Immunochemical studies of type IV and two group-like (Z) carbohydrate antigens of minute streptococci

Analysis of purified fractions of formamide extracts of Z₄IV and 8650 (Z₅) bacteria gave as composition rhamnose, glucose, galactose and N-acetyl-glucosamine in molar ratio’s for Z₄ antigen 9:0:2:5, for type IV antigen 4:4:4:1 and for Z₅ antigen 8:2:2:3. In contradistinction with other polysaccharide type antigens of minute streptococci all type IV reactivity was recovered from the buffer eluate of a DEAE cellulose column. The Z₅ antigen was present in both the water and the buffer eluate. Precipitin and inhibition reactions indicate that the serological reactions between both strains are cross reactions based on the presence of galactose in the determinant groups of type IV, group Z₄ and group Z₅ antigens. Inhibition reactions also suggest a role of β-galactosyl-glucose as immunodominant group of the Z₅ determinant. Partial acid hydrolysis of type IV antigen yielded four oligosaccharides. Analyses and inhibition reactions show that probably both trisaccharides β-galactosyl-glucosyl-galactose and β-galactosyl-glucosyl-rhamnose are determinant groups of the type IV antigen.     

Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).     

Somatic Antigens of Streptococcus Group E: I. Comparison of Extraction Techniques1

Eleven Streptococcus group E strains, representing serotypes I, II, III, IV, V, and "untypable" isolates, were extracted by formamide, trichloroacetic acid, and hydrochloric acid under various conditions in an effort to determine the best method for recovering maximum amounts of group and type antigens. The group antigen was found to be relatively stable, and adequate amounts for identification purposes were recovered by a wide spectrum of conditions. Type-specific antigens were relatively labile, and were destroyed at low pH in acid hydrolysis or by prolonged heating in formamide hydrolysis. The best single procedure for recovering both type and group antigens from Streptococcus group E was formamide hydrolysis for 30 min at 180 C.     

Transformation of type polysaccharide antigen synthesis and hemolysin synthesis in streptococci

Transformation of the ability to synthesize type polysaccharide antigen and beta-hemolysin has been obtained in group F streptococci. Colonies possessing cells transformed to antigen synthesis were detected on the agar surface with fluorescein-labeled anti-type serum. This selection method, in contrast to those with antibiotics, allowed both transformed and nontransformed cells to grow, resulting in sectored colonies. These colonies could be subcultured to further establish the synthesis of antigen. Group F, group A, and group-like z deoxyribonucleic acid (DNA) labeled with type II antigen and hemolysin, and streptomycin resistance transferred each marker to a group F strain lacking a type antigen. DNA from group F and z3 strains labeled with type III antigen, and streptomycin resistance transferred both markers to group F and z3 strains lacking type antigen. A second F strain without type antigen was not transformed with any of these markers. A group H strain was transformed to streptomycin resistance only by the same types of DNA. Transformation to type II antigen synthesis always resulted in the formation of beta-hemolysin. All strains isolated from natural sources contained both markers. A mutant, obtained by nitrosoguanidine treatment of an FII(sr) strain, did not synthesize either the hemolysin or the antigen. This mutant still possessed the group antigen and streptomycin resistance. A close linkage of type II antigen and beta-hemolysin is indicated. The fluorescent-antibody staining of cells containing both group and type antigens showed a more intense ultraviolet adsorption for type than group antigen. A surface location (microcapsular) for the type antigen appeared likely. These results are of interest for studies on antigen biosynthesis, genetics, and classification of the streptococci.     

Immunodiffusion methods of studying the F-fraction antigens obtained from group A streptococci]

F fractions, obtained by the extraction of cultures of group A streptococci with distilled water at different pH, were studied by immunodifusion methods and subjected to chemical analysis. F fractions were shown to contain polyglcerophosphate, antigen E4 and in some cases group polysaccharide. Besides, F fractions were found to contain an antigen insensitive to trypsin and identical to one of the antigens of the thermostable fraction, as well as an antigen sensitive to the action of proteolytic enzymes and common to various types of group A streptococci. The antigen sensitive to the action of proteolytic enzymes were identical to one of the antigens showing no type specificity and contained in HC1 extracts prepared from group A streptococci. In grouping and typing group A streptococci the present of some F fraction antigens unrelated either to polysaccharide or to M substance should be taken into consideration. The antigens of F fraction have no protective properties.     

Serology and immunochemistry ofStreptococcus MG andStreptococcus salivarius

Precipitation and cross adsorption show thatStreptococcus NCTC 8037/50 contains the group antigen F of Lancefield and the type antigen III of Ottens. Sera prepared against this strain contain either anti-F and anti-III antibodies or anti-III antibodies only.The qualitative chemical analysis of the formamide extract ofStreptococcus NCTC 8037/50 and the inhibition of its quantitative precipitation reaction with simple sugars are the same as for a formamide extract of an F III strain.Nineteen strains, classified by Dr. de Moor (Utrecht) or by Dr. Seeleman (Hamburg) asStreptococcus MG, were serologically identical with either F III, L III, or 0 III (meaning zero III) streptococci. Some of these 0 III strains gave slimy colonies on saccharose agar according to Chapman, and on this property could be regarded asStreptococcus salivarius.About 70% of 42 freshly isolatedStr. salivarius strains were shown to contain type III antigen. With one of the strains without type III antigen (strain 51) an antiserum could be prepared which gave precipitation reactions with all non typableStr. salivarius strains and also with some type III positive strains. This was confirmed by cross adsorption tests.Qualitative chemical analyses of the hydrolysates of formamide extracts of F III and bothStreptococcus salivarius strains were compared with each other.The inhibition reaction of the quantitative precipitation of formamide extracts of F III,Str. salivarius N.C.T.C. 8606 andStr. salivarius 51 with the homologous and heterologous sera revealed a-glucosidic endgroup of the determinant groups of the III-andStr. salivarius 8606-antigen to be present. Slight differences in the inhibition pattern showed that these determinant groups were not identical. The determinant group ofStr. salivarius 51 is quite different; the-glucosidic linked sugars gave no inhibition at all. The best inhibitor was in this case rhamnose, although the inhibition was weak.     

Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide

Immunization of rabbits with group B type III streptococcus organisms induces two distinct populations of antibodies with a specificity for determinants on the native capsular polysaccharide antigen of these organisms. Some of the structural and conformational features of the two determinants responsible for the formation of these antibodies were elucidated by (13)C NMR and serological studies on the native type III polysaccharide and some of its structurally modified analogues. The specificity of the determinant corresponding to the major population of antibodies is dependent of the presence of sialic acid residues on the native type III antigen, and although these residues are not an integral part of the determinant, they exert conformational control over it. The carboxylate groups of the sialic acid residues are an important factor in this control mechanism which could possibly involve intramolecular hydrogen bonding. The terminal sialic acid residues control the orientation of the penultimate beta-d-galactopyranose residues with respect to the backbone of the native antigen. The orientation of these residues is critical to the determinant because the determinant is probably small and is located precisely at the junction of the same beta-d-galactopyranose residues with the backbone of the native type III antigen. The determinant corresponding to the other population of antibodies is not sialic acid dependent. This determinant is located on the backbone of the native antigen in the vicinity of the other determinant but on the opposite side to the oligosaccharide branches. In this position, its conformation is unaffected even by the removal of the oligosaccharide branches from the native antigen.     

Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus.

The formation of a nascent peptidoglycan-group-specific antigen of type III group B Streptococcus at the cell membrane level was demonstrated with an M-1 mutanolysin-prepared protoplast system. Protoplasts of group B streptococci in suitably stabilized medium (20% sucrose) readily incorporated [3H]acetate into cell surface macromolecules. Four major polysaccharides were isolated from the protoplast cultural supernatant fluid: the peptidoglycan group-specific antigen polymer, the group B-specific antigen, and the low-molecular-weight and high-molecular-weight forms of the type III polysaccharide antigen. Biosynthesis of all four polymers was not affected by the action of chloramphenicol, indicating protein synthesis was not required for the production of polysaccharide in this system. However, all but the low-molecular-weight type III antigen were inhibited by the action of bacitracin, suggesting that three of the polymers share a common synthesis-assembly site in the membrane. Attachment of the high-molecular-weight antigen to the nascent peptidoglycan-group B antigen complex did not occur in the protoplast system, suggesting that a more complex cell wall matrix may be necessary before linkage of the high-molecular-weight antigen takes place.     

Glycolipids from the cell walls of streptococci

A streptococcus, serologically defined as a Z₃III strain was compared with a mutant strain Z₃ lacking the type III polysaccharide antigen. The loss of type antigen represents a decrease in the carbohydrate content of the cell wall of the mutant and is accompanied by long-chain formation, increased sensitivity to streptomycin and agglutination in saline. Cell-wall preparations can be freed of membrane contamination by treatment with hot sodium dodecylsulfate (SDS). This resulted in a doubling of the ratio of muramic acid to lysine and in the disappearance of phospholipids. It could be shown that the membrane-free cell walls of these strains still contained appreciable amounts of glycolipids which could be identified as monoglucosyl glyceride and diglucosyl-diglyceride.     

Multiple polysaccharide antigens of group B streptococcus, type Ia: emphasis on a sialic acid type-specific polysaccharide.

Group B streptococci, type Ia (strain 090/14/4), were subjected to sequential extraction procedures and the various extracts were fractionated by a combination of DEAE-cellulose chromatography and gel-filtration. Three major antigens were isolated: the conventional group-specific and type-specific carbohydrates and an acidic polysaccharide consisting of galactose, glucose, glucosamine, and sialic acid. Immunochemical data suggest that the acidic antigen, with the exception of the immunodominant sialic acid, is structurally similar to the conventional pH 2.0 extracted type-specific antigen. Removal of the terminal sialic acid residues from the acidic antigen by mild acid hydrolysis resulted in a residual polymer which was chemically and immunologically analogous to the type-specific carbohydrate. Precipitin analysis focused attention to the cross-reactivity between thia acidic antigen and anti-types Ib, Ic, and III sera.     

Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants

Neonatal mice delivered from mothers preimmunized with heated or formalinized whole cell vaccines of type Ia, Ia/c and III/c group B streptococci were infected with each type of bacteria, and then serum antibodies of mothers and neonates who survived the experiments were measured by enzyme-linked immunosorbent assay. The relationship between the protectivity in neonate mice and the antibody titers to the type specific polysaccharide antigens and the protein c antigen of their sera were examined. In the Ia-immunized group which showed high protection against the type Ia infection, anti-Ia IgG antibody titers were low, and anti-protein c IgG antibody was not detected. Type Ia/c and III/c vaccines were highly effective against both type Ia/c and III/c infection, but less effective in type Ia infection. The protein c antigen was identified in both type strains by the double diffusion assay, and the IgG antibodies to the protein c were significantly high in sera of both maternal mice immunized with types Ia/c or III/c organisms and their newborn infants. High titers of the protein c IgG antibody retained 3 to 4 weeks after the last injection of vaccines which corresponded to the period of pregnancy and lactation. Small amounts of IgM antibody to all antigens were detected only in maternal sera. These results suggest that IgG antibodies to the protein c antigen and to the type-specific polysaccharide antigens are equally important protective factors which are transferable from preimmunized mothers to their newborn infants through placenta and/or lactation.     

Relationship between the virulence of a culture and the presence of common (non-type-specific) antigens in strains of group A streptococcus of different types]

In studying common (nontypespecific) antigens sensitivity to trypsin there was shown their wide distribution among the cultures of streptococcus, group A, belonging to different types and containing M-proteins. The antigen No. 1, identical to one of the antigens of the thermostable fraction was found in the cultures, irrespective of the degree of their virulence. The antigen No. 2 was characteristic of only virulent cultures obtained after the increase of the virulence and forming matt-form colonies. Both of the antigens were referred to the category of R-antigens. The presence of the nonspecific antigens in the hydrochloric extracts should be taken into consideration in typing streptococci of group A and determination of M-antigens.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.     

Beta haemolytic streptococci of the Lancefield groups E. P and U:Streptococcus infrequens

The physiological and biochemical characteristics of isolates from swine in Sweden and The Netherlands were compared with those of strains from several culture collections. These characteristics were found to be similar for all three Lancefield groups and they form a well defined pattern distinct from other known streptococcal species. It is suggested that these streptococci be classified together in one species:Streptococcus infrequens.Group and type sera of Group E streptococci have no affinity to Group P and Group U streptococci, and vice versa. None of the sera prepared against group E type strains contains the group antibody. Group P and Group U streptococci have an antigen in common. This common antigen is present in formamide extracts. It is not demonstrable in acid extracts. None of the group P sera tested contains the common antibody. Group P serum has to be considered as a type serum. Group U sera contain the common antibody, and when absorbed with group P cells prove to contain another type antibody, which reacts with extracts of most group U strains.Isolates of all three Lancefield groups were obtained from a variety of pathological conditions in swine.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

Somatic Antigens of Streptococcus Group E: II. Separation and a Partial Physicochemical Characterization1

Antigenic extracts of Streptococcus group E (SGE) were subjected to fractional ethanol precipitation, block (preparative) electrophoresis, and gel filtration for the purpose of separating the type antigen from the group antigen. Ethanol precipitation was ineffective in separating the substances. Block electrophoresis yielded serologically pure group antigen and a mixture of type and group antigen. Serologically pure type antigen was obtained by gel filtration. In some cases, pure group antigen was also recovered; in others, it was contaminated with type antigen. Gel filtration column effluents of antigenic extracts of SGE serotypes, I, II, III, IV, V and "untypable" isolates, collected from the region in which type antigen was eluted, were studied by paper chromatography and infrared spectrophotometry in an effort to develop a nonserological means of detecting type antigen. Hydrolysates of type antigens or suspect type antigens of serotypes I through V contained l-rhamnose, d-glucose, and several unidentified substances. d-Galactose also was present in hydrolysates of serotypes III and V. Untypable isolates and negative controls contained traces of d-glucose only. The data suggested that serotypes I through V contained a type antigen and that the untypable isolates were devoid of type antigen. Infrared absorbance spectra seemed to support the paper chromatography data.     

Isolation of non-type-specific protein antigens of Streptococcus group B

Hydrochloride extracts obtained from group B streptococcal strains of different serotypes have proved to be the source of type-nonspecific protein antigens, precipitated with ethanol and studied by gel chromatography and spectrophotometric scanning in ultraviolet rays. Thus, 2 or 3 antigens, one of them found to be common for streptococci of groups A, B and G, as well as the admixture of group-specific polysaccharide, have been detected. In extracts obtained from group B streptococcal strains of different serotypes a common protein antigen, specific only for group B, has been detected. The suitability of gel chromatography with the use Toyopearl gel HW-55F for the preparative isolation of the specific fraction of protein type-nonspecific antigen with a view to the subsequent study of immune response to group B streptococci has been shown.     

Developmental changes of blood group A-active glycosphingolipids with type 1 and type 2 chains in rat small intestine

Blood group A-active glycosphingolipids of the small intestine, A-6 and A-12, which have been characterized previously in the adult rat [Breimer ME, Hansson GC, Karlsson K-A, Leffler H (1982) J Biol Chem 257:906–12], were found to appear during postnatal development, using immunostaining on thin layer chromatograms with two monoclonal anti-A antibodies, A005 and A581. In this system, A005 was found to be specific for the A determinant based on the type 2 chain, while A581 reacted mainly with the A determinant based on the type 1 chain and only weakly with the A determinant based on the type 2 chain. A-6 Type 1 was detected first at 18 days after birth. Its concentration increased markedly during the fourth week. A-6 Type 2 was detected, at a very low level, in neonates. Its concentration increased between days 15 and 20 and then decreased almost to the neonate level by 28 days. Dodecaglycosylceramide A-12 followed the same pattern of reactivity as A-6 type 1 with A581, and remained strongly reactive with A005 after 20 days. Linear A-6 and branched A-12 appeared simultaneously. Antibodies directed against blood group H determinants based on the type 1 or type 2 chains did not detect any H structure which might have appeared as a precursor of either A-6 or A-12 at the early stages of postnatal development.Abbreviations A-6, A-12, H-5, H-10 etc the glycolipids are abbreviated by giving blood group activity, and number of sugars (see also Fig. 1) - G_M3 G_M3-ganglioside, H³NeuAc-LcCer - PBS phosphate-buffered saline