti-ECA-TT conjugate serum, indicating that the trisaccharide repeating unit represents the ECA epitope. A nonasaccharide (trimer of the ECA repeating unit) inhibited antibody binding better than the hexasaccharide dimer, a finding which suggests that oligosaccharide conformation also makes a contribution to its inhibitory activity.     

Structure of the O-specific polysaccharide for Acinetobacter baumannii serogroup O1.

A polymeric fraction containing D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine was isolated from the lipopolysaccharide produced by the reference strain for Acinetobacter baumannii serogroup O1. By means of NMR spectroscopy, methylation analysis, and chemical degradation, the repeating unit of the polymer was identified as a branched trisaccharide of the following structure. [formula: see text].