INFCTION AND ImmuNITy, July 1977, p. 21-27 Copyright 0 1977 American Society for Microbiology
Vol. 17, No. 1 Printed in U.S.A.
Common (Non-Type-Specific) Antigens of Group A Streptococci I. M. LYAMPERT,* S. G. SHURATOVA, V. V. AKIMOVA, V. P. BUKHOVA, AND V. Y. KOLESNIKOVA Laboratory of Streptococcal Infections, N. F. Gamaleya Institute ofEpidemiology and Microbiology, Academy of Medical Sciences, Moscow 123098, U.S.S.R.
Received for publication 7 July 1976
Two common (non-type-specific) antigens in HCl extracts prepared from group A streptococcal cultures of different types were analyzed by immunodiffusion methods. These antigens are sensitive to trypsin and are apparently related to the cell wall proteins. The common antigens tested belong to a category of nonprotective non-type-specific antigens. The presence of common antigens in the HCI extracts should be taken into consideration when M-proteins are determined. The detection of one of the common antigens may be used as a virulence index, since this antigen is characteristic of the group A streptococcal cultures with enhanced virulence.
Cross-reactions detected in bactericidal and mice protective tests constitute evidence for the presence of a common protective antigen(s) in group A streptococci of different types (11, 14, 18, 22, 25). In our previous studies, cross-reactions were established in bactericidal tests with sera of rabbits continuously immunized with streptococci of types 1, 5, and 29. One or occasionally two trypsin-sensitive antigens shared by these types were detected in hydrochloric acid extracts (HCI extracts) by immunodiffusion methods (14, 20, 22). Data from studies of non-type-specific (NTS) antigens by gel precipitation or capillary tests are not always in agreement with the results of bactericidal tests, indicating the presence of antigens that are common to different types but lack the properties of protective antigens (5, 11, 26). The investigation of NTS antigens related to cell wall proteins of group A streptococci is important not only in view of the search for a common protective antigen(s), but also ofthe development of an improved method for the identification of type-specific M-proteins. Furthermore, NTS antigens can be used for the determination of antibodies and delayed-type hypersensitivity in streptococcal diseases. NTS delayed-type hypersensitivity has been detected with HCI extracts and preparations of M-proteins in animals sensitized by group A streptococci and in human subjects with streptococcal diseases (1, 6, 13, 16). Marked delayed-type hypersensitivity to the antigens of the so-called thermostable fraction (TST fraction) can be demonstrated in human streptococcal infections and in sensitized 21
guinea pigs (13, 16). This fraction, obtained by protein sedimentation from supernatants of streptococcal broth cultures, contains NTS antigens sensitive to trypsin; one of these antigens can be also detected in HCI extracts (16). According to a number of reports, antibodies to M-associated protein, which is an NTS antigen, can be revealed in rheumatic fever and other streptococcal diseases (24). It has been suggested that the admixture of different Massociated proteins in M-protein vaccines might be the cause of toxic reactions (2). The occurrence of NTS antigens in streptococcal cultures ofdifferent types has not been studied, nor have the antigens been compared in detail with other antigens of group A streptococci. The present paper is concerned with an analysis of NTS antigens by immunodiffusion methods. The NTS antigens detected were compared with other streptococcal antigens described earlier. The occurrence of NTS antigens among different types of group A streptococci, belonging to variants obtained before or after an increase in virulence, was studied. The localization of NTS antigens in different structures of a microbial cell and the relation to common protective antigens were also investigated. MATERIALS AND METHODS Strains. Group A streptococcal cultures containing M-substances (Prague collection of 27 strains belonging to 24 types [kindly supplied by J. Rotta]) were used: type 1 (no. 2/49 and 2/55); types 2, 3, 4, 5, 6, 8, 9, 11, 12 (no. 22/59 and 8/56), 13, 14, 15, 17, 18, 19, 22, 23, 24, 26, 27, 29 (no. 62/59 and 15/55), and 44. Two strains of different groups (group C, no. 41/59;
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LYAMPERT ET AL.
INFE:CT. IMMUN.
and group G, no. 22/58) from the same collection; a ative electrophoresis, 20 ml of HCl extract dialyzed strain of group A type 10 (Dochez N-Y-5), the A- against the buffer solution and containing 5 mg of variant strain (T27 A/32/18) (kindly supplied by M. protein per ml was included in the agar. ElectrophoMcCarty); and three newly isolated cultures of resis was carried out for 5 h at a potential gradient of Staphylococcus aureus were also used. The cultures 4 to 6 V/cm and at a power setting of 300 mA (28). Fractions containing different antigens and were grown in combined broth (9 parts of Poup meat broth and 1 part of Marten broth) or in a medium whole HCl extracts (concentrated 5, 10, or 20 times) with casein hydrolysate (in both cases with 0.2% were analyzed by immunodiffusion techniques. Immunoelectrophoresis was performed for 60 to 90 glucose at pH 7.6). The virulence of streptococcal cultures was en- min at a potential gradient of 4 to 6 V/cm and at a hanced through passages in human blood (3). Glossy power setting of 15 to 20 mA. Gel precipitation was variants were selected before and matt variants carried out by a micromodification technique (28). To remove the halos, the plates with the agar gel were selected after enhancement of virulence. Isolation of antigens. HCI extracts were prepared were washed in a 10% NaCl solution for 20 min by the extraction of microbial mass (6.5 ml/10'2 cells) before the reaction was read. Preparation of serum. The immunization scheme or cell walls (6.5 ml per cell walls prepared from 2 x 1012 cells) in a 0.05 N HCI solution and heating at consisted of three to four cycles. Rabbits received the 95°C for 15 min. The cell walls were prepared by increasing doses (from 1 x 109 to 6 x 109) of micromechanical disruption in a Mickel-type apparatus, bial cells (type 1, 5, or 29) killed by heating. Sera lacking antibodies to the cell wall proteins were followed by differential centrifugation. Preparative electrophoresis was used for isolation prepared by immunization with the type 1 culture of different antigens from HCI extracts. Fractions killed by heating and treated with pepsin (19). To eluted from the agar gel were dialyzed and lyophi- produce antibodies to the NTS antigens, 24 rabbits lized. Protein in the fractions was measured by the were immunized with the fractions isolated by premethod of Lowry et al. (12). To determine the sus- parative electrophoresis from the HCl extracts that ceptibility of antigens to trypsin, 10 mg of crystal- had been prepared from cultures of types 1 and 29. line trypsin (Difco) was added to 1 ml of solution Immunization was performed according to the obtained from different fractions and concentrated method of Goudie et al. (8) by inoculation into the 10 times. The mixture was incubated for 30 min at lymph nodes. Preparations containing NTS antiroom temperature and for 5 to 6 h at 37°C. NTS gens with complete or incomplete Freund adjuvant antigens were isolated by preparative electrophore- (800 to 1,000 ,ug of protein) were injected into both popliteal lymph nodes of the hind legs. The same sis in the zone of albumin mobility. Polyglycerophosphate (PGP) was isolated in the dose of protein without adjuvant was inoculated infraction close to the anode (27). Polysaccharide was travenously and intramuscularly in the region of isolated in the zone of pyronin mobility. Fractions the lymph nodes 1 and 2 months later. Control sera containing PGP were hydrolyzed with 0.1 N HCI for were prepared by immunization with normal rabbit 30 min at 100°C (4). The total phosphorus content in sera and complete Freund adjuvant by the same these fractions was determined by a modification regimen. The rabbits were also immunized with the TST (23) of the Fiske-Subbarow method. The PGP-containing fractions were qualitatively fraction by the same method. Sera to membranous analyzed by descending chromatography on and cytoplasm antigens were prepared by Freimer's Schleicher paper in a solution consisting of isopropa- method (7) (sera kindly supplied by 0. I. Vvedennol, acetic acid, pyridine, and water (8:1:8:4). For skaya). Sera containing antibodies to the antigens of identification of phosphate esters, ammonium mo- L-forms of group A streptococci were also used lybdate reagent was used (4). (kindly supplied by G. Y. Kagan). Absorption of sera. To remove antibodies to PGP, Polysaccharide from group A streptococci (A polysaccharide) and peptidoglycan from the A-variant polysaccharide, and E4 antigen, the sera prepared strain were prepared from cell walls by a modified by immunization with whole cells of group A strep(10) formamide method (preparations kindly sup- tococci were absorbed with a culture of type 1, which had been treated with pepsin. Microbial cells (5 x plied by I. I. Rassokhina). The TST fraction was prepared as described else- 101") treated with pepsin (19) were added to 1 ml of where (16). The proteins were sedimented from su- serum, incubated for 2 h at 37°C, and centrifuged; pernatants of a 5-day-old broth culture (strain the precipitate was then discarded. Dochez N-Y-5, type 10) with glacial acetic acid (pH Bactericidal test. The sera of animals immunized 4.0 to 4.2). The sedimented proteins were washed with fractions containing NTS antigens were examwith a 0.85% NaCl solution (pH 4.0), dissolved in ined by an indirect bactericidal test (17) with the borate buffer (pH 8.0), and lyophilized. Antigen E4 blood of a monkey (Macacus rhesus). After their was extracted from whole microbial cells (types 5 virulence had been enhanced (3), streptococci of and 29) with a 0.85% NaCl solution at pH 5.9 to 6.1 types 1, 5, and 29 were used in the bactericidal test. (27). Sera obtained by immunization with NTS antigens Immunodiffusion methods. Preparative electro- prepared from homologous and heterologous types phoresis and immunoelectrophoresis were per- were investigated. In the control test, normal rabbit formed in 1% Difco agar prepared in Veronal-medi- serum and sera to whole cells of streptococci of the nal buffer (pH 8.6; ionic strength, 0.025). For prepar- homologous type were used.
COMMON ANTIGENS OF GROUP A STREPTOCOCCI
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RESULTS Study of HCI extracts by immunodiffusion. When the HCl extracts prepared from streptococci of types 1, 5, and 29 were analyzed by immunoelectrophoresis with sera to whole microbial cells, type-specific antigens that could be destroyed by trypsin were detected in the starting zone. Furthermore, in this system, as well as with sera to the heterologous types, A polysaccharide was revealed in the zone of pyronin mobility (Fig. la). After isolation by preparative electrophoresis, this A polysaccharide was identified by gel precipitation with the A polysaccharide prepared by the formamide method. PGP and E, antigens were detected in some HCl extracts (by immunoelectrophoresis) with unabsorbed sera to whole microbial cells or to cells previously treated with pepsin (Fig. lb). The antigen designated PGP migrated twice as far as the zone of albumin mobility. The former fraction contained 25 ,ug of phosphorus, whereas in the fractions near the origin there was not more than 3 or 4 ,ug of phosphorus per 100 ,tg of protein. The fractions in which PGP was revealed contained a product that migrated in a manner similar to that of glycerophosphate on paper chromatograms. E, antigen localized in the zone between PGP and serum albumin. These antigens could not be destroyed by trypsin. In spite of different electrophoretic mobilities, PGP and E4 antigens are identical (Fig. lb). By agar-gel precipitation, the E4 antigen ex-
albumin
pyronin 4
4
+
23
tracted with 0.85% NaCl was shown to be identical to PGP isolated from HCl extracts by preparative electrophoresis. The precipitin lines formed by PGP and E4 with the corresponding antibodies were undetectable in agar plates after being washed in a 10% NaCl solution. Sera to the whole microbial cells after absorption with the pepsin-treated culture did not contain antibodies to PGP, E, antigen, or A polysaccharide. The same sera, according to the study of HCl extracts in the heterologous system, reacted with one or two NTS antigens with a mobility similar to that of serum albumin (Fig. 2). NTS antigens and their comparison with other streptococcal substances. The fractions isolated, by preparative electrophoresis, in the zone of albumin mobility from the HCl extracts (obtained from types 1, 5, and 29) contained one or two NTS antigens. Both antigens reacted with heterologous sera and were destroyed by trypsin. Precipitation tests revealed the nonidentity of NTS antigens with the A polysaccharide and E4 antigen, as well as with PGP (Fig. 3a and b). Absorption of the sera containing antibodies to both NTS antigens with the peptidoglycan preparation (50 mg/ml) exerted no influence on the reaction with NTS antigens. The sera of animals immunized with NTS antigens gave two lines of precipitation in tests of HCl extracts or fractions containing these antigens. The sera to the NTS antigens did not react with A polysaccharide. Only slight reactions with some sera were obtained with PGP, but these could be removed by washing the plates in a 10% NaCl solution. In control tests, the sera of animals immunized with Freund adjuvant did not react with NTS antigens. Similar results were obtained in the study of NTS antigens in HCl extracts prepared from streptococcal cultures grown in both combined meat broth and the casein hydroly-
0_2t WI
FIG. 1. Reaction of group-specific polysaccharide, PGP, and E4 antigen upon immunoelectrophoresis. Wells: (a) HCl extract prepared from culture type 29; (b) well 1, PGP-containing fraction; well 2, HCI extract prepared from culture type 5 containing PGP and E4 antigen. Troughs: (a) Serum type 1 containing antibodies to the group-specific polysaccharide; (b) serum type 1 containing antibodies to PGP and E4 antigen (immunization with trypsin-treated culture).
FIG. 2. Reaction of NTS antigens upon immunoelectrophoresis with sera to whole microbial cells. Well: HCl extract prepared from culture type 29. Upper trough: Serum type 1 absorbed with pepsintreated culture. Lower trench: Unabsorbed serum type 1.
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LYAMPERT ET AL.
sate medium. The identity of NTS antigens detected in HCl extracts or fractions obtained from streptococci of different types (types 1, 5, 12, and 29) was established (Fig. 4a and b). Also, NTS antigens of type 29 were identical to those of types 9, 17, 23, 24, and others. When NTS antigen-containing fractions were tested in the homologous system (with sera of rabbits immunized with NTS antigens isolated from the homologous type), two antigens were also revealed. These antigens were completely, not partially, identical to the NTS antigens obtained from other types. Moreover, the fraction isolated from type 1 did not give additional lines of precipitation, which could be due to the reaction between the M-substance and type-specific antibodies (Fig. 4b). One of the NTS antigens (the first NTS antigen) was shown to be identical to one of the antigens of the TST fraction (Fig. 4c). The second NTS antigen was not identical to the antigen of the
FIG. 3. Comparison of NTS antigens with E4 antigen. Central wells: Unabsorbed serum type 1 to whole microbial cells. Peripheral wells: (a) No. 1, aqueous extract containing E4 antigen; no. 2, fr-action containing NTS antigens e-xtracted from culture type 29; (b) as above but after a washing in a 10% NaCl
solution.
INFECT. IMMUN.
TST fraction. In further studies of both NTS antigens in different cultures, their identity or nonidentity to the antigen of the TST fraction served as a criterion. NTS antigens did not react with sera to the cytoplasm or membrane antigens as well as to that of the L-forms. Both NTS antigens were found in the HCl extracts prepared from cell walls (Fig. 4b). Study of antisera to NTS antigens by the bactericidal test. The sera of animals immunized with NTS antigens and containing antibodies to both NTS antigens yielded negative results in the bactericidal test with cultures belonging to the heterologous and homologous types. Positive results were obtained in the controls when testing streptococcal cultures with the type-specific sera prepared by immunization with whole cells. NTS antigens in different cultures. The HCl extracts prepared from whole microbial cells (M-positive strains) were analyzed by gel precipitation with sera containing antibodies to both NTS antigens. When 20 cultures were examined before enhancement of virulence, two, one, and zero NTS antigens were revealed in four, twelve, and four cultures, respectively. After enhancement of virulence, two NTS antigens were detected much more frequently (in 11 of 16 cultures), and only 5 of 16 cultures contained one antigen. It should be emphasized that, as a rule, the cultures that contained two common antigens before or after enhancement of virulence produced mainly matt colonies. While testing six cultures before enhancement of virulence (these cultures produced mainly glossy colonies), we did not observe a reaction with the
FIG. 4. Examination of HCI extracts and fractions obtained from streptococci ofdifferent types with serum prepared by immunization with fractions containing NTS antigens. Central wells: Serum to NTS antigens extracted from culture type 1. Peripheral wells: (a) Wells 1 through 4, HCL extracts prepared from cultures type 12 (no. 1), 29 (no. 2 and 3), and 5 (no. 4); (b) well 1, HCl extract prepared from cell walls of streptococcus type 29; wells 2 and 3, fractions containing NTS antigens prepared from types 29 and 1; (c) well 1, HCl extract prepared from the culture type 1; well 2, fraction containing NTS antigens prepared form the culture type 5; well 3, TST fraction.
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two NTS antigens in any of the cases. In five cultures only one of the antigens was revealed. In the same cultures after enhancement ofvirulence (produced mainly matt colonies), two antigens were detected in five of six cases. To examine NTS antigens in glossy variants obtained before enhancement of virulence and in matt variants obtained after enhancement of virulence, both glossy and matt variants from the same strain were studied. Of the matt colonies (types 1, 12, 14, 17, 18, and 29), all six cultures contained two NTS antigens. The glossy variants showed one antigen in four of six cases. It was established that the first antigen, identical to that of the TST fraction, could be detected both before and after the increase in virulence (in glossy and matt variants). After enhancement of virulence, an increase in the amount of the first antigen was shown. The second NTS antigen was detected only after enhancement of virulence in the cultures forming matt colonies or in some cultures forming matt colonies before enhancement of virulence. The first antigen was revealed in streptococci of groups C and G. NTS antigens were not detected in staphylococcal cultures in spite of a 20fold concentration of HCl extracts. In some cases, while HCl extracts prepared from the cultures of streptococci of several types after enhancement of virulence were being tested, additional NTS antigens destroyed by trypsin were detected.
DISCUSSION A number of data indicate that the two trypsin-resistant antigens detected in HCl extracts are PGP and E4 antigen. The following evidence supports this: their electrophoretic mobility to the anode; the identity ofboth antigens in spite of different electrophoretic mobilities (27); the similarity of the E4 antigen obtained by the method of Wilson and Wiley (27) to the PGP isolated by preparative electrophoresis from the HCl extracts; an increased phosphorus content in the fraction containing PGP; and the presence of a substance in the hydrolyzed PGP fraction with a mobility, upon paper chromatography, similar to that of glycerophosphate. In addition to polysaccharide, PGP, and E4 antigen, two NTS antigens sensitive to trypsin were detected, by immunodiffusion methods, in HCl extracts prepared from group A streptococci of different types. These antigens are not identical to PGP, E4 antigen, polysaccharide, or peptidoglycan. NTS antigens presumably are types of cell wall protein because they do not react with the
25
sera of animals immunized with the antigens of membranes, cytoplasm, or L-forms of streptococci group A and are detected in HCl extracts prepared from cell walls. The first NTS antigen identical to one of the antigens of the TST fraction was found in a majority of cultures of different types before and after enhancement of virulence. This antigen was found in cultures producing glossy as well as matt colonies. The second NTS antigen was revealed most frequently after enhancement of virulence by the Becker method (3). Before enhancement of virulence, the second NTS antigen was found only in some cultures producing mainly matt colonies. In the study of the same cultures before and after enhancement of virulence, the second NTS antigen was detected only in cultures possessing increased virulence and forming matt colonies. It is known that streptococci forming glossy colonies are readily phagocytized in human blood, whereas matt colonies are more characteristic of the virulent cultures surviving in human blood (3, 25). Therefore, of interest is the appearance of the second antigen after enhancement of virulence by the Becker method and its occurrence in the cultures forming matt colonies. In this connection, detection of a second NTS antigen in the streptococcal cultures might be used as an index of virulence. The first NTS antigen was detected in strains belonging to groups C and G. Both NTS antigens were not revealed in the staphylococcal cultures. The NTS antigens tested should obviously be assigned to a category of nonprotective antigens according to their inability to stimulate the production of protective antibodies. The NTS antigens under study differ from the trypsin-resistant R-antigen detected by Lancefield (11) in cultures of type 28, but one of the previously described R-antigens (26), as well as the NTS antigens, is sensitive to trypsin. Other authors (21, 24) have described the socalled M-associated protein antigen(s) sensitive to trypsin. The M-associated protein antigen common to type 12 and other types investigated by Vosti (21) differs in electrophoretic mobility from those of the NTS antigens revealed in our work with types 1, 5, and 29. However, NTS antigens ofthe latter types are identical to NTS antigens of type 12. Therefore, it cannot be excluded that the antigen described by Vosti (21), although it has a different electrophoretic mobility, contains an antigenic determinant identical to one of the NTS antigens under study. So far it has not been established whether the M-substance and NTS antigen(s)
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LYAMPERT ET AL.
are different proteins or antigenic determinants of one molecule (21). Therefore the possibility cannot be excluded that the antigen(s) investigated by Vosti (21) and NTS antigens under study are different fragments of the Mprotein. It is likely that the NTS antigens described in our work are fragments lacking type-specific determinants. First, sera reacting with both NTS antigens did not give additional reactions with the type-specific antigen when NTS antigens were tested in the homologous system. Second, sera containing antibodies to both NTS antigens did not show M-antibodies with a culture of the homologous type by the bactericidal test. In addition, the study of HCl extracts obtained from a culture of type 1 revealed that type-specific and NTS antigens had different electrophoretic mobilities. These data do not exclude the possibility that the M-substance and NTS antigens in whole microbial cells are different antigenic determinants of one protein molecule. Moreover, in some cultures, some of the fragments formed upon extraction in HCl are possibly deprived of the type-specific Mdeterninant, whereas other fragments contain it. The HCl extracts obtained from some cultures after enhancement of virulence apparently contain additional NTS antigens. This opens prospects for a further search of common protective antigens as well as other NTS antigens belonging to a nonprotective category. It might be expedient to employ cultures of streptococci or fractions with a large amount of NTS antigens for absorption of sera containing M-antibodies while typing group A streptococci and determining M-proteins. It is likely that delayed-type hypersensitivity to the first NTS antigen develops in human streptococcal infections since an analogous antigen is demonstrated in the TST fraction to which an intensive delayed-type hypersensitivity can be observed in streptococcal diseases and in experimental animals (13, 16). The study of antibodies or delayed-type hypersensitivity to the second NTS antigen is of special interest because this antigen is apparently characteristic of only highly virulent cultures of group A streptococci. One of the crossreactive antigens of streptococci and of human and animal myocardium is contained in streptococcal cultures of different types. This antigen apparently has an electrophoretic mobility similar to that of the NTS antigens (15). In this connection, an investigation into the relationship between NTS antigens and cross-reactive antigens should be undertaken.
INFECT. IMMUN. LITERATURE CITED 1. Beachey, E. H., H. Alberti, and G. H. Stollerman. 1969. Delayed hypersensitivity to purified streptococcal M protein in guinea pigs and in man. J. Immunol. 102:42-52. 2. Beachey, E. H., and G. H. Stollerman. 1973. Mediation of cytotoxic effect of streptococcal M-protein by nontype-specific antibody in human sera. J. Clin. Invest. 52:2563-2570. 3. Becker, C. G. 1964. Selection of group A streptococci rich in M-protein from population poor in M-protein. Am. J. Pathol. 44:51. 4. Burrous, S., F. S. M. Grylls, and J. S. Harrison. 1952. Paper chromatography of phosphoric esters. Nature (London) 170:800-801. 5. Fox, E. N., and M. K. Wittner. 1968. Antigenicity ofthe M proteins of group A hemolytic streptococci. IV. Cross-reactivity between serotypes. J. Immunol. 100:3945. 6. Fox, E. N., M. K. Wittner, and A. Dorfman. 1966. Antigenicity of the M proteins of group A hemolytic streptococci. III. Antibody responses and cutaneous hypersensitivity in humans. J. Exp. Med. 124:11351151. 7. Freimer, E. H. 1968. The protoplast membrane of the group A streptococcus, p. 54-67. In R. Caravano (ed.), Current research on group A streptococcus. Excerpta Medica Foundation, New York. 8. Goudie, R. B., C. H. W. Horne, and P. C. Wilkinson. 1966. A simple method for producing antibody specific to a single selected diffusible antigen. Lancet ii:12241226. 9. Jackson, R. W., and M. Moskowitz. 1966. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2205-2209. 10. Krause, R. M. 1967. Preparation of cell-wall antigens from gram-positive bacteria, p. 34-40 In A. Curtis (ed.), Methods in immunology and immunochemistry, vol. 1. Academic Press Inc., New York. 11. Lancefield, R. C. 1957. Differentiation of group A streptococci with a common R antigen into three serological types with special reference to the bactericidal test. J. Exp. Med. 106:525. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Lyampert, I. M. 1972. Etiology, immunology and immunopathology of rheumatic fever. (In Russian) Medizina, M. 14. Lyampert, I. M. 1973. Virulence factors and protective antigens of the group A streptococcus. (In Russian) Vest. Akad. Med. Nauk SSSR 11:76-83. 15. Lyampert, I. M., and T. A. Danilova. 1975. Immunological phenomena associated with cross-reactive antigens of microorganisms and mammalian tissues. Prog. Allergy 18:423-477. 16. Lyampert, I. M., M. N. Smirnova, and N. Y. Semina. 1967. Delayed-type hypersensitivity and the formation of antibodies in animals sensitized with group A streptococcus antigens. J. Hyg. Epidemiol. Microbiol. Immunol. 11:160-170. 17. Maxted, W. R. 1956. The indirect bactericidal test as a means of identifying antibody to the M antigen of Streptococcus pyogenes. Br. J. Exp. Pathol. 37:415422. 18. Myoda, T. T., G. G. Wiley, and P. N. Bruno. 1973. Cross-reactions among group A streptococci. IV. Extraction, separation and purification of two protective antigens of type G, cocci. J. Immunol. 111:249-259. 19. Osterland, C. K., E. J. Miller, W. W. Karakawa, and R. M. Krause. 1966. Characteristics of streptococcal
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group-specific antibody isolated from hyper-immune rabbits. J. Exp. Med. 123:599-613. Shuratova, S. G., V. V. Akimova, and V. P. Bukhova. 1974. Trypsin-sensitive common antigens in HCI-extracts obtained from group A streptococci of different types. (In Russian). J. Microbiol. Epidemiol. Immunol. 7:11-17. Vosti, K. L. 1975. Characterization of a non-type-specific antigen(s) associated with group A streptococcal type 12 M protein. Infect. Immun. 11:1300-1305. Vvedenskaya, 0. I., I. M. Lyampert, and I. V. Shved. 1972. Cross-reactions in the bactericidal test on studying cultures of the group A streptococci of different types. (In Russian) J. Microbiol. Epidemiol. Immunol. 4:97-100. Weil-Malherbe, H., and R. H. Green. 1951. The catalytic effect of molibdate on the hydrolysis of organic phosphate bonds. Biochem. J. 49:286-292.
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24. Widdowson, J. P., W. R. Maxted, and A. M. Pinney. 1971. An M-associated protein antigen (MAP) of group A streptococci. J. Hyg. 69:553-564. 25. Wiley, G. G., and P. N. Bruno. 1968. Cross reactions among group A streptococci. I. Precipitin and bactericidal cros-reactions among types 33, 41, 43, 52 and Ross. J. Exp. Med. 128:959-968. 26. Wiley, G. G., and P. N. Bruno. 1970. Cross-reactions among group A streptococci. III. The M and R antigens of type 43 and serologically related streptococci. J. Immunol. 105:1124-1130. 27. Wilson, A. T., and G. G. Wiley. 1963. Immunoelectrophoretic studies of the C, M, PGP, E4, F and E antigens of serotype 17 streptococci. J. Exp. Med. 118:527556. 28. Zilber, L. A., and G. I. Abelev. 1962. Virology and immunology of cancer. (In Russian) Meditzina, M.
Vol. 17, No. 1 Printed in U.S.A.
Common (Non-Type-Specific) Antigens of Group A Streptococci I. M. LYAMPERT,* S. G. SHURATOVA, V. V. AKIMOVA, V. P. BUKHOVA, AND V. Y. KOLESNIKOVA Laboratory of Streptococcal Infections, N. F. Gamaleya Institute ofEpidemiology and Microbiology, Academy of Medical Sciences, Moscow 123098, U.S.S.R.
Received for publication 7 July 1976
Two common (non-type-specific) antigens in HCl extracts prepared from group A streptococcal cultures of different types were analyzed by immunodiffusion methods. These antigens are sensitive to trypsin and are apparently related to the cell wall proteins. The common antigens tested belong to a category of nonprotective non-type-specific antigens. The presence of common antigens in the HCI extracts should be taken into consideration when M-proteins are determined. The detection of one of the common antigens may be used as a virulence index, since this antigen is characteristic of the group A streptococcal cultures with enhanced virulence.
Cross-reactions detected in bactericidal and mice protective tests constitute evidence for the presence of a common protective antigen(s) in group A streptococci of different types (11, 14, 18, 22, 25). In our previous studies, cross-reactions were established in bactericidal tests with sera of rabbits continuously immunized with streptococci of types 1, 5, and 29. One or occasionally two trypsin-sensitive antigens shared by these types were detected in hydrochloric acid extracts (HCI extracts) by immunodiffusion methods (14, 20, 22). Data from studies of non-type-specific (NTS) antigens by gel precipitation or capillary tests are not always in agreement with the results of bactericidal tests, indicating the presence of antigens that are common to different types but lack the properties of protective antigens (5, 11, 26). The investigation of NTS antigens related to cell wall proteins of group A streptococci is important not only in view of the search for a common protective antigen(s), but also ofthe development of an improved method for the identification of type-specific M-proteins. Furthermore, NTS antigens can be used for the determination of antibodies and delayed-type hypersensitivity in streptococcal diseases. NTS delayed-type hypersensitivity has been detected with HCI extracts and preparations of M-proteins in animals sensitized by group A streptococci and in human subjects with streptococcal diseases (1, 6, 13, 16). Marked delayed-type hypersensitivity to the antigens of the so-called thermostable fraction (TST fraction) can be demonstrated in human streptococcal infections and in sensitized 21
guinea pigs (13, 16). This fraction, obtained by protein sedimentation from supernatants of streptococcal broth cultures, contains NTS antigens sensitive to trypsin; one of these antigens can be also detected in HCI extracts (16). According to a number of reports, antibodies to M-associated protein, which is an NTS antigen, can be revealed in rheumatic fever and other streptococcal diseases (24). It has been suggested that the admixture of different Massociated proteins in M-protein vaccines might be the cause of toxic reactions (2). The occurrence of NTS antigens in streptococcal cultures ofdifferent types has not been studied, nor have the antigens been compared in detail with other antigens of group A streptococci. The present paper is concerned with an analysis of NTS antigens by immunodiffusion methods. The NTS antigens detected were compared with other streptococcal antigens described earlier. The occurrence of NTS antigens among different types of group A streptococci, belonging to variants obtained before or after an increase in virulence, was studied. The localization of NTS antigens in different structures of a microbial cell and the relation to common protective antigens were also investigated. MATERIALS AND METHODS Strains. Group A streptococcal cultures containing M-substances (Prague collection of 27 strains belonging to 24 types [kindly supplied by J. Rotta]) were used: type 1 (no. 2/49 and 2/55); types 2, 3, 4, 5, 6, 8, 9, 11, 12 (no. 22/59 and 8/56), 13, 14, 15, 17, 18, 19, 22, 23, 24, 26, 27, 29 (no. 62/59 and 15/55), and 44. Two strains of different groups (group C, no. 41/59;
22
LYAMPERT ET AL.
INFE:CT. IMMUN.
and group G, no. 22/58) from the same collection; a ative electrophoresis, 20 ml of HCl extract dialyzed strain of group A type 10 (Dochez N-Y-5), the A- against the buffer solution and containing 5 mg of variant strain (T27 A/32/18) (kindly supplied by M. protein per ml was included in the agar. ElectrophoMcCarty); and three newly isolated cultures of resis was carried out for 5 h at a potential gradient of Staphylococcus aureus were also used. The cultures 4 to 6 V/cm and at a power setting of 300 mA (28). Fractions containing different antigens and were grown in combined broth (9 parts of Poup meat broth and 1 part of Marten broth) or in a medium whole HCl extracts (concentrated 5, 10, or 20 times) with casein hydrolysate (in both cases with 0.2% were analyzed by immunodiffusion techniques. Immunoelectrophoresis was performed for 60 to 90 glucose at pH 7.6). The virulence of streptococcal cultures was en- min at a potential gradient of 4 to 6 V/cm and at a hanced through passages in human blood (3). Glossy power setting of 15 to 20 mA. Gel precipitation was variants were selected before and matt variants carried out by a micromodification technique (28). To remove the halos, the plates with the agar gel were selected after enhancement of virulence. Isolation of antigens. HCI extracts were prepared were washed in a 10% NaCl solution for 20 min by the extraction of microbial mass (6.5 ml/10'2 cells) before the reaction was read. Preparation of serum. The immunization scheme or cell walls (6.5 ml per cell walls prepared from 2 x 1012 cells) in a 0.05 N HCI solution and heating at consisted of three to four cycles. Rabbits received the 95°C for 15 min. The cell walls were prepared by increasing doses (from 1 x 109 to 6 x 109) of micromechanical disruption in a Mickel-type apparatus, bial cells (type 1, 5, or 29) killed by heating. Sera lacking antibodies to the cell wall proteins were followed by differential centrifugation. Preparative electrophoresis was used for isolation prepared by immunization with the type 1 culture of different antigens from HCI extracts. Fractions killed by heating and treated with pepsin (19). To eluted from the agar gel were dialyzed and lyophi- produce antibodies to the NTS antigens, 24 rabbits lized. Protein in the fractions was measured by the were immunized with the fractions isolated by premethod of Lowry et al. (12). To determine the sus- parative electrophoresis from the HCl extracts that ceptibility of antigens to trypsin, 10 mg of crystal- had been prepared from cultures of types 1 and 29. line trypsin (Difco) was added to 1 ml of solution Immunization was performed according to the obtained from different fractions and concentrated method of Goudie et al. (8) by inoculation into the 10 times. The mixture was incubated for 30 min at lymph nodes. Preparations containing NTS antiroom temperature and for 5 to 6 h at 37°C. NTS gens with complete or incomplete Freund adjuvant antigens were isolated by preparative electrophore- (800 to 1,000 ,ug of protein) were injected into both popliteal lymph nodes of the hind legs. The same sis in the zone of albumin mobility. Polyglycerophosphate (PGP) was isolated in the dose of protein without adjuvant was inoculated infraction close to the anode (27). Polysaccharide was travenously and intramuscularly in the region of isolated in the zone of pyronin mobility. Fractions the lymph nodes 1 and 2 months later. Control sera containing PGP were hydrolyzed with 0.1 N HCI for were prepared by immunization with normal rabbit 30 min at 100°C (4). The total phosphorus content in sera and complete Freund adjuvant by the same these fractions was determined by a modification regimen. The rabbits were also immunized with the TST (23) of the Fiske-Subbarow method. The PGP-containing fractions were qualitatively fraction by the same method. Sera to membranous analyzed by descending chromatography on and cytoplasm antigens were prepared by Freimer's Schleicher paper in a solution consisting of isopropa- method (7) (sera kindly supplied by 0. I. Vvedennol, acetic acid, pyridine, and water (8:1:8:4). For skaya). Sera containing antibodies to the antigens of identification of phosphate esters, ammonium mo- L-forms of group A streptococci were also used lybdate reagent was used (4). (kindly supplied by G. Y. Kagan). Absorption of sera. To remove antibodies to PGP, Polysaccharide from group A streptococci (A polysaccharide) and peptidoglycan from the A-variant polysaccharide, and E4 antigen, the sera prepared strain were prepared from cell walls by a modified by immunization with whole cells of group A strep(10) formamide method (preparations kindly sup- tococci were absorbed with a culture of type 1, which had been treated with pepsin. Microbial cells (5 x plied by I. I. Rassokhina). The TST fraction was prepared as described else- 101") treated with pepsin (19) were added to 1 ml of where (16). The proteins were sedimented from su- serum, incubated for 2 h at 37°C, and centrifuged; pernatants of a 5-day-old broth culture (strain the precipitate was then discarded. Dochez N-Y-5, type 10) with glacial acetic acid (pH Bactericidal test. The sera of animals immunized 4.0 to 4.2). The sedimented proteins were washed with fractions containing NTS antigens were examwith a 0.85% NaCl solution (pH 4.0), dissolved in ined by an indirect bactericidal test (17) with the borate buffer (pH 8.0), and lyophilized. Antigen E4 blood of a monkey (Macacus rhesus). After their was extracted from whole microbial cells (types 5 virulence had been enhanced (3), streptococci of and 29) with a 0.85% NaCl solution at pH 5.9 to 6.1 types 1, 5, and 29 were used in the bactericidal test. (27). Sera obtained by immunization with NTS antigens Immunodiffusion methods. Preparative electro- prepared from homologous and heterologous types phoresis and immunoelectrophoresis were per- were investigated. In the control test, normal rabbit formed in 1% Difco agar prepared in Veronal-medi- serum and sera to whole cells of streptococci of the nal buffer (pH 8.6; ionic strength, 0.025). For prepar- homologous type were used.
COMMON ANTIGENS OF GROUP A STREPTOCOCCI
VOL. 17, 1977
RESULTS Study of HCI extracts by immunodiffusion. When the HCl extracts prepared from streptococci of types 1, 5, and 29 were analyzed by immunoelectrophoresis with sera to whole microbial cells, type-specific antigens that could be destroyed by trypsin were detected in the starting zone. Furthermore, in this system, as well as with sera to the heterologous types, A polysaccharide was revealed in the zone of pyronin mobility (Fig. la). After isolation by preparative electrophoresis, this A polysaccharide was identified by gel precipitation with the A polysaccharide prepared by the formamide method. PGP and E, antigens were detected in some HCl extracts (by immunoelectrophoresis) with unabsorbed sera to whole microbial cells or to cells previously treated with pepsin (Fig. lb). The antigen designated PGP migrated twice as far as the zone of albumin mobility. The former fraction contained 25 ,ug of phosphorus, whereas in the fractions near the origin there was not more than 3 or 4 ,ug of phosphorus per 100 ,tg of protein. The fractions in which PGP was revealed contained a product that migrated in a manner similar to that of glycerophosphate on paper chromatograms. E, antigen localized in the zone between PGP and serum albumin. These antigens could not be destroyed by trypsin. In spite of different electrophoretic mobilities, PGP and E4 antigens are identical (Fig. lb). By agar-gel precipitation, the E4 antigen ex-
albumin
pyronin 4
4
+
23
tracted with 0.85% NaCl was shown to be identical to PGP isolated from HCl extracts by preparative electrophoresis. The precipitin lines formed by PGP and E4 with the corresponding antibodies were undetectable in agar plates after being washed in a 10% NaCl solution. Sera to the whole microbial cells after absorption with the pepsin-treated culture did not contain antibodies to PGP, E, antigen, or A polysaccharide. The same sera, according to the study of HCl extracts in the heterologous system, reacted with one or two NTS antigens with a mobility similar to that of serum albumin (Fig. 2). NTS antigens and their comparison with other streptococcal substances. The fractions isolated, by preparative electrophoresis, in the zone of albumin mobility from the HCl extracts (obtained from types 1, 5, and 29) contained one or two NTS antigens. Both antigens reacted with heterologous sera and were destroyed by trypsin. Precipitation tests revealed the nonidentity of NTS antigens with the A polysaccharide and E4 antigen, as well as with PGP (Fig. 3a and b). Absorption of the sera containing antibodies to both NTS antigens with the peptidoglycan preparation (50 mg/ml) exerted no influence on the reaction with NTS antigens. The sera of animals immunized with NTS antigens gave two lines of precipitation in tests of HCl extracts or fractions containing these antigens. The sera to the NTS antigens did not react with A polysaccharide. Only slight reactions with some sera were obtained with PGP, but these could be removed by washing the plates in a 10% NaCl solution. In control tests, the sera of animals immunized with Freund adjuvant did not react with NTS antigens. Similar results were obtained in the study of NTS antigens in HCl extracts prepared from streptococcal cultures grown in both combined meat broth and the casein hydroly-
0_2t WI
FIG. 1. Reaction of group-specific polysaccharide, PGP, and E4 antigen upon immunoelectrophoresis. Wells: (a) HCl extract prepared from culture type 29; (b) well 1, PGP-containing fraction; well 2, HCI extract prepared from culture type 5 containing PGP and E4 antigen. Troughs: (a) Serum type 1 containing antibodies to the group-specific polysaccharide; (b) serum type 1 containing antibodies to PGP and E4 antigen (immunization with trypsin-treated culture).
FIG. 2. Reaction of NTS antigens upon immunoelectrophoresis with sera to whole microbial cells. Well: HCl extract prepared from culture type 29. Upper trough: Serum type 1 absorbed with pepsintreated culture. Lower trench: Unabsorbed serum type 1.
24
LYAMPERT ET AL.
sate medium. The identity of NTS antigens detected in HCl extracts or fractions obtained from streptococci of different types (types 1, 5, 12, and 29) was established (Fig. 4a and b). Also, NTS antigens of type 29 were identical to those of types 9, 17, 23, 24, and others. When NTS antigen-containing fractions were tested in the homologous system (with sera of rabbits immunized with NTS antigens isolated from the homologous type), two antigens were also revealed. These antigens were completely, not partially, identical to the NTS antigens obtained from other types. Moreover, the fraction isolated from type 1 did not give additional lines of precipitation, which could be due to the reaction between the M-substance and type-specific antibodies (Fig. 4b). One of the NTS antigens (the first NTS antigen) was shown to be identical to one of the antigens of the TST fraction (Fig. 4c). The second NTS antigen was not identical to the antigen of the
FIG. 3. Comparison of NTS antigens with E4 antigen. Central wells: Unabsorbed serum type 1 to whole microbial cells. Peripheral wells: (a) No. 1, aqueous extract containing E4 antigen; no. 2, fr-action containing NTS antigens e-xtracted from culture type 29; (b) as above but after a washing in a 10% NaCl
solution.
INFECT. IMMUN.
TST fraction. In further studies of both NTS antigens in different cultures, their identity or nonidentity to the antigen of the TST fraction served as a criterion. NTS antigens did not react with sera to the cytoplasm or membrane antigens as well as to that of the L-forms. Both NTS antigens were found in the HCl extracts prepared from cell walls (Fig. 4b). Study of antisera to NTS antigens by the bactericidal test. The sera of animals immunized with NTS antigens and containing antibodies to both NTS antigens yielded negative results in the bactericidal test with cultures belonging to the heterologous and homologous types. Positive results were obtained in the controls when testing streptococcal cultures with the type-specific sera prepared by immunization with whole cells. NTS antigens in different cultures. The HCl extracts prepared from whole microbial cells (M-positive strains) were analyzed by gel precipitation with sera containing antibodies to both NTS antigens. When 20 cultures were examined before enhancement of virulence, two, one, and zero NTS antigens were revealed in four, twelve, and four cultures, respectively. After enhancement of virulence, two NTS antigens were detected much more frequently (in 11 of 16 cultures), and only 5 of 16 cultures contained one antigen. It should be emphasized that, as a rule, the cultures that contained two common antigens before or after enhancement of virulence produced mainly matt colonies. While testing six cultures before enhancement of virulence (these cultures produced mainly glossy colonies), we did not observe a reaction with the
FIG. 4. Examination of HCI extracts and fractions obtained from streptococci ofdifferent types with serum prepared by immunization with fractions containing NTS antigens. Central wells: Serum to NTS antigens extracted from culture type 1. Peripheral wells: (a) Wells 1 through 4, HCL extracts prepared from cultures type 12 (no. 1), 29 (no. 2 and 3), and 5 (no. 4); (b) well 1, HCl extract prepared from cell walls of streptococcus type 29; wells 2 and 3, fractions containing NTS antigens prepared from types 29 and 1; (c) well 1, HCl extract prepared from the culture type 1; well 2, fraction containing NTS antigens prepared form the culture type 5; well 3, TST fraction.
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COMMON ANTIGENS OF GROUP A STREPTOCOCCI
two NTS antigens in any of the cases. In five cultures only one of the antigens was revealed. In the same cultures after enhancement ofvirulence (produced mainly matt colonies), two antigens were detected in five of six cases. To examine NTS antigens in glossy variants obtained before enhancement of virulence and in matt variants obtained after enhancement of virulence, both glossy and matt variants from the same strain were studied. Of the matt colonies (types 1, 12, 14, 17, 18, and 29), all six cultures contained two NTS antigens. The glossy variants showed one antigen in four of six cases. It was established that the first antigen, identical to that of the TST fraction, could be detected both before and after the increase in virulence (in glossy and matt variants). After enhancement of virulence, an increase in the amount of the first antigen was shown. The second NTS antigen was detected only after enhancement of virulence in the cultures forming matt colonies or in some cultures forming matt colonies before enhancement of virulence. The first antigen was revealed in streptococci of groups C and G. NTS antigens were not detected in staphylococcal cultures in spite of a 20fold concentration of HCl extracts. In some cases, while HCl extracts prepared from the cultures of streptococci of several types after enhancement of virulence were being tested, additional NTS antigens destroyed by trypsin were detected.
DISCUSSION A number of data indicate that the two trypsin-resistant antigens detected in HCl extracts are PGP and E4 antigen. The following evidence supports this: their electrophoretic mobility to the anode; the identity ofboth antigens in spite of different electrophoretic mobilities (27); the similarity of the E4 antigen obtained by the method of Wilson and Wiley (27) to the PGP isolated by preparative electrophoresis from the HCl extracts; an increased phosphorus content in the fraction containing PGP; and the presence of a substance in the hydrolyzed PGP fraction with a mobility, upon paper chromatography, similar to that of glycerophosphate. In addition to polysaccharide, PGP, and E4 antigen, two NTS antigens sensitive to trypsin were detected, by immunodiffusion methods, in HCl extracts prepared from group A streptococci of different types. These antigens are not identical to PGP, E4 antigen, polysaccharide, or peptidoglycan. NTS antigens presumably are types of cell wall protein because they do not react with the
25
sera of animals immunized with the antigens of membranes, cytoplasm, or L-forms of streptococci group A and are detected in HCl extracts prepared from cell walls. The first NTS antigen identical to one of the antigens of the TST fraction was found in a majority of cultures of different types before and after enhancement of virulence. This antigen was found in cultures producing glossy as well as matt colonies. The second NTS antigen was revealed most frequently after enhancement of virulence by the Becker method (3). Before enhancement of virulence, the second NTS antigen was found only in some cultures producing mainly matt colonies. In the study of the same cultures before and after enhancement of virulence, the second NTS antigen was detected only in cultures possessing increased virulence and forming matt colonies. It is known that streptococci forming glossy colonies are readily phagocytized in human blood, whereas matt colonies are more characteristic of the virulent cultures surviving in human blood (3, 25). Therefore, of interest is the appearance of the second antigen after enhancement of virulence by the Becker method and its occurrence in the cultures forming matt colonies. In this connection, detection of a second NTS antigen in the streptococcal cultures might be used as an index of virulence. The first NTS antigen was detected in strains belonging to groups C and G. Both NTS antigens were not revealed in the staphylococcal cultures. The NTS antigens tested should obviously be assigned to a category of nonprotective antigens according to their inability to stimulate the production of protective antibodies. The NTS antigens under study differ from the trypsin-resistant R-antigen detected by Lancefield (11) in cultures of type 28, but one of the previously described R-antigens (26), as well as the NTS antigens, is sensitive to trypsin. Other authors (21, 24) have described the socalled M-associated protein antigen(s) sensitive to trypsin. The M-associated protein antigen common to type 12 and other types investigated by Vosti (21) differs in electrophoretic mobility from those of the NTS antigens revealed in our work with types 1, 5, and 29. However, NTS antigens ofthe latter types are identical to NTS antigens of type 12. Therefore, it cannot be excluded that the antigen described by Vosti (21), although it has a different electrophoretic mobility, contains an antigenic determinant identical to one of the NTS antigens under study. So far it has not been established whether the M-substance and NTS antigen(s)
26
LYAMPERT ET AL.
are different proteins or antigenic determinants of one molecule (21). Therefore the possibility cannot be excluded that the antigen(s) investigated by Vosti (21) and NTS antigens under study are different fragments of the Mprotein. It is likely that the NTS antigens described in our work are fragments lacking type-specific determinants. First, sera reacting with both NTS antigens did not give additional reactions with the type-specific antigen when NTS antigens were tested in the homologous system. Second, sera containing antibodies to both NTS antigens did not show M-antibodies with a culture of the homologous type by the bactericidal test. In addition, the study of HCl extracts obtained from a culture of type 1 revealed that type-specific and NTS antigens had different electrophoretic mobilities. These data do not exclude the possibility that the M-substance and NTS antigens in whole microbial cells are different antigenic determinants of one protein molecule. Moreover, in some cultures, some of the fragments formed upon extraction in HCl are possibly deprived of the type-specific Mdeterninant, whereas other fragments contain it. The HCl extracts obtained from some cultures after enhancement of virulence apparently contain additional NTS antigens. This opens prospects for a further search of common protective antigens as well as other NTS antigens belonging to a nonprotective category. It might be expedient to employ cultures of streptococci or fractions with a large amount of NTS antigens for absorption of sera containing M-antibodies while typing group A streptococci and determining M-proteins. It is likely that delayed-type hypersensitivity to the first NTS antigen develops in human streptococcal infections since an analogous antigen is demonstrated in the TST fraction to which an intensive delayed-type hypersensitivity can be observed in streptococcal diseases and in experimental animals (13, 16). The study of antibodies or delayed-type hypersensitivity to the second NTS antigen is of special interest because this antigen is apparently characteristic of only highly virulent cultures of group A streptococci. One of the crossreactive antigens of streptococci and of human and animal myocardium is contained in streptococcal cultures of different types. This antigen apparently has an electrophoretic mobility similar to that of the NTS antigens (15). In this connection, an investigation into the relationship between NTS antigens and cross-reactive antigens should be undertaken.
INFECT. IMMUN. LITERATURE CITED 1. Beachey, E. H., H. Alberti, and G. H. Stollerman. 1969. Delayed hypersensitivity to purified streptococcal M protein in guinea pigs and in man. J. Immunol. 102:42-52. 2. Beachey, E. H., and G. H. Stollerman. 1973. Mediation of cytotoxic effect of streptococcal M-protein by nontype-specific antibody in human sera. J. Clin. Invest. 52:2563-2570. 3. Becker, C. G. 1964. Selection of group A streptococci rich in M-protein from population poor in M-protein. Am. J. Pathol. 44:51. 4. Burrous, S., F. S. M. Grylls, and J. S. Harrison. 1952. Paper chromatography of phosphoric esters. Nature (London) 170:800-801. 5. Fox, E. N., and M. K. Wittner. 1968. Antigenicity ofthe M proteins of group A hemolytic streptococci. IV. Cross-reactivity between serotypes. J. Immunol. 100:3945. 6. Fox, E. N., M. K. Wittner, and A. Dorfman. 1966. Antigenicity of the M proteins of group A hemolytic streptococci. III. Antibody responses and cutaneous hypersensitivity in humans. J. Exp. Med. 124:11351151. 7. Freimer, E. H. 1968. The protoplast membrane of the group A streptococcus, p. 54-67. In R. Caravano (ed.), Current research on group A streptococcus. Excerpta Medica Foundation, New York. 8. Goudie, R. B., C. H. W. Horne, and P. C. Wilkinson. 1966. A simple method for producing antibody specific to a single selected diffusible antigen. Lancet ii:12241226. 9. Jackson, R. W., and M. Moskowitz. 1966. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2205-2209. 10. Krause, R. M. 1967. Preparation of cell-wall antigens from gram-positive bacteria, p. 34-40 In A. Curtis (ed.), Methods in immunology and immunochemistry, vol. 1. Academic Press Inc., New York. 11. Lancefield, R. C. 1957. Differentiation of group A streptococci with a common R antigen into three serological types with special reference to the bactericidal test. J. Exp. Med. 106:525. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Lyampert, I. M. 1972. Etiology, immunology and immunopathology of rheumatic fever. (In Russian) Medizina, M. 14. Lyampert, I. M. 1973. Virulence factors and protective antigens of the group A streptococcus. (In Russian) Vest. Akad. Med. Nauk SSSR 11:76-83. 15. Lyampert, I. M., and T. A. Danilova. 1975. Immunological phenomena associated with cross-reactive antigens of microorganisms and mammalian tissues. Prog. Allergy 18:423-477. 16. Lyampert, I. M., M. N. Smirnova, and N. Y. Semina. 1967. Delayed-type hypersensitivity and the formation of antibodies in animals sensitized with group A streptococcus antigens. J. Hyg. Epidemiol. Microbiol. Immunol. 11:160-170. 17. Maxted, W. R. 1956. The indirect bactericidal test as a means of identifying antibody to the M antigen of Streptococcus pyogenes. Br. J. Exp. Pathol. 37:415422. 18. Myoda, T. T., G. G. Wiley, and P. N. Bruno. 1973. Cross-reactions among group A streptococci. IV. Extraction, separation and purification of two protective antigens of type G, cocci. J. Immunol. 111:249-259. 19. Osterland, C. K., E. J. Miller, W. W. Karakawa, and R. M. Krause. 1966. Characteristics of streptococcal
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group-specific antibody isolated from hyper-immune rabbits. J. Exp. Med. 123:599-613. Shuratova, S. G., V. V. Akimova, and V. P. Bukhova. 1974. Trypsin-sensitive common antigens in HCI-extracts obtained from group A streptococci of different types. (In Russian). J. Microbiol. Epidemiol. Immunol. 7:11-17. Vosti, K. L. 1975. Characterization of a non-type-specific antigen(s) associated with group A streptococcal type 12 M protein. Infect. Immun. 11:1300-1305. Vvedenskaya, 0. I., I. M. Lyampert, and I. V. Shved. 1972. Cross-reactions in the bactericidal test on studying cultures of the group A streptococci of different types. (In Russian) J. Microbiol. Epidemiol. Immunol. 4:97-100. Weil-Malherbe, H., and R. H. Green. 1951. The catalytic effect of molibdate on the hydrolysis of organic phosphate bonds. Biochem. J. 49:286-292.
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24. Widdowson, J. P., W. R. Maxted, and A. M. Pinney. 1971. An M-associated protein antigen (MAP) of group A streptococci. J. Hyg. 69:553-564. 25. Wiley, G. G., and P. N. Bruno. 1968. Cross reactions among group A streptococci. I. Precipitin and bactericidal cros-reactions among types 33, 41, 43, 52 and Ross. J. Exp. Med. 128:959-968. 26. Wiley, G. G., and P. N. Bruno. 1970. Cross-reactions among group A streptococci. III. The M and R antigens of type 43 and serologically related streptococci. J. Immunol. 105:1124-1130. 27. Wilson, A. T., and G. G. Wiley. 1963. Immunoelectrophoretic studies of the C, M, PGP, E4, F and E antigens of serotype 17 streptococci. J. Exp. Med. 118:527556. 28. Zilber, L. A., and G. I. Abelev. 1962. Virology and immunology of cancer. (In Russian) Meditzina, M.
