Alteration of macromolecular synthesis and membrane permeability by a Streptococcus sanguis bacteriocin.
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Bacterial Proteins↗
Biomedical subjects
H D Slade
Publications and source records attributed to H D Slade.
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.
Adsorption↗
Mechanism of the Adherence of Streptococcus mutans to Smooth Surfaces III. Purification and Properties of the Enzyme Complex Responsible for Adherence.
Enzymes which possess the ability to cause the adherence of Streptococcus mutans cells to a smooth glass surface were purified 1,100 times by chromatography on agarose gel followed by hydroxylapatite gel. During the purification procedures, the enzymes from strain HS6 (group a) were examined for the synthesis of water-soluble and water-insoluble polysaccharide and the ability to produce adherence. The enzyme preparations producing adherence of the S. mutans cells in the presence of sucrose possessed a molecular size of about 400,000 to 2,000,000 and were composed of approximately equivalent amounts of dextran and levan sucrases and 5 to 30% polysaccharide. The most highly purified preparation contained a negligible amount of contaminating protein as judged by polyacrylamide gel electrophoresis, immunoelectrophoresis, and gel diffusion. In these three tests, the location of the enzyme responsible for the synthesis of insoluble polymer was detected by embedding or covering the enzyme-containing gel with a layer of sucrose-containing agarose gel and observing the formation of insoluble polymer. During purification the ability of all fractions to produce adherence was parallel with the enzyme activity responsible for the synthesis of insoluble polysaccharide from sucrose. About two-thirds of the sucrase enzyme complex in the S. mutans culture fluid synthesized water-soluble polymer. This complex, obtained by filtration through agarose gel, was smaller in molecular size, lower in sugar content, and did not produce adherence, in contrast to the enzyme complex which possessed adherence activity. The inhibition of the enzyme complex synthesizing soluble polymer required more anti-synthetase serum than that required to inhibit the synthesis of water-insoluble polymer. It is not known whether the lack of adherence activity in this enzyme was due to its smaller size and lower sugar content or the absence of unknown factors which are essential for its activity. The carbohydrate in these enzyme preparations, composed of glucose, may represent a primer molecule and/or a remnant of the polymer synthesized by the enzyme. The enzyme activity was not inhibited by anti-dextran globulin.
Journal Article↗
Mechanism of adherence of Streptococcus mutans to smooth surfaces. II. Nature of the binding site and the adsorption of dextran-levan synthetase enzymes on the cell-wall surface of the streptococcus.
The mechanism of adsorption of the Streptococcus mutans enzymes responsible for the synthesis of insoluble dextran-levan to the S. mutans cell-wall binding sites has been studied. Certain characteristics of these binding sites are presented. The adsorption of these enzymes to the cell surface occurred rapidly without the addition of a source of energy and over a pH range of 3 to 11. The adsorption was inhibited by soluble dextran, probably due to the strong affinity of the polymer to the enzyme. All other polymers and sugars studied showed little or no inhibition. The adsorption was also inhibited by antibody globulin to the a-d immunologically specific group antigen surface polysaccharide of S. mutans and by anti-dextran globulin. The inhibition by anti-a-d globulin is considered to be due to a restriction of access of enzyme to the binding site of the enzyme which may be located in close proximity to the group antigen. On the other hand, anti-dextran globulin appeared to directly inhibit the adsorption by covering the binding site. Dextranase destroyed the binding site and released glucose from the S. mutans cells. These data indicate that S. mutans grown in media containing glucose possesses a small amount of dextran on the cell surface, and that this dextran is, or is a part of, the binding site for enzymes which synthesize the insoluble dextran-levan polymer. Trypsin inhibited the synthesis of insoluble polysaccharide and the adherence of cells. It is not clear in this case that destruction of the binding sites occurred. These data present a partial explanation of the processes which may be concerned in the formation of dental plaque on the smooth surfaces of teeth.
Adsorption↗
Structure and immunological specificity of the Streptococcus mutans group b cell wall antigen.
The Streptococcus mutans group b antigen of strain FA1 has been defined as to chemical composition and immunological specificity. The antigen in cold trichloroacetic acid extracts was fractionated on diethylaminoethyl-Sephadex A-25 at pH 8.5. Two forms were isolated: a polysaccharide and a mucoprotein. The two polymers reacted as a single substance in agar gel diffusion against specific adsorbed FA1 rabbit antisera but were separated by gel immunoelectrophoresis. No reaction with any other S. mutans or streptococcal group sera occurred. Galactose composed about one-third and galactosamine about 3% of the total weight of each polymer. Rhamnose was a major component of the polysaccharide (47%) but was present only in traces in the mucoprotein. The protein content of the latter was about 40%. No significant quantities of glycerol, phosphorus, or muramic acid were present in either case. Pepsin and trypsin had no effect on the serological specificity of the mucoprotein. d-Galactose and d-galactosamine were strong inhibitors (70%) of the precipitin reaction, whereas d-glucose, d-glucosamine, and N-acetyl-d-glucosamine inhibited between 25 and 35%. The results indicate that the antigen is a major antigenic component of the cell wall and that the specificity of the antigen resides in binding sites which contain both d-galactose and d-galactosamine. Agglutination of whole cells by specific group b antiserum indicates the antibody receptor sites of the polysaccharide antigen are at the surface of the streptococcal cell. The mucoprotein, but not the polysaccharide, was released from the cell by lysozyme. Lysis did not occur. The immunological specificity and other characteristics of the antigen establishes it as the identifying antigen of S. mutans group b.
Agglutination Tests↗
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.
Alkaline Phosphatase↗
Mechanism of adherence of Streptococcus mutans to smooth surfaces. I. Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccharide antigen in plaque formation.
The mechanism of adherence of Streptococcus mutans to smooth glass surfaces has been studied. The results with both viable and heat-killed cells showed that the process required (i) the synthesis of a water-insoluble dextran-levan polymer by cell-bound enzymes and (ii) the participation of a binding site on the surface of the S. mutans cell. Synthesis of the polymer from sucrose in the presence of the cells was required for adherence, and indicates that an "active" form of the polymer was required. Polymer synthesized by cell-free S. mutans enzymes when added to S. mutans cells did not produce adherence. Purified antibody globulin, specific for the a-d site in the polysaccharide S. mutans group a antigen, completely inhibited adherence. Antibody to the second antigen present in the polysaccharide molecule, the a antigen, did not inhibit adherence. The evidence indicates that adherence did not require an antigenic binding site which might be common to all S. mutans strains. The orientation of the synthetase enzyme(s), antigenic binding site, and dextran-levan polymer on the cell surface is under study.
Adsorption↗
Properties of a Streptococcus sanguis (group H) bacteriocin and its separation from the competence factor of transformation.
STH(1), a streptocin elaborated by group H streptococcus strain Challis, is lethal for group H streptococcus strain Wicky and is produced maximally during the exponential growth phase of liquid medium cultures. Crude streptocin preparations are resistant to oxidation and display a biphasic pH stability (stability being maximal at pH 5.0 and 10.0). Survivor studies indicate that streptocin-mediated killing is a "one-hit" phenomenon and proceeds rapidly. The streptocin has been purified 50-fold with (NH(4))(2)SO(4) fractionation and Sephadex G100 chromatography and appears to exist in equilibrium between two molecular weight forms. Low ionic strength and neutral pH buffers favor the isolation of the 110,000 molecular weight form, whereas high ionic strength and alkaline pH conditions facilitate isolation of the 28,000 to 30,000 molecular weight form. These findings suggest an association-dissociation relationship between macromolecules of 28,000 to 30,000 molecular weight. Purified STH(1) has no "competence factor" (CF) activity. In addition, CF has no STH(1) activity and displays no inhibitory effect on exponential-phase Wicky cultures as determined by absorbancy measurements. It appears, therefore, that initiation of the competent state for transformation in strain Wicky is not necessarily accompanied by gross alterations in cell growth.
Ammonium Sulfate↗
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.
Animals↗
Chemical composition and immunological specificity of the streptococcal group O cell wall polysaccharide antigen.
The group O streptococcal group antigen was shown to be a polysaccharide located in the cell wall of the organism. The antigen could be extracted by one of several methods: (i) 0.5 n NaOH at 37 C, (ii) phenol-water (50:50) at 68 C, (iii) 0.2 n HCl at 100 C, or (iv) 10% trichloroacetic acid at 4 C. The last method yielded more polysaccharide with less protein contamination. The polysaccharide was purified on diethylaminoethyl-Sephadex A-25 and Sephadex G-200. It was composed of two-thirds glucosamine and galactosamine, and the remainder glucose plus galactose. Rhamnose, glycerol, ribitol, and muramic acid were absent. Total phosphorus and amino acids were each less than 0.1%. N-Acetyl-beta-d-glucosamine exerted a strong inhibition of the precipitin reaction and is considered the immunodominant sugar. Glucosamine and glucose possessed a partial inhibitory activity. Galactose and galactosamine were essentially negative. No evidence of cross-reactivity was found between the O polysaccharide and group A and L polysaccharides, and group A and Staphylococcus aureus teichoic acids, which posesss N-acetylglucosamine specificity. The release of limited quantities of N-acetyl-glucosamine from its terminal location by enzyme, and glucose by acid hydrolysis, indicates a limited number of side chains in the O antigen. The glucosamine is in acid-stable linkage in the polysaccharide. Glucose was not released by beta-glucosidase and probably does not occupy a terminal position. The O antigen is the only known streptococcal polysaccharide antigen which does not contain rhamnose. The effect of these factors on the immunological specificity is discussed. O serum, after adsorption with the purified polysaccharide, was used to demonstrate the presence of protein antigens in acid extracts of cells from each of the nine strains examined. These antigens may represent type antigens. Two of these strains, originally described as group O, did not contain the O polysaccharide.
Antibodies, Bacterial↗
Immunological and chemical studies of the streptococcal group O protein type antigens.
Two antigens present in group O streptococci have been described and are designated as type antigens. No other antigens are present in extracts of group O cells in significant quantity. Antigen I is a cell wall protein, and II is a nucleoprotein of undetermined cell location. The immunological specificity of the latter resides only in the protein. Both antigens have been extracted from the cell with water or 0.2 n HCl (pH 2) at 100 C, and antigen I can be removed by trypsin. After purification, pH 2 was found to destroy the serological activity of each antigen. Antgens I and II and also the O polysaccharide antigen can be distinguished on a single agar-gel diffusion plate with group O whole-cell antiserum. N- and C-terminal amino acid analyses and total amino acid composition prove that I and II are separate molecular species. Each possesses properties which separate them from the streptococcal M, R, and T proteins. Antigen I separated into three fractions, each possessing serological specificity, on a diethylaminoethyl-Sephadex column at pH 8.6 during an increase in buffer molarity. The protein is considered to possess a multiple unit structure. The results indicate that the O streptococci are limited to two serological types. Group O whole-cell antisera, after adsorption with a strain not containing the O polysaccharide, can now be prepared for the positive identification of the types of group O streptococci.
Alkaline Phosphatase↗
Peptidoglycan composition and taxonomy of group D, E, and H streptococci, and Streptococcus mutans.
The qualitative and quantitative composition of the peptidoglycan from the cell wall of groups D, E, and H streptococci, and Streptococcus mutans, was determined. In group D, S. faecalis and the closely related species S. liquefaciens and S. zymogenes were separated from S. faecium and the closely related species S. durans on the basis of their peptidoglycan composition. A relationship among S. bovis, S. equinus, and some strains of S. mutans was indicated by the presence in each of a similar peptidoglycan containing threonine. Threonine was released from the S. mutans polymer as a threonine-lysine dipeptide. Hydrolysis of the dipeptide at 100 C for 24 hr in 6 n HC1 was required to break the peptide bond. Motile group D streptococci possessed a peptidoglycan of the same composition as S. faecium. Group E and H strains were also similar in the composition of their peptidoglycan. The results demonstrate that peptidoglycan composition can be used to (i) aid in the division of members of an immunological group into subgroups, and (ii) indicate a relationship between members of the same genus which are not related on an immunological basis.
Amino Acids↗
Determination of the rate of transformation from growth curves of transformed streptococci.
A rapid method of determining the rate of transformation of group H streptococci to streptomycin resistance has been developed. The new technique, which involves analysis of the growth response of transformed streptococci in liquid medium containing streptomycin, is independent of chain length fluctuations and is demonstrably more accurate than the standard plating method. The relatively short generation time of streptococci under these conditions permits transformation rates to be estimated in 9 to 14 hr depending on the number of transformants in the inocula as compared to 50 hr by the agar plate procedure.
Agar↗
Bacteriocin production by transformable group H streptococci.
Group H streptococci (strain Challis) which are competent for transformation release a bacteriocin into liquid medium which is bacteriocidal for another group H streptococcus (strain Wicky). The streptocin (STH(1)) is resistant to treatment with deoxyribonuclease and ribonuclease but is sensitive to trypsin, phospholipase C, and alkaline phosphatase. Such enzyme sensitivity experiments indicate that the bacteriocin may be a complex molecule (protein and lipid) containing phosphate groups essential for activity. STH(1), which is readily distinguishable from competence factor and bacteriophage activity, appears to have no role in the initiation of the competent state in strain Wicky. The presence of this factor in Challis culture supernatant fluids indicates that a reevaluation of earlier studies performed with the Challis-Wicky transformation system may be necessary.
Alkaline Phosphatase↗
Group a streptococcal polysaccharide antigens.
Cold trichloroacetic acid extracts of group A, type 3 streptococci were fractionated on Sephadex G50 and ECTEOLA cellulose columns. A glucose-N-acetylglucosamine polymer was isolated and shown by chemical, electrophoretic, and antigenic analysis to be distinct from the group A polysaccharide. The antigenic specificity was due to the glucosamine. A glycerol-phosphate-alanine-glucosamine teichoic acid polymer was isolated. The antigenic specificity was due to N-acetylglucosamine and d-alanine methyl ester, with the former possessing principal responsibility. The glucosamine and ester-linked alanine appear to be labile to the method of extraction and fractionation. Group A streptococcal whole cell antiserum, after adsorption with cells of a heterologous serological group and group A polysaccharide antigen, contained antibodies against both polymers.
Journal Article↗
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.
Journal Article↗
Binding of deoxyribonucleic acid by cell walls of transformable and nontransformable streptococci.
Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.
Alkalies↗
Factors affecting the adsorption of esterified streptococcal group A and E cell wall polysaccharide antigens to red blood cells and their effect on antibody measurement.
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Adsorption↗