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The role of D-alanine in the serological specificity of group A streptococcal glycerol teichoic acid

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... The application of immunoelectrophoresis to extracts of group A streptococci showed that in addition to polyglycerophosphate, a slower-moving component (E4) was present, and the fusing of the lines indicated a serological relationship (300). Subsequent work (178) showed that component E4 contained D-alanine residues, thus accounting for its lower mobility, whereas the crossreaction depended on the common glycerol phosphate backbone. Immunoelectrophoresis has also been used to separate teichoic acid, lipoteichoic acid, and lipoteichoic acid-protein complexes from lactobacilli and to show by appropriate absorptions that only antibodies to the teichoic acid component can be detected (294). ...
... Whether this generalization can be extended to teichoic acids will depend on whether more knowledge on the shapes of the polymers is obtained, but it might be expected that the bulky 2-acetamido grouping of the N-acetyl-glucosaminyl-substituted ribitol teichoic acids of S. aureus and the disaccharide substituents of the Streptococcus faecalis glycerol teichoic acid would hamper steric flexibility; in each case these substituents are the antigenic determinants. Because antibodies are generally formed against the substituents on the polyribitolor polyglycerol-phosphate backbone, it has frequently been concluded that such substituents mask the backbone and thus prevent the formation of antibodies to the glycerol or ribitol components (43,152,178,179). However, McCarty had noted that certain group A streptococcal sera were specific for the glycerol phosphate backbone and reacted with substituted glycerol teichoic acids. ...
... conflicting conclusions on whether group D streptococcal teichoic acid cross-reacts with antisera to group A streptococci (78,179). D-Alanine as an Antigenic Determinant In contrast to the observation that antibodies are frequently formed against the carbohydrate components of teichoic acids, there are only a few instances of antibodies with specificity for the D-alanine substituents, namely, certain rabbit antisera to group A streptococci (178,188) and S. aureus phage type 187 (152). This lack of immunogenicity may be related to the marked lability of the alanine ester linkage, for D-alanine components of peptidoglycan (246) and poly-D, L-alanyl-proteins (244) have been shown to be immunodominant. ...
... The application of immunoelectrophoresis to extracts of group A streptococci showed that in addition to polyglycerophosphate, a slower-moving component (E4) was present, and the fusing of the lines indicated a serological relationship (300). Subsequent work (178) showed that component E4 contained D-alanine residues, thus accounting for its lower mobility, whereas the crossreaction depended on the common glycerol phosphate backbone. Immunoelectrophoresis has also been used to separate teichoic acid, lipoteichoic acid, and lipoteichoic acid-protein complexes from lactobacilli and to show by appropriate absorptions that only antibodies to the teichoic acid component can be detected (294). ...
... Whether this generalization can be extended to teichoic acids will depend on whether more knowledge on the shapes of the polymers is obtained, but it might be expected that the bulky 2-acetamido grouping of the N-acetyl-glucosaminyl-substituted ribitol teichoic acids of S. aureus and the disaccharide substituents of the Streptococcus faecalis glycerol teichoic acid would hamper steric flexibility; in each case these substituents are the antigenic determinants. Because antibodies are generally formed against the substituents on the polyribitolor polyglycerol-phosphate backbone, it has frequently been concluded that such substituents mask the backbone and thus prevent the formation of antibodies to the glycerol or ribitol components (43,152,178,179). However, McCarty had noted that certain group A streptococcal sera were specific for the glycerol phosphate backbone and reacted with substituted glycerol teichoic acids. ...
... conflicting conclusions on whether group D streptococcal teichoic acid cross-reacts with antisera to group A streptococci (78,179). D-Alanine as an Antigenic Determinant In contrast to the observation that antibodies are frequently formed against the carbohydrate components of teichoic acids, there are only a few instances of antibodies with specificity for the D-alanine substituents, namely, certain rabbit antisera to group A streptococci (178,188) and S. aureus phage type 187 (152). This lack of immunogenicity may be related to the marked lability of the alanine ester linkage, for D-alanine components of peptidoglycan (246) and poly-D, L-alanyl-proteins (244) have been shown to be immunodominant. ...
... The studies of Wagner (1959), Sterzl, Kostka and Lanc (1962), Landy and Weidanz (1964), and Ikari (1964) supported Springer's contention that 'natural' antibodies result from inapparent environmental stimuli. These ubiquitous antigens are frequently cross-reacting, prominent examples being the Forssman antigen (reviewed by Buchbinder, 1935), pneumococcal polysaccharides (Heidelberger, 1967), streptococcal glycerol-teichoic acid (McCarty, 1959McCarty, , 1964), the 'B-like' antigen of certain strains of Escherichia coli (Springer, 1959), and the 'common antigen' of Gram-negative bacteria (Bradhage, 1962; Kunin, 1963). Many of these antibodies are quite specific for the determinants with which they react (Heidelberger, 1967; McCarty, 1967; Decker, Chorpenning and Frederick, 1972). ...
... These ubiquitous antigens are frequently cross-reacting, prominent examples being the Forssman antigen (reviewed by Buchbinder, 1935), pneumococcal polysaccharides (Heidelberger, 1967), streptococcal glycerol-teichoic acid (McCarty, 1959McCarty, , 1964), the 'B-like' antigen of certain strains of Escherichia coli (Springer, 1959), and the 'common antigen' of Gram-negative bacteria (Bradhage, 1962; Kunin, 1963). Many of these antibodies are quite specific for the determinants with which they react (Heidelberger, 1967; McCarty, 1967; Decker, Chorpenning and Frederick, 1972). Yet few studies have involved the cell-mediated responses to such antigens. ...
... The bound antigens may then provoke immunocytotoxic effects to produce local tissue injury. Several studies have demonstrated that LTA is exposed on the surfaces of gram-positive bacteria (6,11,20). The surface LTA appears to play a central role in the binding of group A streptococci to epithelial cells of mucosal surfaces (3,6,14) as well as of the skin (1). ...
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The spontaneous binding of group A streptococcal lipoteichoic acid (LTA) to mammalian cell membranes was studied in isolated membranes of human erythrocytes. The binding of radiolabeled LTA to erythrocyte membranes was dependent on membrane concentration and time. Binding approached a maximum within 30 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. The displaced LTA was eluted from a column of Sepharose 6B in a position identical to that of authentic LTA, suggesting that binding did not alter the size of the molecule. A dissociation constant of 42 micrometers was calculated, and only one population of approximately 5.5 X 10(6) binding sites per erhtyrocyte membrane was detected. Since these results suggested that erythrocyte membranes possess specific binding sites for LTA, an attempt was made to localize the putative receptors to the outside or the inside surface of the erhtyrocyte membrane. Assays of the binding of LTA to resealed right-side-out and inside-out membrane ghosts demonstrated that the outside surface was able to bind over 10 times more LTA than the inside surface. These results support the concept that the membranes possess specific binding sites for LTA and inciate that these binding sites are located almost entirely on the outside surface of erythrocyte membranes.
... This indicates that the change in electrophoretic mobility is a convenient, simple marker for estimating the D-alanyl ester content of an LTA preparation. Although implied by the work of McCarty (12), this observation has not been used in physiological studies of LTA production. In contrast to the change of mobility in classic immunoelectrophoresis, the distance migrated by LTA in rocket immunoelectrophoresis with anti-PGP serum was essentially independent of the presence or absence of the D-alanyl ester substituents. ...
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The content and D-alanyl ester complement of lipoteichoic acid from stationary-phase culture filtrates of Streptococcus mutans (strains BHT and GS-5; serotypes b and c) were determined chemically and serologically. A third less lipoteichoic acid was obtained from strain GS-5 than from strain BHT. This lipoteichoic acid had an increased mobility on immunoelectrophoresis after exposure overnight at pH 8 and a 10-fold greater content of alanine per mole of glycerol.
... However, resistant spheroplasts from strain X14 can be rendered sensitive to the lytic agent by suspension in acetate buffer at pH 6. In view of the fact that polyglyceiol phosphate is removed from streptococci by acetate buffer (11), it is probable that similar treatment removed the inhibitory teichoic acid from the spheroplasts. ...
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Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Effect of teichoic acid on resistance to the membrane-lytic agent of Streptococcus zymogenes. J. Bacteriol. 92 1623–1631. 1966.—The resistance of Streptococcus zymogenes to its own lytic agent has been shown to be due to the production of a specific, inhibitory teichoic acid. A survey of streptococcal strains showed that only strains resistant to the lytic agent produced the specific inhibitor. In addition, the inhibitor can be removed from spheroplasts of resistant strains, thereby making them sensitive to the lysin. Throughout the early part of the growth cycle, the inhibitor is associated with the cell and cannot be found in the medium. During late logarithmic phase, however, the inhibitor is released into the medium by the cells, and therefore is a contributing factor to the apparent lability of the lytic agent. The purified, inhibitory teichoic acid contains ribitol, phosphate, glucose, and d-alanine. The alkaline lability of the biological activity of the teichoic acid was correlated with the hydrolysis of the d-alanine. A streptococcal strain which is sensitive to the membrane-lytic agent produced an inactive ribitol teichoic acid which lacks the ester-linked d-alanine, whereas a lysin-resistant mutant of this strain produces a teichoic acid which contains d-alanine and which has inhibitory activity.
... imilar to that isolated from one particular strain. However, the results of the present study indicate that such a conclusion is not necessarily valid because of the cross-reaction of teichoic acids differing in structure. With regard to the observations on serological cross-reactions, the situation may be analogous to that discussed by Kabat (1966). McCarty (1964 had noted that certain group A streptococcal antisera reacted equally well with streptococcal glycerol ...
Article
Both wall and membrane teichoic acids from Lactobacillus helveticus NCIB 8025 are glycerol phosphate polymers partially substituted with α-D-glucosyl residues. The membrane teichoic acid, isolated as a complex with lipid (lipoteichoic acid), was antigenic when injected into rabbits with Freund's adjuvant. The α-D-glucosyl substituents are primarily responsible for the serological specificity of the membrane antigen, and account for the reaction of wall teichoic acid with antisera to the membrane teichoic acid. Glycerol teichoic acids either differing in or lacking sugar substitution may cross-react and the significance of these observations is discussed.
... was extracted from the intact surface of untreated or pep 5.8 streptococci with acetate buffer at pH 6.0 as described by McCarty (17). The anti-LTA reactivity and spontaneous binding to erythrocytes of the LTA extracted from pep 5.8 organisms was similar to that extracted from untreated organisms, indicating that LTA was retained on the surface of the organisms aider they had been denuded of M protein. ...
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Group A streptococci were treated with various enzymatic and chemical agents in an attempt to dissociate the type-specific M protein from intact surface "fimbriae." Mild peptic digestion at pH 5.8, which was previously shown to extract serologically active M antigen from intact streptococci had little visible effect on the fimbriae even though virtually all of the M protein was removed as demonstrated by (a) increased susceptibility to phagocytosis, (b) lack of opsonic effect of homologous M antibody on the treated streptococci, and (c) loss of HCl-extractable M protein. These fimbriated streptococci which lacked M protein adhered to human oral mucosal cells equally as well as untreated, fimbriated organisms which retained their M protein. Removal of both fimbriae and M protein by digesting organisms with HCL at pH 2.0 at 94 degrees C. or with trypsin abolished their ability to bind mucosal cells. Electron microscopy of streptococci bound to epithelial cells demonstrated fimbriae radiating from the surface of the organisms to the membrane of the epithelial cells. It is apparent, therefore, that the determinants of streptococcal fimbriae involved in resistance to phagocytosis can be dissociated from those involved in epithelial cell binding. These results are consistent with our previous studies which suggested that fatty acids ester linked with glycerol teichoic acid rather than M protein of streptococci binds the organisms to epithelial cells.
Article
A stabilized L-form of Streptococcus pyogenes continues to synthesize glycerol teichoic acid. This polymer was obtained from S. pyogenes and its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid of d-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1,3-phosphodiester-linked glycerophosphate polymer substituted with d-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.
Article
A teichoic acid antigen consisting of polyglycerophosphate, essentially free of alanine or other side groups, was shown to adsorb spontaneously to the erythrocytes of several species, thus potentiating their activity in passive hemagglutination (PHA) by specific antisera. The adsorption of this antigen was influenced by time, temperature, pH, ion concentration and antigen concentration. The bond appeared to be weak and a reverse reaction occurred, which may account for differences in PHA results reported by others. A recommended procedure for coating red cells with teichoic acid is presented.
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Teichoic acid-like material extracted by cold trichloroacetic acid from lyophilized whole cells of streptococci from groups A,D,E,O, and T was shown to give a positive precipitin reaction with group antisera. Similar material from cells of groups B,C,F,G,H,K,L,M,N,P,Q,R, and S did not give a positive reaction with group antisera. The group A material also reacted with anti-E serum; however, the opposite did not occur. A similar result was also obtained on the group T material and anti-O serum. The group A teichoic acid was purified by Sephadex column chromatography, and was shown to be free of cell wall peptidoglycan and polysaccharide, and ribitol teichoic acid. It was composed of glycerol, phosphate, alanine, and glucosamine. Alkaline hydrolysis showed the presence of ester-linked alanine and glucosaminylglycerol. Phosphorus was released from ester linkage by alkaline phosphatase. N-acetylglucosamine produced a 72% inhibition of the precipitin test at a level of 10 mumoles, and d-alanine methyl ester was significantly stronger than the l-alanine ester. A single precipitin band was seen with group A serum. The data indicate that teichoic acid of group A streptococci is a polymer composed of glycerol phosphate and containing N-acetylglucosamine and alanine. Antisera to these streptococci contain antibodies specific for the alanine and the glucosamine linkages. The use of serum containing antibodies to alanine-polyglycerophosphate shows that the occurrence of this type of teichoic acid is widespread among the streptococci.
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Antibodies directed against mucopeptide were obtained by immunization of rabbits with Bacillus licheniformis N. C. T. C. 6346 cell walls extracted chemically to remove the teichoic acids. The teichuronic acid component present in whole walls or alkali extracted walls of the organism was not significantly immunogenic in rabbits. The mucopeptide antisera reacted strongly with sonicates of Bacillus licheniformis and Bacillus subtilis mucopeptides and the homologous reaction was inhibited by soluble fragments of low molecular weight derived from mucopeptide. The best inhibition of the homologous reaction was obtained with fragments containing peptide and hexosamine material isolated from the soluble products of lysozyme treatment of Bacillus licheniformis mucopeptide. Immune sera obtained from rabbits immunized with intact Bacillus licheniformis N. C. T. C. 6346 walls containing mucopeptide, teichuronic acid and teichoic acids contained antibodies directed mainly against the teichoic acids. The immune response to the mucopeptide component was greatly reduced compared to that obtained when wall preparations lacking teichoic acids were used as immunizing antigens.
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1. Streptococci causing neonatal infection in piglets have been identified serologically as belonging to group D. 2. Glycerophosphate, glucose and xylose have been identified as components of the group substance isolated from these micro-organisms. 3. The streptococci isolated from piglets with this disease in England appear to belong to a single serological type characterized by a capsular polysaccharide. 4. Glucose, glucosamine and galactosamine have been identified as components of the type-specific substance. 5. It is suggested that these streptococci should be designated Capsular Type 1 in Streptococcus suis , a new subgroup in group D.
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A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.
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Native substitution with the D-alanine ester of lipoteichoic acids (LTAs) affects their immunological properties, the capacity to bind divalent cations, and LTA carrier activity. In this study we tested the influence of the D-alanine ester on anti-autolytic activity, using extracellular autolysin from Staphylococcus aureus and nine LTAs with alanine/phosphorus molar ratios of between 0.23 and 0.71. The inhibitory activity, highest with alanine-free LTA, exponentially decreased with increasing alanine content, approaching zero at substitutions of greater than 0.6. Correspondingly, dipolar ionic phospholipids were not inhibitory, in contrast to negatively charged ones. Glycosylation of LTA up to an extent of 0.5 did not depress inhibitory activity, and even at a degree of 0.8 the effect was comparatively small. On comparison of LTAs from various sources, differences in lipid structures and chain lengths were without effect. The inhibitory activity drastically decreased when the glycolipid carried a single glycerophosphate residue or the hydrophilic chain had the unusual structure [6 leads to Gal(alpha 1--6)Gal(alpha 1--3)Gro-(2 comes from 1 alpha Gal)-P]n, in which digalactosyl moieties connect the alpha-galactosylated glycerophosphate units. Principally, the same results were obtained with the more complex system of autolysis of S. aureus cells. We hypothesize that the anti-autolytic activity of LTA resides in a sequence of glycerophosphate units and that the negative charges of appropriately spaced phosphodiester groups play a crucial role. The alanine ester effect is discussed with respect to the putative in vivo regulation of autolysins by LTA.
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The ability of Streptococcus pyogenes lipoteichoic acid and palmitic acid to bind to purified human plasma fibronectin was investigated. Initial studies indicated that intact fibronectin formed soluble complexes with lipoteichoic acid, resulting in a change in the mobility of fibronectin in an electrical field. Fibronectin covalently linked to agarose beads bound radiolabeled lipoteichoic acid in the acylated form but not in the deacylated form. An 18-M excess of fibronectin inhibited binding of lipoteichoic acid to the immobilized protein by 92%. Fibronectin-bound [(3)H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid, as well as by fatty acid-free serum albumin. Serum albumin, which is known to contain fatty acid-binding sites capable of binding to the lipid moieties of lipoteichoic acid, inhibited the binding of lipoteichoic acid to fibronectin in a competitive fashion. The fibronectin-bound lipoteichoic acid could be eluted by 50% ethanol and various detergents but not by 1.0 M NaCl, various amino acids, or sugars. Similarly, radiolabeled palmitic acid adsorbed to fibronectin could be eluted with 50% ethanol but not with 1.0 M NaCl. Fibronectin adsorbed to a column of palmityl-Sepharose was eluted with 50% ethanol in 0.5% sodium dodecyl sulfate but not with 1.0 M NaCl or 1% sodium dodecyl sulfate alone. The binding of lipoteichoic acid to fibronectin followed first-order kinetics and was saturable. A Scatchard plot analysis of the binding data indicated a heterogeneity of lipoteichoic acid-binding sites similar to that previously found for serum albumin. Nevertheless, fibronectin contains at least one population of high-affinity binding sites for lipoteichoic acid. The binding affinity (nKa approximately 250 muM(-1)) is 2 orders of magnitude greater than the binding affinity of serum albumin. These data suggest that human plasma fibronectin contains specific binding sites for fatty acids and that lipoteichoic acid binds to these sites by way of its glycolipid moiety.
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The contribution of lipoteichoic acid (LTA) to the hydrophobic surface properties of group A streptococci was investigated in aqueous dextran-polyethylene glycol two-phase systems. Enzymatic digestions were performed to characterize the hydrophobic surface structure. The results obtained indicated that LTA is a major factor responsible for the hydrophobic character of the cell surface of group A streptococci. This was further supported by the similarity of partition in polymer two-phase systems between whole group A streptococci and tritiated LTA extracted from a group A streptococcal strain. Surface LTA was also determined on intact organisms by a new method measuring the adsorption of antibodies to LTA to the bacterial surface. A correlation was found between the content of surface LTA and the hydrophobicity of the group A streptococci. We conclude that surface-associated LTA is the major factor determining surface hydrophobicity of group A streptococci.
Article
The influence of D-alanine and carbohydrate substitution of lipoteichoic acids (LTAs) on the binding of antibody directed to the polyglycerol phosphate (PGP) portion was found to be at least partially dependent upon the mode of presentation of the antigen. There were greater differences in binding of anti-PGP immunoglobulins to substituted and unsubstituted LTAs in solution (micellar presentation) than when the same LTAs were adsorbed to the erythrocyte surface, which suggests that there is greater hindrance of access to the PGP chain, possibly as a result of closer packing, in a vesicle or micelle than when LTA is bound to a membrane surface. Although the difference in binding of anti-PGP immunoglobulins was nearly 20-fold between unsubstituted and highly substituted LTAs, rocket heights by rocket immunoelectrophoresis were only 4-fold different for the most highly substituted LTA and unsubstituted LTA. However, unsubstituted LTA clearly bound more immunoglobulin molecules than was reflected in the rocket height alone, since the resulting immunoprecipitates were much more prominent or intense, both before and after staining, than those of highly substituted LTAs. Differences between lightly and moderately substituted LTAs were less than twofold, indicating that under most circumstances estimates of LTA concentrations in samples where the composition is unknown will be within approximately twofold of the estimate.
Article
Naturally occurring precipitins with 2 different specificities were found in 45 per cent of human sera from male and female subjects whose average age was 24.6 yr. One specificity was for a factor from Actinomyces species and strains (A+) and the other for a factor from streptococci, staphylococci and lactobacilli (S+). Serologically identical factors were found in soluble extracts of dental plaque. The S, but not the A factor, was rendered inactive by treatment with mild alkali. The serum precipitins, uncharged at pH 8.2, were four times less reactive after IgG had been removed with Staphylococcal Protein A.
Article
Jackson, Robert W. (Purdue University, Lafayette, Ind.), and Merwin Moskowitz. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91 2205–2209. 1966.—A method for purifying a streptococcal antigen which sensitizes red cells to agglutination by antiserum is described. The antigen, when purified by this method, is almost exclusively composed of glycerophosphate and d-alanine. The ratio of alanine to glycerophosphate varies from 1:5 to 1:3. The glycerophosphate is polymerized and is thus a teichoic acid. The polyglycerophosphate appears to be the antigenic determinant for agglutination. d-Alanine is readily removed by mild base and appears to be necessary for the attachment of the teichoic acid to red cells. Quantitative removal of alanine does not affect the ability of the polymer to absorb antibody from serum.
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Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts. J. Bacteriol. 91 595–600. 1966.—The effect of streptolysin S, the group D hemolysin, and phospholipase C (the α toxin of Clostridium perfringens) on whole cells and spheroplasts or protoplasts of three strains of streptococci and Micrococcus lysodeikticus was tested. Viability, C¹⁴-glycine uptake, and lysis were measured. The group D hemolysin and phospholipase C were active against whole bacteria; streptolysin S was not. All three substances were active on spheroplasts. A partially resistant mutant derived from a strain sensitive to the group D hemolysin was also partially resistant to streptolysin S and phospholipase C. Antimycin A protected spheroplasts from streptolysin S but not from the group D hemolysin.
Chapter
Glycoglycerolipids, phosphoglycolipids, and lipoteichoic acids are characteristic constituents of the cytoplasmic membrane of a large number of gram-positive bacteria (Fischer, 1981; Ishizuka and Yamakawa, 1985). These lipid amphiphiles are structurally and biosynthetically related to each other. Glycolipids are the lipid moiety in phosphoglycolipids and lipoteichoic acids. Phosphoglycolipids carry a glycerophosphate, a di(glycerophosphate) or a phosphatidyl residue; the common types of lipoteichoic acid carry a 1,3-phosphodiester-linked poly(glycerophosphate) chain. By contrast, glycophospholipids are derived from phospholipids containing phosphatidylglycerol, cardiolipin, or phosphatidylinositol to which monosaccharides or oligosaccharides are glycosidically linked (for a review, see Ishizuka and Yamakawa, 1985).
Chapter
Nach der Beschreibung der Methoden, mit deren Hilfe die Antigen-Antikörper- und die Hapten-Antikörper-Wechselwirkungen erfaßt werden, sollen nun die Natur der antigenen Determinante sowie Struktureigenschaften und Dimension des komplementären Bindungsbereiches am Antikörper betrachtet werden. Dies sind lediglich andere Aspekte der gleichen Fragestellung, welche ein sehr wichtiges Problem der Immunchemie behandelt.
Article
Protoplasts of a group A streptococcal strain were shown to contain enzymatic activity capable of converting lipoteichoic acid (LTA) to deacylated lipoteichoic acid (dLTA). The enzyme(s) appear to be located mainly in the membrane, although activity was also found in the cytoplasm. Determination of the sites of cleavage within the LTA molecule was approached by comparing the chemical composition of LTA and native dLTA. Native dLTA, as distinguished from chemically deacylated LTA, was isolated from buffer in which live streptococci had been resuspended and incubated. The chemical data suggest that the enzyme(s) was(were) lipolytic in nature; that is, the conversion of LTA to dLTA was the result of cleavage of the ester linkages between the fatty acids and the remainder of the LTA molecule.
Article
This chapter provides an account of the present state of the studies on the glycoglycerolipids (GGroLs) focusing on eubacterial, archaebacterial, and animal kingdoms. Glycoglycerolipids include several classes of glycoconjugates ranging from simple monogalactosyldiacylglycerols to highly complex polymers. About half of the glycoglycerolipids contain one or two of the acidic groups—sialic acids, sulfate, phosphate or phosphono groups, and carboxyl or uronic acids. GGroLs are anchored with two “legs” in the lipid bilayer of cell surface membranes. These “legs” of diacylglycerol (DAG) are replaced, in glycosphingolipids, by ceramide—a sphingoid base and a fatty acid linked to the sphingoid through an amide bond. GGroLs usually have two ester-linked long-chain fatty acids and a glycerol (Gro) molecule forming a lipophilic domain: sn-1,2-DAG, of which the position sn-3 of Gro is occupied with carbohydrate (the head). DAG is less polar than ceramide, and GalDAG and GalDAG sulfate migrate faster than GalCer and GalCer sulfate, respectively, on silica gel TLC. There are also less common types of legs, in which an alk-1-enyl (in alk-1-enyl-2-O-acyl-sn-Gro) or an O-alkyl group (in l-O-alkyl-2-O-acyl-sn-Gro) is substituted for an acyl ester at position 1. These are called the plasmenic acid (plasmalogen) type or alkylacyl type GGroLs, respectively.
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The precipitin reaction between peptidoglycan solubilized by ultrasonic treatment and the sera of rabbits immunized with Group A variant streptococci was inhibited with various amino acid derivatives and synthetic peptides. The peptides were synthesized by the solid phase method. The following conclusions were drawn from these experiments: 1. The pentapeptide l-Ala-d-Glu-γ-l-Lys-d-Ala-d-Ala is the antigenic determinant of the peptide moiety of the peptidoglycan. 2. The antibodies are directed against the COOH-terminal portion of the pentapeptide. 3. The terminal d-alanyl-d-alanine is the immunodominant site of the antigen. Thus, the antibodies are directed against the "growing" site of the peptidoglycan. The antibodies bind to the same site as the enzyme transpeptidase, which catalyzes the last step in the biosynthesis of the peptidoglycan. Penicillin, a structural analogue of the COOH-terminal d-Ala-d-Ala and thus an inhibitor of the transpeptidase reaction, did not inhibit the precipitin reaction, an indication that the antibodies distinguish between the structure of penicillin and d-Ala-d-Ala. 4. The contribution of the other amino acids to the antigenic site of peptidoglycan is of less importance (l-Lys, d-Glu) or is insignificant (l-Ala). 5. The upper limit of the combining site of the antibodies is complementary to that of a peptide ranging between the sizes of a tetrapeptide and a pentapeptide.
Article
Human leukocyte extracts, egg white lysozyme, cationic proteins, polymyxin, colimycin, and phenol are capable of releasing lipoteichoic acids (LTA) from group A streptococci andStrep, mutans. While the extraction of LTA by phenol is optimal at pH 4.7, the release of LTA from streptococci by the other agents is optimal at pH 7.4. LTA released by all agents was found to have the same sensitizing abilities, as determined by passive hemagglutination, and to have a similar chemical composition, as shown by thin-layer chromatography and radioactive scanning. The LTA-releasing capacity of all the agents is strongly inhibited by normal human serum. The possible role played by LTA released by leukocyte factors in the pathogenesis of tissue damage during bacterial infections is discussed.
Article
Human leukocyte extracts, egg white lysozyme, cationic proteins, polymyxin, colimycin, and phenol are capable of releasing lipoteichoic acids (LTA) from group A streptococci and Strep. mutans. While the extraction of LTA by phenol is optimal at pH 4.7, the release of LTA from streptococci by the other agents is optimal at pH 7.4. LTA released by all agents was found to have the same sensitizing abilities, as determined by passive hemagglutination, and to have a similar chemical composition, as shown by thin-layer chromatography and radioactive scanning. The LTA-releasing capacity of all the agents is strongly inhibited by normal human serum. The possible role played by LTA released by leukocyte factors in the pathogenesis of tissue damage during bacterial infections is discussed.
Article
Immunoelectrophoresis revealed in phenol extracts from S. faecalis and S. faecium a mixture of free and lipid-bound teichoic acids, both reactive with Group D antisera. In phenol extracts from S. suis only lipid-bound teichoic acid, also reactive with Group D antiserum, was seen. This difference probably accounts for the low yield of Group D antigen from S. suis as compared with S. faecalis and S. faecium when heating at pH 2 is used for extraction. When phenol is used good yields are obtained from S. suis as well as from S. faecalis and S. faecium. Lipoteichoic acids from S. faecalis and S. faecium have a backbone structure the same as or similar to that of Group A streptococcal teichoic acid. Lipoteichoic acid from S. suis has a structure differing from that of S. faecalis and S. faecium, e.g., possibly in the attachment of its glucosyl substituents. Precipitation reactions between S. suis lipoteichoic acid and Group D antisera were specifically inhibited by glucose. Reactions between S. bovis phenol extracts and some Group D antisera were also specifically inhibited by glucose, but extracts from S. faecalis and S. faecium were not. This may indicate a monosaccharide glucosyl substituent in teichoic acid from S. suis and S. bovis instead of the di- or trisaccharide previously postulated as the glucosyl substituent in the teichoic acid of S. faecalis.
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Rabbits immunized with group B, type Ia streptococci produce two distinct populations of protective antibodies. Evidence is presented indicating that these antibodies are directed against two major immunodeterminants which coexist in the specific type Ia antigen. Immunochemical data, using purified antibody preparations, indicate that the type substance, a sialic acid polymer consisting of galactose, glucose, glucosamine, and sialic acid, possesses two distinct immunodominant determinants, terminal sialic acid residues and a galactosyl oligosaccharide. Antibodies directed against either of these determinants were shown to possess in vivo and in vitro opsonic capabilities.
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Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts of Streptococcus sanguis by alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation of S. sanguis. The polysaccharides were composed of l-rhamnose, d-glucose, and N-acetyl d-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar is l-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.
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The interaction of group A streptococcal lipoteichoic acid (LTA) with mammalian cell membranes was studied in human platelets. The binding of LTA to platelets was platelet concentration and time dependent. Binding approached a maximum within 10 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. An association constant of 1.9 X 10(-7) M was calculated, and only one population of binding sites was detected. Immuno-ferritin labeling of LTA-treated platelets demonstrated a patchy distribution of LTA binding sites on the platelet surface. LTA inhibited collagen- and alpha1 chain-induced platelet aggregation, but not the platelet release reaction, suggesting that the LTA and collagen binding sites on human platelets are distinct. Apparently, LTA binds to platelets and interferes with collagen-induced aggregation although collagen is still able to attach to binding sites to trigger the release reaction.
Article
Protoplasts of a group A streptococcal strain were shown to contain enzymatic activity capable of converting lipoteichoic acid (LTA) to deacylated lipoteichoic acid (dLTA). The enzyme(s) appear to be located mainly in the membrane, although activity was also found in the cytoplasm. Determination of the sites of cleavage within the LTA molecule was approached by comparing the chemical composition of LTA and native dLTA. Native dLTA, as distinguished from chemically deacylated LTA, was isolated from buffer in which live streptococci had been resuspended and incubated. The chemical data suggest that the enzyme(s) was(were) lipolytic in nature; that is, the conversion of LTA to dLTA was the result of cleavage of the ester linkages between the fatty acids and the remainder of the LTA molecule.
Article
The teichoic acids, which are found in the cell walls of many gram-positive bacteria, are polymers of phosphates of polyhydroxyalcohols and sugars. Their name is derived from the Greek word teichos, “the wall”. It was in 1951 [108] when the possibility that such components may exist in bacteria was suggested for the first time by detection of considerable phosphorus excess in the staphylococcal cells, as compared to the nucleic acids, phospholipids, and other phosphorus-containing compounds known at that time. In the next years Armstrong, Baddiley, Archibald and their co-workers published a number of papers elucidating the structure of teichoic acids in many bacterial species.
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It is evident that characterization of any serum with respect to its specificity, and class of antibody showing that specificity, is essential for consistent serological investigations. It must also be emphasized that a particular regimen of injection of the same immunogen preparation into the same animal species will not always produce the same immunologic response.
Article
The widespread occurrence of lipoteichoic acids and their assumed structural homogeneity, together with their singular location on the bacterial membrane, led to the hypothesis that they may play an essential part in the cell physiology of Gram-positive bacteria. In the meantime, deviant lipoteichoic acid structures have been detected and, more importantly, glycosyl and alanyl substituents have been found to affect considerably the biological activities of the classical poly (g1ycerophosphate) lipoteichoic acids. Mutants defective in lipoteichoic acid biosynthesis have not yet been described. However, loss of rather substantial fractions of cellular lipoteichoic acid, caused for example by phosphate limitation, osmotic shock, or treatment with penicillin can apparently be tolerated by certain bacteria without significantly affecting the ability of lipoteichoic acid-depleted cells to survive. This does not preclude that a minor fraction of lipoteichoic acid may be indispensable or, that its presence may provide selective advantages to the organism.
Article
Glycerophosphate-containing lipoteichoic acids (LTAs) interact with the first component of the classical complement pathway (C1). This resulted in the activation of the classical complement pathway in serum, shown by the consumption of C1, C2, and C4. The dose-dependent interaction of LTAs with purified C1 and C1q was dependent on the negative charges of the phosphate groups of LTA. It was reduced by charge compensation through D-alanine ester substituents and by sterical hindrance through di- and trihexosyl residues linked to position 2 of the glycerol moieties. The charge density of LTA may also play a role: poly(digalactosylglycerophosphate) LTAs, in which the phosphate groups are in a greater distance from each other, were less effective, and the loss of micellar organization by deacylation of LTA drastically reduced the complement activation capacity.
Article
The successful classification of Group A streptococci by the capillary precipitin technique requires a complete series of M type antisera which are sufficiently potent and specific to give unequivocal type-specific reactions with all the serotypes. Specific antisera for this purpose have been prepared by absorption with heterologous streptococci. Unabsorbed antisera have been employed here in the Ouchterlony double-diffusion agar-gel test to identify the M type of streptococci. Techniques have been developed for making this method of M typing fully reliable. The results reported here confirm and amplify the original findings of Michael and Massell (3). With crude HCl extracts and unabsorbed M type antisera, a precipitin line due to the M protein and another to the group-specific carbohydrate are the two major reactions observed. These reactions, however, are usually readily distinguishable. There was a surprising lack of cross-reactive precipitin lines due to non-type-specific protein antigens in the extracts. Although many of the unabsorbed M type antisera can be employed in the double-diffusion tests, the group-specific antibody must be removed from some of the unabsorbed antisera to avoid confusing cross-reactions. Absorption of these antibodies has been achieved by means of a specific immunoabsorbent column prepared from para-aminophenyl-ß-N-acetylglucosamine and cyanogen bromide-activated Sepharose. Excellent agreement was observed between the M typing results obtained on 117 field strains by the conventional capillary precipitin method and the Ouchterlony double-diffusion method.
Article
Alanine ester hydrochlorides, which have been used in studying the specificity of antibodies to teichoic acids, were shown to cause non-specific inhibition of the precipitin reaction. Non-specific inhibition was also caused by lysine and glucosamine hydrochlorides and inorganic salts. The marked effect of divalnent cations would be consistent with their forming complexes with teichoic acids. It is concluded that the effect is due to an increase in the ion concentration in general rather than to a decrease in pH.
Article
Decreases in electrophoretic mobilities of intracellular lipoteichoic acid, intracellular deacylated lipoteichoic acid, and extracellular deacylated lipoteichoic acid were observed during inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation. Increased carbohydrate content, and thus an increased mass-to-charge ratio, rather than changes in ester alanine content or novel fatty acid substitutions, appeared to account for the decreased electrophoretic mobilities. The increase in carbohydrate content, as judged from mobility measurements, was progressive over time and appeared to occur on biosynthetically new lipoteichoic acid as well as on lipoteichoic acid made before inhibition of protein synthesis.
Article
The immunoelectrophoretic characteristics of the known cellular antigens of serotype 17, Group A streptococci have been presented. These include C, M, T, and polyglycerophosphate. In addition, three hitherto undescribed antigens of serotype 17 have been encountered. The F antigen occurs in most serotype 17 strains, has a faster electrophoretic mobility than M, appears in acid, distilled water, and other extracts of harvested cells, is released from the cells in large quantity into the culture medium during growth when the pH is maintained at 7.3 or over, is probably protein in nature, and may play a minor role in mouse virulence of serotype 17 strains. Its antibodies do not confer bactericidal power on human blood. The E antigen is serotype-specific and is closely associated with the M antigen. The suggestion is made here that E is a part of the M molecule, acquiring independent electrophoretic mobility when separated from the rest of the M molecule by acid hydrolysis and carrying an antigenic determinant serologically distinct from the determinant on the rest of the M molecule. E can be recognized only by immunoelectrophoresis. Its role, if any, in virulence has not been established. The third antigen, E4, is a non-serotype-specific antigen found in most serological groups and types of hemolytic streptococci. It is serologically related to polyglycerophosphate, but its chemical nature has not been determined. It appears to be unrelated to virulence.
Article
A bacterial substance has been described which gives a precipitin reaction with certain antisera to Group A streptococci. The precipitating antigen is present in various Gram-positive bacteria, including most hemolytic streptococci, staphylococci, and aerobic sporulating bacilli. It is not present in any of the Gram-negative species examined or in pneumococci, clostridia, or corynebacteria. Analysis of purified preparations obtained from Group A streptococci indicates that the antigen is a simple polymer of glycerophosphate. The identification has been confirmed by immunochemical studies, including precipitin tests and specific inhibition with synthetic polyglycerophosphates. In addition, the infrared spectra of bacterial and synthetic polyglycerophosphate are shown to be closely similar. Immunochemical analysis suggests that the amount of polyglycerophosphate present in Group A streptococci and staphylococci is approximately 1 per cent of the dry weight of the cells. The cellular localization and function of the polyglycerophosphate have not been established.
Article
DURING a taxonomic study of several Streptomyces cultures, most of which were isolated from soil, experiments were performed on the production of soluble melanoid pigments on two gelatin media-plain 15 per cent gelatin and 15 per cent gelatin plus 2 per cent glucose.
Article
A method for the preparation of d-amino acid oxidase as the iron-free crystalline flavoprotein is described. The enzyme has a typical flavoprotein spectrum and exhibits one component in the ultracentrifuge. The presence of a transient intermediate on reduction with alanine is reported and its significance in the mechanism of the enzyme discussed.
Article
THE immunological specificity of carbohydrates in the cell walls of streptococci characterizes groups A, C, E and G in Lancefield's classification of these micro-organisms1-3. Group D is different. Here the cell wall carbohydrate is type-specific, and a component the chemical nature of which has hitherto been in doubt characterizes the group4. The results of the present work indicate that this substance, the group D antigen, is probably a polymer of glucosyl glycerophosphate.
Article
SUMMARY The ‘intracellular’ teichoic acids from two strains of group D streptococci (strains 8191, 39) were purified and their serological and chemical properties examined. Both compounds reacted serologically with group D streptococcal antiserum. Chemical analysis showed them to be polymers of glycerol phosphate containing a high proportion of glucose and bearing alanine ester residues on the sugar; it is likely that the glucose comprises di- and tri-saccharide residues attached to most or all of the glycerol units. Analysis of group antigen preparations from three other strains of group D streptococci previously studied indicated that they are serologically identical with and chemically similar to the teichoic acid from strain 39. The difference in serological activity between the intracellular teichoic acid from strain 8191 and that from the other four strains is probably associated with small differences in chemical composition, particularly with respect to glucose content.
Article
The immunoelectrophoretic characteristics of the known cellular antigens of serotype 17, Group A streptococci have been presented. These include C, M, T, and polyglycerophosphate. In addition, three hitherto undescribed antigens of serotype 17 have been encountered. The F antigen occurs in most serotype 17 strains, has a faster electrophoretic mobility than M, appears in acid, distilled water, and other extracts of harvested cells, is released from the cells in large quantity into the culture medium during growth when the pH is maintained at 7.3 or over, is probably protein in nature, and may play a minor role in mouse virulence of serotype 17 strains. Its antibodies do not confer bactericidal power on human blood. The E antigen is serotype-specific and is closely associated with the M antigen. The suggestion is made here that E is a part of the M molecule, acquiring independent electrophoretic mobility when separated from the rest of the M molecule by acid hydrolysis and carrying an antigenic determinant serologically distinct from the determinant on the rest of the M molecule. E can be recognized only by immunoelectrophoresis. Its role, if any, in virulence has not been established. The third antigen, E4, is a non-serotype-specific antigen found in most serological groups and types of hemolytic streptococci. It is serologically related to polyglycerophosphate, but its chemical nature has not been determined. It appears to be unrelated to virulence.
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