STUDIES ON BACTERIOPHAGES OF HEMOLYTIC STREPTOCOCCI : I. FACTORS INFLUENCING THE INTERACTION OF PHAGE AND SUSCEPTIBLE HOST CELL

The host ranges of bacteriophages for group A, types 1, 6, 12, and 25 and group C streptococci have been determined. The findings indicate that the susceptibility to these phages is primarily a group-specific phenomenon, although it is modified by several factors such as the hyaluronic acid capsule, lysogeny, and possibly the presence of surface proteins. Phage antibody studies indicate that while the group A phages are antigenically related, they are distinct from the group C phage. This is in agreement with the observation that group A phages are not specific for their homologous streptococcal types. The purified group C carbohydrate inactivates group C phage but not the group A phages, thus suggesting that the carbohydrate, a component of the cell wall, may serve as the phage receptor site. It has not been possible to inactivate the group A phages with group A carbohydrate. Phage lysis of groups A and C streptococci is accompanied by fragmentation of the cell wall since the C carbohydrate has been identified serologically and chemically in the supernate of centrifuged lysates. The immediate lysis of groups A and C hemolytic streptococci and their isolated cell walls by an accesory heat-labile lytic factor in fresh group C lysates is also described

VARIATION IN THE GROUP-SPECIFIC CARBOHYDRATE OF GROUP C HEMOLYTIC STREPTOCOCCI

Certain strains of Group C hemolytic streptococci, termed Group C-intermediate, contain a group-specific carbohydrate antigen which gives a precipitin cross-reaction with A-variant antiserum. The carbohydrate antigens of these strains have a rhamnose:hexosamine ratio ranging from 2.4 to 2.6 whereas the ratio of typical Group C strains varies between 1.1 and 1.7. N-acetylgalactosamine, the major hexosamine in all of these strains is the principle determinant of Group C specificity. The high concentration of rhamnose in the C-intermediate carbohydrate suggests that a portion of the rhamnose oligosaccharide side chains are devoid of terminal N-acetylgalactosamine and thus react with Group A-variant antiserum. This view is supported by the fact that the induced variant enzyme, which destroys A-variant carbohydrate reactivity with the liberation of rhamnose oligosaccharides, has a similar action upon the Group C-intermediate carbohydrate. C-intermediate carbohydrate, after treatment with variant enzyme which removed approximately 25 per cent of the rhamnose, does not react with A-variant antisera

STUDIES ON THE CHEMICAL STRUCTURE OF THE STREPTOCOCCAL CELL WALL : I. THE IDENTIFICATION OF A MUCOPEPTIDE IN THE CELL WALLS OF GROUPS A AND A-VARIANT STREPTOCOCCI

Lysis of trypsinized Group A streptococcal cell walls with phage-associated lysin releases into solution dialyzable and non-dialyzable mucopeptide fractions composed of N-acetylglucosamine, N-acetylmuramic acid and alanine, glutamic acid, lysine, and glycine in addition to the characteristic group-specific carbohydrate. The latter substance contains appreciable amounts of N-acetylmuramic acid and the amino acids as well as N-acetylglucosamine and rhamnose. Hot formamide extraction of the cell walls results in a soluble fraction of group-specific carbohydrate and an insoluble residue. The Group A carbohydrate in this instance is composed of rhamnose and N-acetylglucosamine. The composition of the insoluble residue is similar to that of the mucopeptide fractions released from the cell wall by phage-associated lysin. This residue was shown by electron microscopy to be composed of discrete discs which appear similar in structure to the intact cell wall. The specific carbohydrate obtained by hot formamide extraction of Group A-variant cell walls was composed almost exclusively of rhamnose. The residue fraction was similar to that of Group A. The residue of cell walls extracted with hot formamide is extensively solubilized not only by phage-associated lysin and S. albus enzyme, but also by lysozyme, which has no measurable effect on the intact streptococcal cell wall

STUDIES ON THE CHEMICAL STRUCTURE OF THE STREPTOCOCCAL CELL WALL : II. THE COMPOSITION OF GROUP C CELL WALLS AND CHEMICAL BASIS FOR SEROLOGIC SPECIFICITY OF THE CARBOHYDRATE MOIETY

The trypsinized cell walls of Group C streptococci contain two components, the group-specific carbohydrate and a mucopeptide polymer. Hot formamide extraction of Group C cell walls results in a soluble group-specific carbohydrate fraction and an insoluble mucopeptide residue. This mucopeptide, similar in composition to that of Groups A and A-variant streptococci, contains N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, lysine, and glycine. It is dissolved by the muralytic enzymes, including lysozyme, which does not attack the whole cell wall. Lysis of the cell wall by phage-associated lysin results in the release of soluble fragments composed of the elements of mucopeptide. Group C carbohydrate extracted with formamide is composed primarily of N-acetylgalactosamine and rhamnose. Serological studies suggest that the specificity of Group C carbohydrate is determined by the N-acetylgalactosamine

GROUP-SPECIFIC CARBOHYDRATE OF GROUP C-VARIANT HEMOLYTIC STREPTOCOCCI

The trypsinized cell walls of Group C hemolytic streptococci are composed of the specific carbohydrate antigen and a mucopeptide matrix. Certain phage resistant strains of Group C streptococci, isolated from organisms which survive exposure to Group C bacteriophage also possess carbohydrate and mucopeptide fractions, but the carbohydrate gives a precipitin reaction with Group A-variant antiserum and not with Group C antiserum. These strains have been termed Group C-variant. The C-variant carbohydrate contains 86 per cent rhamnose and only 2 per cent galactosamine, and is thus chemically and immunologically similar to Group A-variant carbohydrate. The evidence suggests that the antigenic determinants of Groups A-variant and C-variant carbohydrates are rhamnose-rhamnose linkages. These results strongly support the hypothesis that the carbohydrates of Groups A and C streptococci are composed of a similar rhamnose moiety but that the determinant amino sugar terminal to the rhamnose-rhamnose linkages in the case of Group A is N-acetylglucosamine whereas that of Group C is N-acetylgalactosamine

IMMUNOCHEMICAL STUDIES ON THE SPECIFIC CARBOHYDRATE OF GROUP G STREPTOCOCCI

Group G hemolytic streptococcal cell walls which have been treated with trypsin are composed of a group-specific polysaccharide moiety and a mucopeptide matrix. The mucopeptide contains N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, lysine, and glycine, a composition similar to that of other groups of streptococci. The Group G carbohydrate is composed of rhamnose, N-acetylgalactosamine, and galactose. Serological studies suggest that the monosaccharide of L-rhamnose is a major component of the determinant of antigenic specificity

THE INDIVIDUAL ANTIGENIC SPECIFICITY OF ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

Although a single electrophoretically uniform antibody component with specificity for the group carbohydrate may comprise the bulk of the γ-globulin in rabbits immunized with streptococcal vaccines, this is not always the case. Not infrequently, electrophoresis may reveal multiple antibody components. Nevertheless, it has been feasible by various preparative procedures to isolate from a single antiserum at least two antibody components with similar reactivity for the carbohydrate both of which are electrophoretically monodisperse. Light chains from such antibodies reveal a restricted pattern when examined by disc electrophoresis. Antibodies to streptococcal carbohydrates have been examined for their individual antigenic specificity. Goats were immunized with isolated Group C and Group A-variant antibodies raised in rabbits. Individual antigenic specificity of these antibodies was brought out by absorption of the goat anti-antiserum with Fr II of pooled normal rabbit sera. Additional absorption of the goat anti-antisera with Fr II diminished but did not eliminate the reactivity for the homologous antibody. Immunoelectrophoretic studies with papain fragments of purified streptococcal antibodies localized the specificity to the Fab fragment. Specificity was not confined to the isolated light chains of the antibody

STUDIES ON THE IMMUNOCHEMISTRY OF STREPTOCOCCAL MUCOPEPTIDE

Streptococcal mucopeptide, solubilized by either ultrasonic treatment or lysozyme, gave a precipitin reaction with rabbit antimucopeptide serum. A haptenic inhibitor of this reaction, which was composed of alanine, glutamic acid, and lysine in a mole ratio of 4:1:1, was isolated from a Streptomyces albus enzymes digest of Group D cell walls by ion exchange chromatography. When selected antisera were employed, greater than 90% inhibition of the mucopeptide quantitative precipitin reaction was achieved with 2 mg/ml of this inhibitor, whereas a hexosamine fraction with minimal concentrations of amino acid residues was inactive in this respect. These results suggest that the peptide moiety is an antigenic determinant of mucopeptide. Preliminary results indicate that the hexosamine polymer of the mucopeptide is a secondary antigenic determinant

ANTIGENIC RELATIONSHIPS BETWEEN GROUPS B AND G STREPTOCOCCI

Trypsinized cell walls of Group B hemolytic streptococci are composed of a group-specific carbohydrate and mucopeptide. The carbohydrate extracted with hot formamide is composed of rhamnose, N-acetylglucosamine, and galactose. Quantitative precipitin inhibition studies have shown that L rhamose is the significant component of the antigenic determinant. The cross-reactivity between B and G carbohydrates is dependent upon the fact that Lrhamose is a determinant sugar in both antigens

IMMUNOCHEMICAL STUDIES ON THE GROUP AND TYPE ANTIGENS OF GROUP F STREPTOCOCCI AND THE IDENTIFICATION OF A GROUPLIKE CARBOHYDRATE IN A TYPE II STRAIN WITH AN UNDESIGNATED GROUP ANTIGEN

Two antigens, the group-specific carbohydrate and the Type II carbohydrate, have been isolated by cellulose column chromatography from a formamide extract of a Group F streptococcus. Chemical and immunologic analyses indicate that both antigens are free of other cellular components. Both antigens are components of the cell wall although the Type II antigen is probably more superficial than the group antigen. The Type II antigen is composed of rhamnose, glucose, galactose, and galactosamine. The Group F antigen is composed of rhamnose, glucose, galactosamine and a small percentage of glucosamine. A grouplike carbohydrate and the Type II carbohydrate have been isolated from a streptotoccal strain which lacks a serologically detectable streptococcal group antigen. This grouplike carbohydrate, which does not cross-react immunologically with Group F serum, is composed of rhamnose, galactose, and glucosamine. No chemical or immunological differences were observed between the Type II antigen isolated from the Group F strain and the Type II antigen isolated from the nongroupable strain