Native Polysaccharide Research Articles

Structural Elucidation of the O‐Antigen Lipopolysaccharide from two Strains of Plesiomonas Shigelloides that Share a Type‐Specific Antigen with Shigella Flexneri 6, and the Common Group 1 Antigen with Shigella Flexneri spp and Shigella Dysenteriae 1

Sugar and methylation analyses of native polysaccharides together with one-dimensional 1H- and 13C-NMR spectroscopy revealed that the two polysaccharides from strains 22074 and 12254 of Plesiomonas shigelloides are identical. The structure of the polysaccharide from strain 22074 was deduced from a uronic acid degradation and by NMR spectroscopy where heteronuclear multiple bond connectivity and two-dimensional nuclear Overhauser effect spectroscopy experiments established the pentasaccharide repeating unit as-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->.

Polysaccharides at interfaces 2. Surface potential of adsorbed cholesteryl-pullulan monolayers at the solution-air interface

The surface potential of adsorbed monolayers of cholesteryl-pullulan (CHP) derivatives has been determined by the ionizing differential electrode method. It has been found that this potential is highly dependent on the degree of cholesterol grafted onto pullulan, and that the native polysaccharide displays neither surface activity nor surface potential. As the disordered structure of the non-ionic polysaccharide unit generates a random orientation of intrinsic dipole moments, it has been considered that its contribution to the measured surface potential is rather small, compared to the cholesteryl group dipolar contribution. The surface densities of cholesteryl groups of adsorbed CHP molecules have been determined from the relationship between the surface potential and the surface density of spread cholesterol molecules. The assessment of these quantities was essential, as the determination of the surface tension data for the CHP derivatives with low cholesteryl content (CHP 45−0.6 and CHP 50−0.9) was difficult to achieve (Part I of this work [B. Deme´, V. Rosilio and A. Baszkin, Colloids Surfaces B: Biointerfaces, 4 (1995) 357]). These results complement those from the surface tension measurements, and confirm that in the surface layer of the adsorbed polysaccharide the ordered cholesteryl groups are oriented towards the air phase and the disordered polysaccharide is immersed in the aqueous subphase. Proposed models for semi-organized adsorbed CHP layers are discussed.

Isolation of β‐galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β‐Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear (Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β‐galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β‐galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo‐hexane‐trans‐1,2‐diamine tetraacetic acid‐, Na2CO3‐, guanidine thiocyanate‐ and KOH‐soluble fractions) and arabinogalactan (from larch wood). All the β‐galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH‐soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane‐trans‐1,2‐diamine tetraacetic acid‐, Na2CO3‐ and guanidine thiocyanate‐soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS‐PAGE.

Isolation of beta-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

Isolation of beta-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

Immunogenicity of meningococcal B polysaccharide conjugated to tetanus toxoid or CRM197 via adipic acid dihydrazide

Vaccine development against Group B Neisseria meningitidis is complicated by the nature of the capsular polysaccharide, which is α 2-8-linked poly-sialic acid, identical in structure to the poly-sialic acid found in many mammalian tissues during development. To test the feasibility of a vaccine based on this polysaccharide, we synthesized several conjugates of meningococcal B polysaccharide linked to a carrier protein (tetanus toxoid or diphtheria CRM197), via an adipic acid dihydrazide (ADH) spacer. All conjugates induced a strong immune response. However, most of the antibodies were not directed against the Meningococcus B polysaccharide and could not be inhibited by the purified polysaccharide alone. Further investigations showed that the antibodies recognized an epitope composed by the junction between the spacer and the polysaccharide and protein, that is not present in the native polysaccharide and is generated during the coupling reaction. This epitope becomes immunodominant with respect to the poorly immunogenic polysaccharide. While the majority of the immune response is directed against the above epitope, the conjugates induced also an immune response against the Meningococcus B polysaccharide. The anti-Meningococcus B antibodies elicited are of the IgM and IgG class and are inhibitable by the polysaccharide. Moreover, they are bactericidal, thus suggesting that they would induce protection against disease.

Structural properties of native and sonicated cinerean, a β-(1 → 3)(1 → 6)- d-glucan produced by Botrytis cinerea

Cinerean, the extracellular β-(1 → 3)(1 → 6)- d-glucan of the fungus Botrytis cinerea was studied. Electron micrographs of the native polysaccharide revealed quasi-endless fibrils with an estimated diameter of ca. 1.5 nm. A particle mass of 10 9–10 10 daltons was determined from dilute solutions by low-angle laser light scattering. Sonication of increasing duration led to fragmentation of the native polymer with an approximately exponential decrease of mass in the range of average molecular masses between 250 000 and 50 000 daltons. Shadowed by platinum, cinerean fibril fragments with a weightaverage molecular mass of 172 000 ± 3000 daltons could be characterized from electron micrographs as a distribution of rods of most probable length of 45 nm and an average length of 72 nm. Smallangle X-ray scattering confirmed the fibrillar structure of the native cinerean and the rodlike structure of sonicated cinerean. A rod diameter of 1.9±0.2 nm and a mass per unit length of 2250±490 daltons/nm were found. The latter is in agreement with the value of 1830 daltons/nm calculated from the length distribution determined from the electron micrographs. These data — especially the mass per unit length — suggest a quaternary structure for the polysaccharide. Such a structure would explain the rigidity of the rods which, in turn, is responsible for the characteristic phase separation behaviour in aqueous solutions observed by nephelometry and viscometry.

Pneumococcal conjugate vaccines: review and update.

Microbial Drug ResistanceVol. 1, No. 1 Pneumococcal Conjugate Vaccines: Review and UpdateDAVID L. KLEINDAVID L. KLEINSearch for more papers by this authorPublished Online:29 Jan 2009https://doi.org/10.1089/mdr.1995.1.49AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByPolyester as Antigen Carrier toward Particulate Vaccines24 May 2019 | Biomacromolecules, Vol. 20, No. 9Decrease in Hospitalizations for Pneumonia in Children under Five Years of Age in an Indian Reservation in Panama after the Introduction of the Heptavalent Pneumococcal Conjugate Vaccine (PCV7)International Journal of Pediatrics, Vol. 2013The viridans group streptococciReviews in Medical Microbiology, Vol. 21, No. 4Glycoconjugate vaccines and immune interference: A reviewVaccine, Vol. 28, No. 34Automation of the Anthrone Assay for Carbohydrate Concentration Determinations2 February 2010 | Analytical Chemistry, Vol. 82, No. 5PNEUMOCOCCAL INFECTIONSComparison of secular trends in pneumococcal serotypes causing invasive disease in Denver, Colorado (1971–2004) and serotype coverage by marketed pneumococcal vaccinesClinical Microbiology and Infection, Vol. 12, No. 11Serotypes of carriage and invasive isolates of Streptococcus pneumoniae in Brazilian children in the era of pneumococcal vaccinesClinical Microbiology and Infection, Vol. 12, No. 1Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic PneumolysinJournal of Clinical Microbiology, Vol. 44, No. 1Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSDAnalytical Biochemistry, Vol. 347, No. 2Heptavalent pneumococcal conjugate vaccine: current and future impact9 January 2014 | Expert Review of Vaccines, Vol. 2, No. 57. Vaccine4 December 2016 | Annals of Otology, Rhinology & Laryngology, Vol. 111, No. 3_supplThe Prevention of Pneumococcal Disease in ChildrenNew England Journal of Medicine, Vol. 345, No. 16Minimizing Potential Resistance: A Population Dynamics ViewClinical Infectious Diseases, Vol. 33, No. s3The Prevention of Pneumococcal Disease by Vaccines: Promises and ChallengesInfectious Disease Clinics of North America, Vol. 15, No. 1Role of communicable disease control measures in affecting the spread of resistant pneumococci: the Swedish modelClinical Microbiology and Infection, Vol. 5Carriage of resistant pneumococci by children in southern Israel and impact of conjugate vaccines on carriageClinical Microbiology and Infection, Vol. 5Treatment options for resistant pneumococcal infectionsClinical Microbiology and Infection, Vol. 5Development and testing of Streptococcus pneumoniae conjugate vaccinesClinical Microbiology and Infection, Vol. 5The epidemiology of pneumococcal infection in children in the developing worldPhilosophical Transactions of the Royal Society of London. Series B: Biological Sciences, Vol. 354, No. 1384The burden of pneumococcal disease: the role of conjugate vaccinesVaccine, Vol. 17, No. 13-14Vaccines Against Streptococcus pneumoniaeSerotyping and antibiotic resistance of Streptococcus pneumoniae isolated from pediatric infections in central GreeceClinical Microbiology and Infection, Vol. 4, No. 12Antimicrobial susceptibility of Streptococcus pneumoniae from children attending day-care lefts in a central Italian cityClinical Microbiology and Infection, Vol. 4, No. 11Emergence of Penicillin Resistance in Recurrent Pneumococcal Endocarditis in an HIV-Infected Patient CHRISTOPHER GONZAGA and LISA L. DEVER29 January 2009 | Microbial Drug Resistance, Vol. 4, No. 1Limiting the Spread of Penicillin-Resistant Streptococcus pneumoniae: Experiences from the South Swedish Pneumococcal Intervention Project KARL EKDAHL, HANS BERTIL HANSSON, SIGVARD MÖLSTAD, MARGARETA SÓDERSTRÖM, MATS WALDER, and KRISTINA PERSSON29 January 2009 | Microbial Drug Resistance, Vol. 4, No. 2Vaccination Against Middle-ear Bacterial and Viral PathogensAnnals of the New York Academy of Sciences, Vol. 830, No. 1 Immunologic DSafety and immunogenicity of tetravalent pneumococcal vaccines containing 6B, 14, 19F and 23F polysaccharides conjugated to either tetanus toxoid or diphtheria toxoid in young infants and their boosterability by native polysaccharide antigensThe Pediatric Infectious Disease Journal, Vol. 16, No. 11Reduction of pneumococcal nasopharyngeal carriage in early infancy after immunization with tetravalent pneumococcal vaccines conjugated to either tetanus toxoid or diphtheria toxoidThe Pediatric Infectious Disease Journal, Vol. 16, No. 11Safety and Immunogenicity of Three Doses of a Five-Valent Pneumococcal Conjugate Vaccine in Children Younger Than Two Years With and Without Human Immunodeficiency Virus Infection1 April 1997 | Pediatrics, Vol. 99, No. 4Effect of Antimicrobial Use and Other Risk Factors on Antimicrobial Resistance in Pneumococci KARL G. KRISTINSSON29 January 2009 | Microbial Drug Resistance, Vol. 3, No. 2Epidemiology of Pneumococcal Serotypes and Conjugate Vaccine Formulations JAY C. BUTLER29 January 2009 | Microbial Drug Resistance, Vol. 3, No. 2Combination VaccinesAntimicrobial Resistance of Invasive Streptococcus pneumoniae in Slovenia, 1993-19958 July 2009 | Scandinavian Journal of Infectious Diseases, Vol. 29, No. 3CURRENT PERSPECTIVES ON MULTIDRUG-RESISTANT BACTERIAInfectious Disease Clinics of North America, Vol. 10, No. 4Infections Due to Drug-Resistant Pneumococci, Enterococci, and Mycobacterium tuberculosisPediatric Annals, Vol. 25, No. 9Comparison of the safety and immunogenicity of a pneumococcal conjugate with a licensed polysaccharide vaccine in human immunodeficiency virus and non-human immunodeficiency virus-infected childrenThe Pediatric Infectious Disease Journal, Vol. 15, No. 3 Volume 1Issue 1Jan 1995 To cite this article:DAVID L. KLEIN.Pneumococcal Conjugate Vaccines: Review and Update.Microbial Drug Resistance.Jan 1995.49-58.http://doi.org/10.1089/mdr.1995.1.49Published in Volume: 1 Issue 1: January 29, 2009PDF download

Structure of the Type VI Group BStreptococcusCapsular Polysaccharide Determined by High Resolution NMR Spectroscopy

Abstract The structure of the capsular polysaccharide of the recently characterized type VI Group B Streptococcus (GBS) has been established using one- and two-dimensional homo- and heterocorrelated 1H and 13C NMR spectroscopy. The native polysaccharide contains D-glucose, D-galactose and sialic acid in the molar ratio 2:2:1. The structure of its repeating unit is:

Purification and properties of an exo-(1→3)-β- d-galactanase from Aspergillus niger

An exo-(1→3)-β- d-galactanase was purified by six chromatographic steps from a culture supernatant of Aspergillus niger. Its apparent molecular mass was 66 kDa, as estimated by SDS-PAGE analysis. The purified enzyme had no detectable activity on various p-nitrophenyl glycosides and on native plant polysaccharides but exhibited a high activity on a (1→3)-β- d-linked galactan backbone obtained after partial acid hydrolysis and two Smith degradations of gum arabic. The optimum conditions were pH 4.5 and 40–50°C. The enzyme had a Michaelis constant ( K m) of 1.9 mg/mL for the β-(1→3)- d-galactan with a maximum reaction velocity ( V max) of 1380 nkat/mg. The study of the reaction products obtained after enzyme treatment of two galactans derived from gum arabic through one or two Smith degradations showed that it was an exo-(1→3)-β- d-galactanase able to by-pass the branching points of galactan backbones and thus to release the side-chains of type II arabinogalactans in an undegraded form.

Structural investigation of the capsular polysaccharide of Escherichia coli O101 : K103 : H − using bacteriophage degradation and NMR spectroscopy

NMR spectroscopy was performed on the depyruvated capsular antigen of E. coli K103 and on the oligosaccharide obtained by depolymerisation of the native polysaccharide with a viral-borne endoglycanase. This capsular polysaccharide is the only one to be co-expressed with O group 101 and joins a small group of unusual capsular polysaccharides which possess pyruvic acid as the only acidic function. The primary structure was shown to be composed of the repeating unit: ▪

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.

Synthesis and two-dimensional nuclear magnetic resonance analysis of a tetra- and a hexa-saccharide fragment of the O-specific polysaccharide of Shigella dysenteriae type 1

The synthesis of the tetra- and hexa-saccharide methyl glycosides α- d-Gal p-(1 → 3)α- d-Glc pNAc-(1 → 3)-α- l-Rha p-(1 → 3)-α- l-Rha p-OMe ( 1), and α- l-Rha p-(1 → 3)-α- l-Rha p-(1 → 2)-α- d-Gal p-(1 → 3)-α- d-Glc pNAc-(1 → 3)-α- l-Rha p-(1 → 3)α- l-Rha p-OMe ( 3) is described, which represent various epitopes of the O-specific polysaccharide of Shigella dysenteriae type 1. The following monosaccharide intermediates were used: 1,3-di- O-acetyl-2- O-benzoyl-4- O-benzyl-α- l-rhamnopyranose ( 6α), methyl 2,4-di- O-benzyl-α- l-rhamnopyranoside ( 7), methyl 2,4-di- O-benzoyl-1-thio-α- l-rhamnopyranoside ( 8), 2,3,4-tri- O-benzoyl-α- l-rhamnopyranosyl bromide ( 9), methyl 3,4,6-tri- O-benzyl-2- O-(4-methoxybenzyl)-1-thio-β- d-galactopyranoside ( 13), methyl 2,3,4,6-tetra- O-benzyl-1-thio-β- d-galactopyranoside ( 16), and 2-azido-4,6- O-benzylidene-3- O-bromoacetyl-2-deoxy-β- d-glucopyranosyl chloride ( 19). A detailed analysis of the 1H and 13C NMR spectra of oligosaccharides 1 and 3 confirmed that the hexasaccharide 3 better approaches the conformation of the native polysaccharide, than either 1 or the homologous pentasaccharide 41.

Fractionation of Heparin, Dermatan Sulfate, and Chondroitin Sulfate by Sequential Precipitation: A Method to Purify a Single Glycosaminoglycan Species from a Mixture

Purified heparin, dermatan sulfate, and chondroitin sulfate in mixtures were fractionated by sequential precipitation with increasing volumes of acetone and analyzed by agarose-gel electrophoresis and for Mr, charge density, constituent disaccharides, and anticoagulant activity (for heparin). Purified glycosaminoglycans are generally utilized for pharmaceutical purposes and show physicochemical properties of glycosaminoglycans used as drugs. Heparin is the first glycosaminoglycan to precipitate at low percentages of acetone. The relative amount of slow moving and fast moving components, the Mr and charge density, and the disaccharide pattern of fractionated heparin depend on the percentage of solvent. The activated partial thromboplastin time activity of fractions composed of heparin decreases with the charge density and Mr. Dermatan sulfate is precipitated by acetone over a narrow range (0.6-0.7 vol, 37-41%), and one of these fractions is constituted by 100% of this polysaccharide. These species of dermatan sulfate have different percentages of constituent disaccharides compared to the native polysaccharide. Nonsulfated disaccharide and disaccharide-6-sulfate are enriched. The dermatan sulfate species precipitated by acetone are also enriched in disaccharide-4,6-disulfated. Chondroitin sulfate is the most soluble glycosaminoglycan in mixed acetone/water solvent. It begins to precipitate at 0.8 vol (44%) of acetone. Different species of chondroitin sulfate can be recovered by precipitation at different percentages of solvent, and they show a decrease in Mr and charge density depending on the percentage of acetone. The chondroitin sulfate species fractionated are also enriched in disulfated disaccharides compared to native polysaccharide. A different distribution of the three disulfated disaccharides can be pointed out in the fractionated chondroitin sulfate. Sequential precipitation performed by carefully increasing acetone percentages can help obtain purified species of glycosaminoglycan with desired properties from a mixture and tissue extracts, and achieve savings in time.

Structural Studies on a Cell Wall Polysaccharide Preparation of Lactococcus Lactis Subspecies Cremoris H414

Abstract A cell wall polysaccharide preparation of Lactococcus lactis subsp. cremoris H414 was isolated by nitrous acid extraction, and purified by gel filtration and anion-exchange chromatography. Oligosaccharide fragments, obtained by partial acid hydrolysis or alkaline hydrolysis, were studied by means of monosaccharide analysis, methylation analysis, FAB-MS, EI-MS, and 1H NMR spectroscopy. In addition 1H, 13C, and 31P NMR spectroscopy, including two-dimensional homo- and heteronuclear measurements, were performed on the native polysaccharide. Combination of the various results demonstrated that in the polysaccharide preparation two repeating units with the structures [→2)-α-l-Rhap-(1→3)-α-d-Galp-(1→]n and [→6)-[α-d-Galp-(1→4)]-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→1)-Ribitol-(4-phosphate→]m, respectively, can be recognized. So far, a covalent linkage between both polymers could not be confirmed nor be excluded.

Reaction of germinal centers in the T-cell-independent response to the bacterial polysaccharide alpha(1-->6)dextran.

Primary immunization of BALB/c mice with alpha(1-->6)dextran (DEX), a native bacterial polysaccharide, induces an unexpected pattern of splenic B-cell responses. After a peak of antibody-secreting B-cell response at day 4, deposition of dextran-anti-dextran immune complexes, as revealed by staining with both dextran and antibodies to dextran, occurs and persists in splenic follicles until at least the fourth week after immunization. Antigen-specific B cells appear and proliferate in such follicles, leading by day 11 to development of DEX-specific germinal centers as characterized by the presence of distinct regions of DEX+ peanut agglutinin-positive (PNA+) cells. At this time, fluorescence-activated cell sorter analysis also reveals the appearance of a distinct population of DEX+ PNA+ splenic B cells. In contrast, DEX+ PNA- cells, characterized by intense cytoplasmic staining, are present outside of splenic follicles, peak at day 4 to day 5, and persist until at least day 28. The frequency of these cells correlates with DEX-specific antibody-secreting cells, as detected by the ELISA-spot assay. Thus, in addition to the expected plasma cellular response, the typical T-cell-independent type II antigen, DEX, surprisingly also elicits the formation of antigen-specific germinal centers. These observations raise fundamental questions about the roles of germinal centers in T-cell-independent immune responses.

On the Specificity of a Bacteriophage-Borne Endoglycanase for the Native Capsular Polysaccharide Produced by Klebsiella pneumoniae SK1 and Its Derived Polymers

The specificity of the endoglycanase associated with the bacteriophage φSK1 particles was tested on the native capsular polysaccharide produced by Klebsiella pneumoniae serotype SK1 and on three chemically modified polymers derived from it. The primary structure of the SK1 capsular polysaccharide is: [formula] and the β1-3 linkage between the glucose and the galactose residues is the one cleaved by the phage enzyme. The enzyme activity was assayed on the deacetylated polysaccharide and on two derivatives obtained by removal of both the side-chain sugars and of only the α-D-galactosyl unit, respectively. The endoglycanase was more active on the deacetylated polysaccharide than on the native one, suggesting that the presence of the acetyl groups interferes with the enzyme-polysaccharide interaction. A possible role of the acetyl groups in the control of the polysaccharide chain length and hence on the rheological behaviour of the capsule cannot be ruled out, as already indicated for other bacterial polysaccharides. On the contrary, the removal of the side chains, either complete or selective, caused the modification of the recognition site in such a way that the enzymatic depolymerization no longer occurred. Therefore, it can be inferred that the φSK1 endoglycanase requires the presence of both the side chain sugars to exhibit its cleaving activity, although this latter is in the main chain.

Involvement of phospholipid end groups of group C Neisseria meningitidis and Haemophilus influenzae type b polysaccharides in association with isolated outer membranes and in immunoassays.

There are several bacterial polysaccharides (PSs) which contain a terminal lipid moiety. It has been postulated that these terminal lipid moieties anchor the PSs to the outer membrane of the bacteria. Our studies have shown that incubation of native PS from group C Neisseria meningitidis or Haemophilus influenzae type b with isolated outer membrane vesicles results in association of a portion of the PS with the vesicles. Removal of the terminal lipid from the PS by treatment with phospholipase A2 or phospholipase D eliminates this association. In other studies, it was shown that delipidated PSs are not suitable as solid-phase antigens in a currently used enzyme-linked immunosorbent assay (ELISA). Measurement of antibody units in the reference sera by using delipidated PSs as antigens in an ELISA yielded negligible absorbance compared with native PSs when methylated human serum albumin was used to coat the PSs to the plate. Nevertheless, phospholipase A2 and phospholipase D treatment did not noticeably affect antigenic epitopes, since soluble group C PS without the terminal lipid bound antibody as effectively as the native PS did, as measured by a competitive inhibition assay. Both hydrophobic and electrostatic interactions are important for the binding of group C N. meningitidis PS to the ELISA plate, while charge interactions seem to be sufficient for binding the more negatively charged H. influenzae type b PS.

Structure and physical properties of the extracellular polysaccharide PS-P4 produced by Sphingomonas paucimobilis P4 (DSM 6418)

A new strain, Sphingomonas paucimobilis P4 (DSM 6418), was found during a screening programme for exopolysaccharide-producing bacteria. The highly viscous fermentation broth yields a polysaccharide (up to 10 kg/m 3), named PS-P4, and shows thixotropic flow behaviour. In the presence of phosphate ions, PS-P4 forms aqueous gels after heating and cooling at alkaline pH. After isolation and purification of the exopolysaccharide, structural analysis by 1D and 2D 1H NMR spectroscopy and mass spectrometry was performed. The deacylated exopolysaccharide has the following repeating trisaccharide structure: → 4)-β- dGlc p-(1 → 4)-α- l-Rha p-(1 → 3)-β- d-Glc p(1 → Additionally, the presence of ester-bound acetic acid, d-glyceric acid, and ( R)-3-hydroxybutyric acid in the native polysaccharide was demonstrated.

Structural elucidation of the specific capsular polysaccharide of Rhodococcus equi serotype 7

Structural analysis of the specific capsular polysaccharide produced by Rhodococcus equi serotype 7 indicated it to be a high-molecular-weight polymer consisting of equal molar amounts of d-galactose, d-mannose, l-rhamnose, and pyruvic acid. By employing a combination of chemical and NMR techniques, it was established that the polysaccharide is composed of the linear repeating trisaccharide units, ▪ →3)-α- d-Gal p-(1→3)-α- d-Man p-(1→3)-α- l-Rhap p-(1→, in which the cyclic pyruvic acid acetal groups bridging the O-4 and O-6 positions of the α- d-Man p residues have the S-configuration. The 1H and 13C NMR spectra of the native and pyruvic acetal-free polysaccharides were fully assigned.

Immunogenicity of tetanus toxoid conjugates of anti-idiotypes that mimic Pseudomonas aeruginosa surface polysaccharides.

Pseudomonas aeruginosa continues to be a serious pathogen in humans, and there is no commercially available vaccine. We have previously developed monoclonal anti-idiotypic antibodies that mimic two surface polysaccharide epitopes of pseudomonas--the high-molecular-weight polysaccharide of the O side chain of immunotype 1 lipopolysaccharide and mucoid exopolysaccharide. We now show that conjugation of the anti-idiotypes to the carrier tetanus toxoid is not necessary to induce antipolysaccharide antibodies of equal or greater titer than the native polysaccharide antigen in mice.