A structural investigation of the capsular polysaccharide of Klebsiella K69

The structure of the capsular polysaccharide isolated from Klebsiella serotype K69 has been investigated by a combination of chemical and spectroscopic methods. The repeating structure of the deacetylated polysaccharide is shown to be of the "3 + 1 + 1" type, and it carries a 1-carboxyethylidene acetal at positions 4 and 6 of a terminal galactosyl group. The location of acetyl groups in the polysaccharide has not been established. The repeating unit of the deacetylated polysaccharide has the following structure. (Formula: see text).     

Immunologic properties of purified and complex preparations of group-B meningococcal polysaccharide

The complex preparations of group B meningococcal polysaccharide have been found to be capable of inducing primary immune response in mice, while purified group B polysaccharide has proved to be immunologically inert. As revealed in this investigation, the intravenous injection to mice of the optimum doses of the complex preparation of group B polysaccharide leads to the increased number of specific B-antibody-forming cells in their spleens and to a rise in B-antibody titers in their sera; besides, the time course of the process has been studied. Both preparations have been found capable of forming the immunological memory in mice if booster immunization is made with the complex preparation of group B polysaccharide. The immunological inertness of purified group B polysaccharide is attributed, supposedly, to the action of some specific suppressor mechanism. Considering the pronounced antigenic activity of the complex preparation of group B polysaccharide and the insignificant admixture of endotoxin in this preparation, the suitability of its future use as vaccine for the prophylaxis of meningitis caused by group B meningococcus is indicated and the tentative immunization schedules are discussed.     

Molecular origin of two polysaccharides of Campylobacter jejuni 81116

The nature of the polysaccharide molecules of the human enteric pathogen Campylobacter jejuni has been the subject of debate. Previously, C. jejuni 81116 was shown to contain two different polysaccharides, one acidic (polysaccharide A) and the other neutral (polysaccharide B), occurring in a 3 : 1 ratio, respectively. The aim of this study was to determine the molecular origin of these polysaccharides. Using a combination of centrifugation, gel permeation chromatography, chemical assays, and (1)H-NMR analysis, polysaccharide B was shown to be derived from lipopolysaccharide and polysaccharide A from capsular polysaccharide. Thus, C. jejuni 81116 produces both lipopolysaccharide-like molecules and capsular polysaccharide.     

The phosphate diester linkage of the peptidoglycan polysaccharide moieties of Micrococcus lysodeikticus cell wall

The external polysaccharide is a major component of Micrococcus lysodeikticus cell wall and displays distinct composition. The complete structure of the external polysaccharide had been elucidated as a basis for investigation of the cell wall structure-function relation. However, the mode of attachment of the polysaccharide to the peptidoglycan through a phosphodiester was not clear due to limitations in structural and biosynthetic studies. The present study describes purification of a lysozyme-resistant nondialyzable high-molecular-weight fragment of cell wall and identifies the sugar, D-glucose, as the point of external polysaccharide attachment to the peptidoglycan through a phosphate diester. Kinetic studies for the acid-catalyzed release of external polysaccharide from the peptidoglycan were performed in parallel with synthetic [methyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-alpha-D- glucopyranoside-6-yl]-alpha-D-glucopyranosyl phosphate and alpha-D-glucopyranosyl phosphate and showed the presence of a phosphodiester linkage between external polysaccharide and peptidoglycan. In addition, type of phosphate residue and cross-linking between muramic acid and protein part have been determined.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

Distribution of Enzymes Forming Polysaccharide from Sucrose and the Composition of Extracellular Polysaccharide Synthesized by Streptococcus mutans

The distribution of polysaccharide-forming activity from sucrose was investigated in cultures of three strains of Streptococcus mutans by using an assay which conveniently determines total polysaccharide. The enzymatic activity for polysaccharide formation from sucrose is almost exclusively extracellular. The ratio of the fructan to glucan in the polysaccharide differs among the three strains investigated. The enzymatic activity for the formation of polysaccharide from sucrose has been shown to be bound to the cell-free polymer itself.     

Rheological studies of specific cation forms of kappa carrageenan gels

Measurements have been made of the shear modulus of calcium, potassium and sodium kappa carrageenate gels as a function of polysaccharide concentration and temperature. Under the experimental conditions used the efficiency of the cations in gelling the polysaccharide has been found to be Ca²⁺>K⁺>Na⁺. The relative gelling efficiencies of the cations is attributed to their extent of hydration which controls the solubility of the salt form of the polysaccharide. Gelation is attributed to ‘microcrystallite’ formation at localised sites on adjacent polysaccharide chains. The sharp decrease of the shear modulus on heating is attributed to localised melting of these ordered regions.     

Oxidation of the capsular polysaccharide of pneumoccal type IX by periodate

1. The pneumococcal type IX polysaccharide (polysaccharide S IX) has been oxidized by sodium metaperiodate and reduced by sodium borohydride. Of the constituent monosaccharides, N-acetylglucosamine and N-acetylmannosamine remain unaltered, whereas 40% of the glucose and 90% of the glucuronic acid are oxidized. 2. The effect of oxidation and subsequent reduction on the precipitation of polysaccharide S IX in anti-(pneumococcal) sera is described and interpreted in structural terms. 3. Oligosaccharides produced by oxidation, reduction and hydrolysis with dilute acid have been isolated and partially characterized. 4. The results in this paper and the preceding one (Higginbotham et al., 1972) are used to postulate a possible structure for polysaccharide S IX.     

Acetylated methylmannose polysaccharide of Streptomyces.

A polysaccharide composed of 3-O-methyl-D-mannose and D-mannose in a molar ratio of approximately 10:1 and containing 3 to 4 esterified acetyl residues has been isolated from Streptomyces griseus. This acetylated methylmannose polysaccharide (AMMP) is similar to the methylmannose polysaccharide (MMP) of Mycobacterium smegmatis (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) in its size and composition, the absence of acidic or basic groups, and the lack of a reducing end. It is different, however, in its content of esterified acetyl residues, and it is slightly different in its structure and in its gel filtration properties. The structure of AMMP has been established by proton magnetic resonance spectroscopy, and by combinations of methylation analysis and Smith degradation utilizing non-radioactively labeled polysaccharide and [3H]methyl-labeled polysaccharide obtained from cells grown in the presence of L-[methyl-3H]methionine. It is concluded that AMMP is a linear, nonreducing, neutral polysaccharide composed of a terminal D-mannose residue linked alpha(1 leads to 4) to a chain of 10 consecutive alpha(1 leads to 4)-linked 3-O-methyl-D-mannose residues. The reducing terminal 3-O-methyl-D-mannose residue exists, at least in part, as its alpha-methyl glycoside. The positions of attachment of the ester residues have not been established.     

The structure of the O-specific polysaccharide chain of the lipopolysaccharide of Yersinia pseudotuberculosis serovar VII

An O-specific polysaccharide of Yersinia pseudotuberculosis serovar VII has been isolated and characterized. The polysaccharide consists of colitose, D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratio 1 : 2 : 2. From the results of methylation analysis, partial acid hydrolysis, 1H and 13C NMR spectroscopy the structure of the repeating unit of the O-specific polysaccharide is deduced as follows:     

Structural studies of the capsular polysaccharide of Klebsiella type 52

The structure of the capsular polysaccharide from Klebsiella Type 52 has been investigated. Methylation analysis, characterization by gas-liquid chromatography-mass spectrometry of oligosaccharide derivatives obtained on partial hydrolysis of the methylated polysaccharide with acid, and specific degradation of the methylated polysaccharide by successive treatments with base and acid followed by characterization of the product, were the principal methods used. The polysaccharide is composed of hexasaccharide repeating-units containing D-glucuronic acid, D-galactose, and L-rhamnose, in the ratios 1:3:2. A structure for these units, disregarding the anomeric natures of the sugar residues, is proposed.     

Mucilage from a fresh-water red alga of the genus Batrachospermum

The gelatinous polysaccharides of a Batrachospermum species have been extracted from the alga. The major polysaccharide is acidic and has been separated from neutral polysaccharides by chromatography on DEAE-cellulose. The constituent sugars of the acidic polysaccharide include d- and l-galactose, d-mannose, d-xylose, l-rhamnose, d-glucuronic acid, and two O-methyl sugars, which have been characterized as 3-O-methyl-l-rhamnose (l-acofriose and 3-O-methyl-d-galactose. Partial acid hydrolysis of this polysaccharide has given a complex mixture of neutral and acidic oligosaccharides. The two preponderant acidic oligosaccharides contained galactose and glucuronic acid in 1:1 ratio, suggesting the presence of a repeating sequence of these two residues as a major structural feature of the polysaccharide.     

Properties of the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei have been separated by mild conditions of acid hydrolysis. 2. Removal of the polysaccharide renders the mucopeptide susceptible to lysozyme. 3. The mucopeptide and polysaccharide components have been analysed and the results compared with those obtained previously. 4. The polysaccharides responsible for group specificity have a terminal reducing N-acetylgalactosamine residue substituted on C((3)) by the adjacent sugar; estimation of this component gave an indication of the molecular weight of the polysaccharides. 5. Evidence has been obtained for the presence of rhamnosyl-(1-->3)-N-acetylgalactosamine among the products of acid hydrolysis of the group B polysaccharide.     

Development and validation of an NMR-based identity assay for bacterial polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.     

Structural studies of the capsular polysaccharide from streptococcus pneumoniae type 37

The structure of the capsular polysaccharide elaborated by Streptococcus pneumonia type 37 has been investigated; methylation analysis, Smith degradation, and n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of disaccharide repeating-units having the following structure.

This comb-like structure is very crowded, which influences the n.m.r. spectra of the polysaccharide.     

病原细菌多糖疫苗和多糖结合疫苗研究进展

在许多病原细菌中,荚膜多糖和O抗原多糖能够刺激机体产生保护性抗体,因此利用病原细菌的多糖制成的疫苗能有效预防传染病,同时避免了病原细菌耐药性的出现。此类疫苗包括多糖疫苗和多糖结合疫苗。细菌体内糖基化的发现,使得利用生物法生产多糖结合疫苗成了多糖结合疫苗生产的热门方向。我们简要综述了多糖疫苗和多糖结合疫苗在研究和应用方面的主要进展。     

The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei

1. The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei is rapidly hydrolysed under mild acid-hydrolysis conditions. 2. The release of the polysaccharide is accompanied by the hydrolysis of an N-acetylhexosaminide linkage. The N-acetylhexosamine residue readily forms chromogen and it is concluded that it is substituted on C₍₃₎ by the adjacent sugar. 3. Continued heating of the polysaccharide in acid results in a slower release of reactive N-acetylhexosamine due to the hydrolysis of glycosidic linkages within the polysaccharide. 4. After the linkage between the polysaccharide and mucopeptide has been hydrolysed, acid phosphatase will release approx. 40% of the total phosphorus as inorganic phosphate. 5. It is concluded that the polysaccharide component of the cell wall is joined through its reducing end group to a phosphate grouping in the mucopeptide.     

Isolation and characterization of a novel polysaccharide fromBacillus licheniformis NCIB 11634

Summary A polysaccharide producing strain ofBacillus licheniformis was isolated from exudate of raffia palm,Raffia vinifera. The optimum conditions for growth and polysaccharide production have been investigated and established. No appreciable polysaccharide was formed on glucose. It grew best in Czapek-Dox media with sucrose as the carbon source. The polysaccharide has been characterized as a heteropolymer containingd-glucose,d-mannose andd-xylose.     

Structure and conformation of a novel genetically engineered polysaccharide P2

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.