Some biological properties of Pneumococcus type 37 and the chemistry of its capsular polysaccharide.

Pneumococcus Type 37, like pneumococcus Type 3, is characterized by the production of large mucoid colonies on the surface of solid media and by its very large capsule. It differs from the highly virulent pneumococcus Type 3 in that it is only slightly virulent for mice and rats and is isolated infrequently from man. A study of the behavior of pneumococcus Type 37 in systems comparable to those used in the study of pneumococcus Type 3 and an examination of the chemical structure of the capsule of pneumococcus Type 37 are described. The capsular polysaccharide of pneumococcus Type 37 is a viscous, optically inactive polymer composed of 95% hexose. Glucose is obtained in 88% yield upon acid hydrolysis. Periodate oxidation studies and the behavior of the polysaccharide on acid hydrolysis suggest that the molecule consists of a core of repeating units of 1,3 glucosyl-glucose to which short chains of glucose are attached at frequent intervals. Isolation of a disaccharide, the properties of which are identical with those of sophorose (beta1 --> 2 glucosyl-glucose), and of a trisaccharide are described. A tentative structure for the capsular polysaccharide of pneumococcus Type 37 is proposed.

Lipid composition of aminopterin-resistant and sensitive strains of Streptococcus pneumoniae. Effect of aminopterin inhibition.

The polar lipids of Streptococcus pneumoniae wild type and aminopterin-resistant strains were analysed. The membrane contained only two acid phospholipids, phosphatidylglycerol and cardiolipin, and a large amount of two glycolipids, glucosyldiglyceride and galactosylglucosyldiglyceride. The unsaturated acyl chains ranged from 58 to 87% of total fatty acids, depending on the strain and on growth conditions. No relation could be established between aminopterin resistance and polar lipid or fatty acid compositions. However, in the presence of bacteriostatic concentrations of aminopterin, the wild type and the resistant mutant did not have the same behavior. The resistant strain maintained its fatty acid composition and a normal [32P]phosphate distribution among phospholipids while the wild type shifted to a higher content in unsaturated fatty acids and to a high relative cardiolipin labelling. Such a differencein [32P] distribution was not observed when bacteriostatic concentrations of chloramphenicol were used, or when growth was stopped after amino acid deprivation induced by high concentrations of isoleucine. The biochemical basis of the aminopterin resistant character of the amiA mutants are not yet well understood but the present study establishes that the mutation confers a certain insensitivity of the lipid metabolism to aminopterin.

The specific capsular polysaccharide of Streptococcus pneumoniae type 15F.

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 15F (American type 15) is composed of D-galactose (3 parts), D-glucose (1 part), 2-acetamido-2-deoxy-D-glucose (1 part), phosphate (1 part) and O-acetyl (2 parts). Methylation, periodate oxidation, nitrous acid deamination, optimal rotation and nuclear magnetic resonance studies showed that the polysaccharide is a high mol. wt linear polymer of a pentasaccharide repeating unit having the structure.

Automatic analysis of neutral sugar components in glycoproteins and complex carbohydrates.

A simple and sensitive automated method has been developed to separate monosaccharides by reverse-phase chromatography using the amino acid analyzer column in the Li+ form with 90% alcohol as eluent. The column can be regenerated with LiBr and 90% ethanol to achieve highly reproducible resolution of sugar components. Glycoproteins and polysaccharides are hydrolyzed by a combination of methanesulfonic acid/Dowex 50 X 8 resin in water. Glucose and mannose are distinguished by treatment of the hydrolysate with glucose oxidase prior to analysis.

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 1.

The capsular polysaccharide from Streptococcus pneumoniae type 1 is composed of D-galactopyranosyluronic acid residues and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranosyl residues. The latter sugar, previously unknown in Nature, was not isolated but was identified from the products obtained on deamination of the polymer. Using n.m.r. spectroscopy, methylation analysis, and Smith degradation as the principal methods of structural investigation, it is concluded that the polysaccharide is composed of trisaccharide repeating-units having the structure: leads to 3)-alpha-Sugp-(1 leads to 4)-alpha-D-GalpA-(1 leads to 3)-alpha-D-GalpA-(1 leads to, in which Sug denotes the new sugar.

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 2, a reinvestigation.

The structure of the capsular polysaccharide from Streptococcus pneumoniae type 2 has been reinvestigated, specific degradations and n.m.r. spectroscopy being the main methods used. It is concluded that the polysaccharide is composed of hexasaccharide repeating-units having the following structure, which differs from that previously proposed. formula; see text)

Structural determination of the capsular polysaccharide of Streptococcus pneumoniae type 19A (57).

The structure of the Pneumococcus type 19A (57) capsular polysaccharide has been reinvestigated by using methylation analysis and n.m.r. spectroscopy. It is composed of residues of 2-acetamido-2-deoxy-D-mannose, D-glucose, L-rhamnose, and phosphate in the molar ratios of 1:1:1:1. The polysaccharide is linear, and is composed of these components in a repeating unit of the following structure. ---- 4)-beta-D-ManpNAc-(1 ---- 4)-alpha-D-Glcp-(1 ---- 3)-alpha-L- Rhap-(1-PO4-) ---- The type 19A polysaccharide (Na+ salt) was depolymerized by heating it in water at 100 degrees, conditions that also hydrolyzed the newly formed phosphoric monoesters.

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 7A.

Application of methylation analysis, specific degradations, and n.m.r. spectroscopy to the capsular polysaccharide elaborated by Streptococcus pneumoniae type 7A indicates a hexasaccharide repeating-unit with the structure (Formula; see text).

G.L.C.-M.S. of N-(1-deoxyalditol-1-yl)octadecylamine derivatives in the analysis of methanolysates of neoglycolipids obtained by reductive amination.

Hydrophobic conjugates of a series of aldoses have been prepared by reductive amination with octadecylamine and sodium cyanoborohydride, as model compounds for the analysis of reductively aminated oligosaccharides derived from capsular polysaccharides of Streptococcus pneumoniae. In the context of the methanolysis procedure for sugar analysis, g.l.c. and g.l.c.-m.s. (e.i.-mode) studies were carried out on the N-(1-deoxyalditol-1-yl)octadecylamine derivatives obtained after treatment with methanolic HCl, and subsequent N-acetylation and trimethylsilylation.

Application of high-resolution n.m.r. spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F.

The specific capsular polysaccharide of Streptococcus pneumoniae type 7F (American type 51) is a high-molecular-weight neutral polymer composed of 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, L-rhamnose, and 2-O-acetyl-L-rhamnose residues. N.m.r. spectroscopy (1H and 13C), in conjunction with composition and methylation analyses, and periodate oxidation data, showed the polysaccharide to be a branched polymer with a repeating heptasaccharide unit having the following structure. (formula; see text)

The structure of capsular polysaccharide of the Pneumococcus type II.

The pneumococcus type II capsular polysaccharide (SII) is composed of singly-branched hexasaccharide repeating units, for which three alternative structures have been proposed. The correct structure has now been determined by consecutive eliminations of the sugar residues in the side chain. The terminal D-glucuronic acid group was eliminated by treating the fully methylated and esterified SIIpolysaccharide first with base, and then with weak acid. The hydroxyl group at C-6 in the penultimate D-glucose residue released by this elimination was transformed into the 6-deoxy-6-tosyl derivative, and the residue thereafter eliminated by treatment with base. As the side-chains were eliminated by these reactions, it is considered that they contain only two sugar residues, which thus excludes two of the three alternative structures. Structure 1 was further confirmed by subjecting SII to a Smith degradation, which yielded the tetrasaccharide L-Rhap-(1 yields 3)-L-Rhap-(1 yields 3)-L-Rhap-(1 yields 2-erythritol, characterised by methylation analysis.

The structure of the repeating oligosaccharide unit of the pneumococcal capsular polysaccharide type 18C.

The structure of the repeating oligosaccharide of the pneumococcal capsular polysaccharide type 18C has been investigated. The repeating oligosaccharide, isolated from an aqueous hydrofluoric acid hydrolyzate of the polysaccharide, was shown, by fast atom bombardment-mass spectrometry, to have a molecular weight of 928, and to contain an O-acetyl group and a glycerol residue. Information about the sequence in the per-O-methylated oligosaccharide was derived from electron-impact mass spectrometry. Supporting data were obtained from methylation analysis, periodate and chromium trioxide oxidations, and enzymic and acid hydrolyses of the oligosaccharide. These studies indicated that the polysaccharide consists of the following pentasaccharide repeating unit. (formula see text)

[Immunochemical study of the type-specific polysaccharides of pneumococcus]. / Immunokhimicheskoe izuchenie tipospetsificheskikh polisakharidov penvmokokka.

Polysaccharide-containing preparations obtained from pneumococci, serovars 3, 6B, 9N, V and 23F, were studied by the methods of immunodiffusion and rocket immunoelectrophoresis (RIE). The tests were carried out with the use of rabbit antisera to formol vaccines prepared from serovars 3, 6, 9, 10, 12, 14, 18, and 23. The highly purified preparations of each of these serovars were shown to possess strict serological specificity. The results of RIE and chemical analysis permitted the selection of the preparations which served as reference antigens for 5 above-mentioned pneumococcal serovars. The calibrating curves plotted with the use of the reference antigens helped calculate the relative quantitative content of specific polysaccharide in preparations of different batches. The antigenic preparations were shown to differ in the content of specific polysaccharide and the degree of their purification. The authors recommend that double immunodiffusion and RIE should be used in combination in the study of the serological specificity of pneumococcal polysaccharide-containing preparations. The latter may be used as a rapid method for the standardization of the above-mentioned pneumococcal preparations and their mixtures. To achieve greater objectivity in the analysis of the mixtures, the use of sectional RIE, a variant of RIE proposed by the authors, is probably expedient. The present work also shows the possibility of using RIE for evaluating the level of the accumulation of specific polysaccharide during the cultivation of pneumococci in a liquid culture medium.

[Physicochemical and immunochemical characteristics of the polysaccharides of Streptococcus pneumoniae serotype 3]. / Fiziko-khimicheskaia i immunokhimicheskaia kharakteristika polisakharidov Streptococcus pneumoniae serologicheskogo tipa 3.

Capsular polysaccharides were isolated from Str. pneumoniae (serotype 3), grown on solid culture media, by the method of acetone sedimentation and subsequent deproteinization with phenol. The preparations thus obtained possessed serological type specificity, contained more than 40% of carbohydrates and insignificant amounts of protein and nucleic substances. The isolated polysaccharides contained a high-molecular fraction with the coefficient of distribution not exceeding 0.1, thus corresponding to the requirements for highly immunogenic preparations.

[The detection of streptococcal cell-wall proteins that form complexes with human macroglobulins]. / Vyiavlenie belkov kletochnoi stenki streptokokkov, obrazuiushchikh kompleksy s makroglobulinami cheloveka.

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.