Chemical structure and immunological specificity of the streptococcal group e cell wall polysaccharide antigen

The streptococcal group E cell wall polysaccharide antigen was extracted from strain K129 cells with hot trichloroacetic acid and purified. It contained rhamnose and glucose in a 2:1 molar ratio, 2% protein, 1% phosphorus, and was free of muramic acid and glycerol. No type polysaccharide antigen was present. The reaction of specific group E rabbit antiserum with the polysaccharide was effectively inhibited by d-glucose and beta-glucosides such as 1-methyl-beta-d-glucose, cellobiose, and gentiobiose. The 1-methyl-alpha-d-glucose was one-half as effective as the beta isomer. l-Rhamnose and N-acetyl-d-glucosamine were ineffective. Partial acid hydrolysis of the antigen followed by chromatographic separation of the oligosaccharides resulted in the isolation and analysis of five fractions. These fractions were di-, tri-, and tetrasaccharides. A study of these fractions by chemical analysis, reduction with borohydride, inhibition of the antigen-antibody reaction, release of glucose by beta-glucosidase, and other evidence indicate that beta-d-glucose is the immunodominant sugar in the antigen. A glucose-rhamnose trisaccharide (1:2 molar ratio) was the most effective inhibitor of the precipitin reaction; the glucose was readily released by beta-glucosidase, and one-half of the rhamnose was reduced with borohydride. This trisaccharide is considered to be a repeating unit in the native polysaccharide and probably has the following structure: O-beta-d-glucosyl-(1-2)-O-alpha-l-rhamnosyl- (1-4)-l-rhamnose. A glucose-rhamnose disaccharide in which the hexose and pentose are linked as in the trisaccharide was an effective inhibitor of the precipitin reaction. Strain K129 cells do not appear to contain a type polysaccharide antigen.     

Chemical composition and immunological specificity of cell wall polysaccharide group antigens of streptococcal groups P and U.

The group antigens of streptococcal groups P and U were extracted with formamide and purified on diethylaminoethyl-Sephadex A-25 and Sephadex G-200. The antigens were shown to be polysaccharides located in the cell walls of the organisms. In a precipitin test, the P and U group antigens did not cross-react with homologous sera of each other, nor with specific antiserum of the group antigen of group E streptococci. The polysaccharide comprising the group P antigen contained rhamnose, glucose, galactose, glucosamine, and galactosamine; the group U antigen was similar in composition but lacked galactosamine and contained more galactose.     

Immunochemistry of the streptococcal group R cell wall polysaccharide antigen

The group R streptococcal group antigen has been shown to be a polysaccharide located at the surface of the cell wall of the organism. The antigen was extracted from cell walls in 0.05 n HCl or 5% trichloracetic acid at 100 C, from whole cells at room temperature in 0.85% NaCl or 0.1 m acetate (pH 5.0), and by sonic oscillation. The antigen is largely destroyed when extracted from whole cells in 0.05 n HCl at 100 C. Acetate is recommended for routine extraction. The antigen extracted by sonic treatment was separated into six immunologically active fractions on diethylaminoethyl-Sephadex. The fractions were found to possess a common antigen which exhibited similar properties on immunodiffusion and immunoelectrophoresis. The purified antigen did not react with any other streptococcal group antisera. Adsorption of group R serum with the antigen removed all antibodies against whole cell antigen extracts of R cells. Chemical and enzymatic analysis of three fractions showed that the antigen was composed of d-glucose, d-galactose, rhamnose, and glucosamine. No significant quantities of phosphorus, glycerol, ribitol, or muramic acid were present. Significant inhibition of the quantitative precipitin determination by d-galactose and stachyose indicated that galactose in terminal alpha linkage was the immunodominant hexose in the antigen. d-Glucose and d-glucosamine possessed a partial inhibitory activity. N-acetyl-d-glucosamine and l-rhamnose did not produce significant inhibition. The results indicate that the R antigen is an immunologically specific structure which serves as a reliable means of identification of these streptococci as a serological group.     

Chemical composition and immunological characterization of polysaccharide antigen from Eubacterium saburreum T18.

Two types of polysaccharide were obtained from the oral microorganism Eubacterium saburreum T18 by formamide extraction and subsequent gel filtration and ion-exchange chromatography. One polysaccharide, which was composed of D-glycero-D-galacto-heptose, had antigenic activity in an immunoprecipitation reaction with rabbit anti-T18 serum due to immunoglobulin M antibodies. The second polysaccharide was composed of D-glycero-D-manno-heptose and L-rhamnose, but it did not have immunoprecipitation activity. These polysaccharide antigens were not alkali labile and differed from E. saburreum L44 and T27 antigens, which were composed of D-glycero-D-galacto-heptose.     

Purification and characterization of Streptococcus mutans group d cell wall polysaccharide antigen

The Streptococcus mutans group d antigen of strain B13 has been purified and characterized with respect to chemical composition and immunochemical properties. The antigen was extracted from lyophilized cells or cell walls by using 5% trichloroacetic acid at 5 C for 16 h. The antigen could also be extracted with water or 0.01 N HCl at 100 C for 20 min. The antigen was purified by ion-exchange and gel chromatography and was found to contain 96% carbohydrate, 1.6% protein, and 0.3% phosphorus. Characterization by gas chromatography indicated that the polysaccharide was composed of galactose and glucose in a 2:1 ratio. The antigen contained two serologically active sites: one site specific for group d and a second site common to both group d and group a strains. Agar diffusion and immunoelectrophoresis indicated that the two sites existed on a single molecule. The immunological specificity of the group d polysaccharide site depended on a terminal d-galactose. The purified B13 antigen did not react with antisera specific for the glycerol teichoic acid from streptococci. Anti-d serum rapidly agglutinated whole cells, indicating that the antibody receptor sites of the polysaccharide antigen were at the surface of the streptococcal cell.     

Extraction, purification, and chemical and immunological properties of the Streptococcus mutans group "a" polysaccharide cell wall antigen

An antigen of Streptococcus mutans has been extracted from HS6 (group "a") whole cells and repeatedly fractionated by Sephadex chromatography. The antigen is shown to be a polysaccharide and contains the S. mutans group "a" antigenic site and also a second antigenic site which is common to "a" strains and 2 of 3 group "d" strains. Immunological electrophoretic and chromatographic data indicate that the two sites exist in a single molecule. The polysaccharide has a molecular weight of 107,000 and is composed of glucose, galactose, glucosamine, and galactosamine. No significant quantities of lipid, phosphorus, glycerol, or ribitol are present. Immunological specificity of the group "a" polysaccharide site depends primarily on a d-glucose . d-glucose sequence, the "a-d" site on a terminal d-galactose. Water at 100 C and pepsin (pH 2.5) at room temperature are very effective in extracting the polysaccharide from lyophilized S. mutans cells. Trypsin and lysozyme are less effective. The antigen-antibody combining site appears to be located at the cell wall surface. A small quantity of enzyme-resistant protein (5%) is firmly linked to the antigen and is considered to be a remnant of a protein to which the polysaccharide is attached in the cell wall. The composition of the protein does not identify it as a part of the peptidoglycan. No reaction to the purified polysaccharide is obtained with antisera specific for teichoic acid glycerophosphate polymers from streptococci, staphylococci, or lactobacilli.     

Chemical and immunological properties of the type f polysaccharide antigen of Streptococcus mutans.

The type-specific cell wall polysaccharide antigen was extracted, purified, and characterized from type f Streptococcus mutans strain OMZ175 and MT557. The antigen was extracted from lyophilized cells with 5% trichloroacetic acid at 85 C for 15 min or saline at 120 C for 30 min. The trichloroacetic acid antigen was chromatographically separated into three antigenic fractions on a diethylaminoethyl-Sephadex A-25 column. Antigen 1 (Ag1P), which was specific for type f antiserum, was further purified by passing through carboxymethyl-Sephadex C-25 and Sephadex G-200 columns. It was a polysaccharide composed of 49% rhamnose and 47% glucose. No reaction was obtained with anti-polyglycerophosphate (PGP) serum. Antigen 2 was reactive with both type f and PGP antisera and contained significant amounts of protein and phosphorus. Antigen 3 was reactive only with PGP antiserum and had no type specificity. The polysaccharide antigen gave a single precipitin band against type-specific antiserum on immunodiffusion and immunoelectrophoresis. The presence of alpha-1,6-glucosidic linkages was indicated by a 90% inhibition of the precipitin reaction by isomaltose and alpha-methyl-D-glucopyranoside, adsorption to and release from a concanavalin A-Sepharose column, and reaction with an S. mutans (type e) glucan antiserum. This antiserum was used to show that the type f polysaccharide antigen did not contain free glucan. An analysis of the antigen released from the antigen-glucan antiserum complex showed the presence of rhamnose and glucose. This released antigen also reacted with an f antiserum, which did not react with commercial dextran. The results show that the type f polysaccharide antigen is the first of those S. mutans type-specific polysaccharides identified to be immunologically related to an S. mutans glucan.     

Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed.     

Monoclonal antibody to Streptococcus mutans type e cell wall polysaccharide antigen.

A monoclonal antibody against the polysaccharide antigen of Streptococcus mutans serotype e was prepared. It was found that beta-methyl-D-glucopyranoside and cellobiose markedly inhibited the precipitin reaction, whereas maltose showed no inhibition. The beta-glucosyl moiety of the type e polysaccharide seems to be the predominant antigenic determinant of the antigen.     

Sensitivity and specificity of rapid diagnostic tests for detection of group B streptococcal antigen in bacteremic neonates.

Latex particle agglutination (LPA) testing for antigen to group B streptococcus (GBS) has been useful in the diagnosis of GBS sepsis in newborns. However, recent reports have demonstrated that the sensitivity of LPA assays may be as low as 27 to 54%. The purposes of the present study were to directly compare the abilities of four urine antigen assays to detect GBS antigen with clinical urine samples from neonates with GBS bacteremia and to evaluate the effect of the urine concentration on the sensitivities and specificities of these assays. Urine samples were collected serially from neonates with blood cultures positive for GBS or on admission from healthy full-term infants. One milliliter of urine was removed, and the remainder was concentrated to a volume of 1 ml. Unconcentrated samples were serially diluted with normal saline and were assayed to determine the highest dilution which would produce a positive test result. The Wellcogen, Bactigen, and Directigen LPA tests and ICON immunoassay were directly compared by using concentrated and unconcentrated urine specimens and urine specimens with known titers. A total of 94 urine specimens, including 61 concentrated and 75 unconcentrated specimens, from bacteremic infants were available for sensitivity testing, and 220 urine specimens from uninfected infants were available for specificity testing. There were significant differences in sensitivity among the four assays when they were performed on concentrated urine specimens, as follows: Directigen, 98%; Bactigen, 92%; ICON, 89%; Wellcogen, 68%. When the assays were performed on unconcentrated urine specimens, the Directigen (84%) and Bactigen (76%) assays were each significantly more sensitive than the ICON (59%) or Wellcogen (43%) assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

A group a streptococcal polysaccharide stimulating nonspecific cytotoxic reactions in an autologous spleen cell system

N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. V. Prozorovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 2, pp. 167–169, February, 1990.     

Mast cell activation by group A streptococcal polysaccharide in the rat and its role in experimental arthritis.

Acute edematous responses were induced in Sprague-Dawley rats by the intravenous injection of group-specific polysaccharide (PS) isolated from group A streptococci. Thirty minutes after the intravenous injection of PS there was marked degranulation of subcutaneous and periarticular mast cells in all 4 feet, carbon particle labeling of adjacent venules, and an 8-fold increase in Evans blue dye content of the extremities. This acute reaction to PS was completely blocked by pretreatment with compound 48/80, but the polyarticular relapsing arthritis following the systemic injection of an arthropathic dose of streptococcal cell wall fragments containing large, covalently bound peptidoglycan-polysaccharide (PG-PS) was not blocked.     

Interactive effect of Gm and Km allotypes on cellular immune responses to streptococcal cell wall antigen

Serum samples from 121 unrelated, healthy Japanese individuals were typed for several Gm and Km(1) allotypes. Peripheral blood lymphocytes from these subjects were cultured with streptococcal cell wall (SCW) antigen and the incorporation of 3H-thymidine into T lymphoblasts was measured. Log-linear analysis showed a significant interactive effect of Gm1,17;13,16,21 and Km(1) on the cellular immune response to group A SCW antigen.     

Chemical composition and biological functions of Listeria monocytogenes cell wall preparations.

A crude Listeria cell wall fraction, a purified fraction (PF) with demonstrated biological activity, as well as a third fraction of base-hydrolyzed PF (BHPF) were analyzed for chemical composition and activities not previously described. Listeria cell wall fraction and PF contained significant quantities of lipid, whereas BHPF was lipid depleted. Fatty acid compositions were typical of gram-positive bacteria. PF and BHPF were depleted in protein. Alanine, glutamic acid, diaminopimelic acid, glucosamine, and muramic acid were found in all fractions, in enhanced concentration in PF and BHPF, and with molar ratios typical of bacterial peptidoglycans. Major neutral sugars were rhamnose, ribose, ribitol, and glucose. The concentrations of rhamnose, ribose, and glucose were increased in BHPF. Differences in chemical composition of the fractions reflected differences in their biological activities: Listeria cell wall fraction induced resistance to Listeria infection, whereas PF did not. Mitogenic and adjuvant activities were demonstrated for Listeria cell wall fraction and PF but were lost in BHPF. BHPF retained the ability to induce macrophage-mediated tumoricidal activity and decrease resistance to Listeria infection.     

Nonspecific and specific immunological mitogenicity by group A streptococcal pyrogenic exotoxins.

Group A streptococcal pyrogenic exotoxin (SPE) types A, B, and C induced lymphocyte proliferation both specifically and nonspecifically, and the responses showed characteristics associated with both types of stimulation. Guinea pig lymphocytes from animals presensitized to SPE A displayed immunologically specific proliferation in response to SPE A; control lymphocytes showed little activity in the presence of SPE A. Lymphocytes from guinea pigs not presensitized to SPE responded nonspecifically to SPE types B and C. Guinea pig lymphocytes from SPE A-presensitized animals showed enhanced proliferation over controls when treated with SPE B, suggesting that a degree of cross-reactivity between SPE types may exist, though they are serologically distinct. Mouse splenic lymphocytes exhibited low-level responsiveness to all SPE types, as would be expected for an antigen-specific proliferative response. Unlike mouse splenic lymphocytes, rabbit spleen cells and human cord blood, lymphocytes responded nonspecifically to all SPE types. Although rabbit spleen cells and human cord blood lymphocytes responded nonspecifically, the maximum response occurred at day 4 or 5, comparable to an antigen-specific system rather than a day 2 or 3 such as that with the nonspecific thymus-derived cell mitogen, concanavalin A.     

Guanine-plus-cytosine composition of five group B streptococcal serotypes.

The percent guanine-plus-cytosine content of deoxyribonucleic acid of each of the five serotypes of group B streptococci was determined by thermal denaturation. The range of guanine-plus-cytosine content was 35.1 to 36.9%, with a mean value of 35.9%. These values suggest a genetic homogeneity to the serotypes of the group B streptococci.