Streptococcus suis serotype 3 and serotype 18 capsular polysaccharides contain di-N-acetyl-bacillosamine.

Streptococcus suis serotype 3 is counted among the S. suis serotypes causing clinical disease in pigs. Yet, limited information is available on this serotype. Here we determined for the first time the chemical composition and structure of serotype 3 capsular polysaccharide (CPS), a major bacterial virulence factor and the antigen at the origin of S. suis classification into serotypes. Chemical and spectroscopic data gave the repeating unit sequence for serotype 3: [4)D-GlcA (ß1-3)d-QuiNAc4NAc(ß1-]n. To the best of our knowledge, this is the first report of di-N-acetyl-d-bacillosamine (QuiNAc4NAc) containing polysaccharides in Streptococci and the second time this rare diamino sugar has been observed in a Gram-positive bacterial species since its initial report. This led to the identification of homologues of UDP-QuiNAc4NAc synthesis genes in S. suis serotype 18. Thus, the repeating unit sequence for serotype 18 is: [3)d-GalNAc(α1-3)[d-Glc (ß1-2)]d-GalA4OAc(ß1-3)d-GalNAc(α1-3)d-QuiNAc4NAc(α1-]n. A correlation between S. suis serotypes 3 and 18 CPS sequences and genes of these serotypes' cps loci encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit was tentatively established. Knowledge of CPS structure and composition will contribute to better dissect the role of this bacterial component in the pathogenesis of S. suis serotypes 3 and 18.

Ancillary Activity: Beyond Core Metabolism in Immune Cells.

Immune cell function and fate are intimately linked to engagement of metabolic pathways. The contribution of core metabolic pathways to immune cell bioenergetics has been vigorously investigated in recent years. However, precisely how other peripheral metabolic pathways support immune cells beyond energy generation is less well understood. Here we survey the literature and highlight recent advances in our understanding of several ancillary metabolic pathways and how they support processes beyond ATP production and ultimately contribute to protective immunity.

A novel cancer immunotherapy based on the combination of a synthetic carbohydrate-pulsed dendritic cell vaccine and glycoengineered cancer cells.

Immune tolerance to tumor-associated carbohydrate antigens (TACAs) has severely restricted the usefulness of most TACAs. To overcome this problem, we selected a sialylated trisaccharide TACA, GM3, as a target antigen, and tested a new immunotherapeutic strategy by combining metabolic bioengineering with dendritic cell (DC) vaccination. We engineered cancer cells to express an artificial structure, N-phenylacetyl-D-neuraminic acid, in place of the natural N-acetyl-D-neuraminic acid of GM3 by using N-phenylacetyl-D-mannosamine (ManNPhAc) as a biosynthetic precursor. Next, we selectively targeted the bioengineered cancer cells by vaccination with DCs pulsed with the GM3 N-phenylacetyl derivative. Vaccination with GM3NPhAc-KLH-loaded DCs elicited robust GM3NPhAc-specific T cell-dependent immunity. The results showed that this strategy could significantly inhibit FBL3 tumor growth and prolong the survival of tumor-bearing mice; B16F10 lung metastases could also be reduced. These findings lay out a new strategy for overcoming immune tolerance to TACAs, such as GM3, for the development of effective tumor immunotherapies.

Synthesis and preliminary biological evaluation of carba analogues from Neisseria meningitidis A capsular polysaccharide.

The Gram-negative encapsulated bacterium Neisseria meningitidis type A (MenA) is a major cause of meningitis in developing countries, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine against MenA is greatly hampered by the poor hydrolytic stability of its capsular polysaccharide, consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to get a carbocyclic analogue leads to the loss of the acetalic character of the phosphodiester and consequently to the enhancement of its chemical stability. Here we report the synthesis of oligomers (mono-, di- and trisaccharide) of carba-N-acetylmannosamine-1-O-phosphate as candidates for stabilized analogues of the corresponding fragments of MenA capsular polysaccharide. Each of the synthesized compounds contains a phosphodiester-linked aminopropyl spacer at its reducing end to allow for protein conjugation. The inhibition abilities of the synthetic molecules were investigated by a competitive ELISA assay, showing that only the carba-disaccharide is recognized by a polyclonal anti-MenA serum with an affinity similar to a native MenA oligosaccharide with average polymerization degree of 3.

Mannosamine-biotin as a novel masking agent for coating IgG for immune response silencing and augmentation of antibody-antigen interaction.

A variety of protein-coating procedures are used to modify proteins' properties. The principle coating agent used is PEGylation, in which proteins are coated by conjunction to polyethylene glycol (PEG). In the present study, we describe a novel approach that makes use of small molecules with multifunctional groups as the protein-coating agent. The new coating molecule was produced by reacting two endogenous molecules, mannosamine and biotin, to form mannose-biotin adducts (MBA). hIgG was coated with MBA at various MBA/protein ratios. The immunogenicity of MBA-coated hIgG was tested in chickens. A dose-responsive effect of MBA/hIgG ratio on immune response suppression was detected, with an optimal masking effect at a 12:1 ratio. The immune response to MBA-coated hIgG was about eightfold lower than that to PEG-coated hIgG. MBA also increased antibody-antigen-binding affinity, and decreased recognition of the Fc domain of MBA-coated hIgG by Fc receptor and secondary antibodies. While the PEG molecule consists of inert repeating units of ethylene oxide with no additional functional group to allow for potentially desirable modifications, the MBA has several functional groups, including vicinal hydroxyls, which can easily be converted to active residues such as aldehydes or carboxyls. This may be of importance for developing passive immunizations or for achieving tolerance of the immune response to an immunogenic molecule or virus. In summary, we developed a new protein-coating molecule with the ability to mask foreign antigens and in the case of antibodies, to enhance activity.

Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis.

The (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate meningococcal capsule of serogroup A Neisseria meningitidis is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene, ctrA, and galE, encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from ctrA by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to ctrA and that transcription of the ORFs was initiated from the intergenic region by a sigma-70-type promoter that overlapped the ctrA promoter. The first ORF exhibited 58% amino acid identity with the UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) 2-epimerase of Escherichia coli, which is responsible for the conversion of UDP-GlcNAc into UDP-N-acetyl-D-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A N. meningitidis-specific operon located between ctrA and galE.

Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide.

Brucella native haptens (NHs) extracted with hot water from smooth (S)-type B. abortus and B. melitensis were purified to high levels of serological activity and compared with the polysaccharide obtained by acid hydrolysis (PS) of the S lipopolysaccharide (S-LPS). By 13C nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of alpha-1,2- or alpha-1,2- plus alpha-1,3-linked 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine) previously reported for the LPS O chain. However, while PS contained up to 0.6% 3-deoxy-D-manno-2-octulosonate, this LPS-core marker was absent from NH. High performance liquid chromatography and thin-layer chromatography showed heterogeneity in NH purified from whole cells but not in PS. By immunoprecipitation, polysaccharides indistinguishable from NH were demonstrated in extracts obtained with phenol-water, saline at 60 degrees C, and ether-water treatments, and none of these treatments caused S-LPS hydrolysis detectable with antibodies to the O chain and lipid A. Two lines of evidence showed that NH was in the cell surface. First, NH became biotinylated when B. abortus live cells were labelled with biotin-hydrazide, and the examination of cell fractions and electron microscopy sections with streptavidin-peroxidase and streptavidin-coloidal gold, respectively, showed that labelling was extrinsic. Moreover, whereas only traces of NH were found in cytosols, the amount of NH was enriched in cell envelopes and in the outer membrane blebs spontaneously released by brucellae during growth. Interactions between NH and S-LPS were observed in crude cell extracts, and such interactions could be reconstituted by using purified NH and LPS. The results demonstrate that NH is not a hydrolytic product of S-LPS and suggest a model in which LPS-independent O-type polysaccharides (NH) are intertwined with the O chain in the outer membrane of S-type brucellae.

Carbohydrate-specific antibodies in normal human sera. II. Structural definition of antigenic determinants.

IgG and IgM antibodies against several sugars have been characterized in sera of normal donors by passive hemagglutination and a quantitative hemagglutination inhibition test. These antibodies distinguish between the equatorial and axial OH groups at C2, C3, or C4 positions of the glycopyranose configuration, differences between the anomers, linkage types, changes in the primary alcohol group at C6, and OH substitution. In the examples of antibodies to mannose, galactose, and glucose investigated, specificities were usually directed against the beta-anomers. In disaccharides, the antibodies appeared to react only with 1 of the 2 sugar subunits, but unlike monosaccharides, the glycosidic linkages also seemed to be a part of the reaction site. Thus, the reacting moiety in gentiobiose was beta-D-glucopyranosyl with 1----6 linkage, in cellobiose with beta-D-glucopyranosyl with 1----4 linkage, in meliboise was alpha-D-galactopyranosyl with 1----6 linkage, and in lactose the reaction was directed against beta-D-galactopyranosyl with 1----4 linkage. In the maltose-dependent hemagglutination, alpha-D-glucose appeared to be the main reaction site. ManNAc exemplified the specificity determined by OH group substitution. Antibody to D-Fucose represented example of specificity evolving from substitution of the primary alcohol.

Immunological activity of the peptidoglycan.

Studies on the immunology of peptidoglycan received impetus from the initial observation that the rabbit Group A-variant streptococcal antisera were a rich source of antibodies to peptidoglycan. Indeed, quantitative precipitin studies revealed concentrations as high as 7-10 mg/ml of antiserum. The amount of antibody after Group A-variant streptococcal immunization is much greater than the amount in the sera following immunization of rabbits with the Group A or C streptococci. Furthermore, earlier studies had shown that the purified peptidoglycan obtained as a residue following extraction of streptococcal cell walls with hot formamide was a poor antigen. Both the hexosamine polymer and the peptide moiety are antigenic. Use of the solid phase synthesized pentapeptide L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala and related similar peptides facilitated the determination of the fine structure of the immunodominant site of pentapeptide. The evidence points to the C-terminal D-Ala-D-Ala as the immunodominant determinant. Because of the similarity of the peptidoglycans of a number of different bacteria, it would be anticipated that they would cross-react immunologically, and this has been shown to be the case. The biological and medical significance of antibodies to peptidoglycan has yet to be determined. Certainly the exposure to this ubiquitous substance which occurs in all the indigenous bacteria of the respiratory and the gastrointestinal tract must mean that from an early age and through advancing years there is a constant stimulation of the immune response to peptidoglycan. Because the immunochemistry of these substances is now firmly established, there is a scientific basis for proceeding with the medical and biological implications of peptidoglycan immunity.

Penicillin immunogenicity in the presence of different pharmaceutical adjuvants.

Preliminary results in the variations of the penicillin immunogenicity in the presence of aminosugars, polysaccharides and terpenic substances are presented and the decreasings of the potential antigenicity of the penicillin molecule appeared in the presence of hexosamine molecules are estimated. It might be possible as a consequence of modifications in the early immunogenic interactions between antibiotic and serum proteins of the hipersensitive animals.