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Group A streptococcal strains were isolated from the throats of 46 children suffering from scarlet fever. For detection of erythrogenic toxins (ETs), the culture supernatants were concentrated 100 times by ethanol precipitation and solubilisation in acetate buffer. ELISA was used to identify ETA and double immunodiffusion to identify ETB and ETC. The presence of the ETA gene was detected by a specific DNA probe. ETA (alone or in combination with ETB and/or ETC) was found in 51.9% of the strains, ETB (alone or in combination with ETA and/or ETC) in 76.9% and ETC (in combination with ETA and ETB) in 28.9%. Only 5.8% of strains did not produce any detectable ET. In SDS-PAGE, supernatants of ETB-producing strains showed a pronounced band in either the region of the proteinase zymogen or the active proteinase. There was no correlation between the type of erythrogenic toxin and the serological M or T type of the producing strain. The mitogenic potency of culture supernatants did not differ significantly irrespective of the toxin type(s) present. Culture supernatants of strains without a detectable amount of the known ETs were highly mitogenic, indicating the production of other streptococcal mitogens. A correlation with clinical symptoms was determined with regard to exanthema and fever. Strains producing two or three toxins caused a more intense exanthema. Patient temperature was higher (greater than or equal to 38 degrees C) when the infecting strain produced ETB. The toxin-producing patterns of the strains of this study were compared with those isolated during the last epidemic outbreak of scarlet fever in East Germany.

The production of type A erythrogenic toxin was determined in 11 strains of Streptococcus pyogenes (serological group A). All strains produced this toxin, at quantities varying between 16 mg/l (strain "Smith") and 0.03 mg/l (strain T18). The erythrogenic toxins of 5 strains were purified by adsorption to silicate, elution, precipitation with ammonium sulfate, treatment with calcium phosphate, CM-Sepharose chromatography and gel filtration on Sephacryl S200. The preparations were found to be homogenous in SDS electrophoresis and isoelectric focusing (m.w. between 27 000 an 28 000; i.p. 52). They were serologically identical by immunodiffusion (Ouchterlony), and no spur formation was seen with crude concentrates of the toxins produced by the remainder of the strains.

Reference strains of Streptococcus pyogenes and strains from recent epidemics and sporadic cases of scarlet fever were examined for their ability to produce erythrogenic toxin type A (ET A) by ELISA and double immunodiffusion (Ouchterlony) using an anti-ET A antibody purified by affinity chromatography. Of the reference strains (most of them isolated before 1945) 16/51 produced more or less ET A (Table 1). ET A synthesis is strain-specific, but not type-specific. Well-known toxin producers like the strains NY-5; 594 or "Smith" produce up to 16.000 micrograms/l under optimal culture conditions. Type 3 strains isolated from scarlet fever patients during the outbreak 1972/73 seem to belong to one clone as evidenced by the uniform SDS-PAGE pattern: They were found to produce 5-200 micrograms/l (mean 68 micrograms/l) ET A only. Type 3 strains from sporadic cases, isolated 10 years later, produced 0-138 micrograms/l (mean 30 micrograms/l). Strains of the type 1 clone, causing the epidemic in 1982/83 produced only 0.75-10 micrograms/l (mean 8 micrograms/l) ET A (Table 3). Only a few strains of S. pyogenes isolated 1984 or later synthesized ET A but they were found more often to produce ET B (proteinase precursor) in batch cultures. S. pyogenes strains seem to have lost their ability to produce large amounts of ET A during the last decades. Because this toxin must be considered as a pathogenicity factor the decrease in toxin production may be one reason for the present mild form of scarlet fever.

Infection of rabbits with erythrogenic toxin producing streptococcal strains caused a marked increase of humoral antibodies, which was detected by immunoprecipitation and ELISA. An antibody response directed towards the erythrogenic toxin type A was demonstrated by fused rocket immunoelectrophoresis. All toxinogenic reference strains produced ET type A under in vivo conditions despite that this toxin was not always demonstrated under in vitro conditions. The infection resulted in an increase of mitogenic response of peripheral lymphocytes to the initial nonspecific mitogenic erythrogenic toxins, whereas the Con A stimulation was depressed starting 14 days after infection and lasting during a period of 90 days. Since a normal antibody response was evoked, it seems likely that the T helper cell function was not affected.

M proteins of Streptococcus pyogenes types 1 and 12 were purified by immunochromatography on immobilized type-specific opsonizing antibodies. The M proteins were characterized serologically and biochemically. They absorb after immobilization opsonizing antibodies and only precipitate with homologous antisera in immunodiffusion. In SDS PAGE they show one main band, corresponding to mol. weights of 5 × 10 4 (type 1) and 5.4 × 10 4 (type 12), accompanied by faint lines, which form with the main band a precipitation line of identity in SDS-crossed immunoelectrophoresis. Both M proteins aggregate blood platelets and clot fibrinogen.

Summary Some in vivo biological activities of a purified erythrogenic toxin preparation from strain NY-5 of group A streptococci (ET NY-5) were studied. After intracutaneous administration in a dose of minimally 5 × 10−8 mg, the toxin provoked a skin reaction in old guinea pigs. No skin reaction could be elicited in young adult rabbits. ET NY-5 was pyrogenic for rabbits; the MPD-4 amounted to 0.017 μg/kg intravenously. The pyretic response had a delayed onset and then rose gradually up to hour 5 postinjection. Intravenous administration led to changes in circulating leukocyte counts, viz. brisk acute granulocytopenia and protracted lymphopenia. The method of pyrogenic cross tolerance revealed that ET NY-5 contains two types of pyrogenic exotoxin, A and C.

Summary The “classical” method for purification of erythrogenic toxin type A results in two products: erythrogenic toxin type A and a low molecular weight protein, m. w. 10.000 (LMP-10k) with mitogenic activities. LMP-10k was purified from culture supernatants of S. pyogenes (group A) by CM-Sepharose CL-6B chromatography and Sephacryl S-200 and Sephadex G75 gel filtration to a high degree of purity with minimal amounts of residual erythrogenic toxin A. The isoelectric point of LMP-10k is the same as for erythrogenic toxin A: 5.2. The immunogenic acitivty is low, only one of two rabbits produced anti-LMP-10k-antibodies after a prolonged course of immunization. On the other hand it is possible to induce antierythrogenic toxin A-antibodies by immunization with LMP-10k preparations contaminated with small amounts of erythrogenic toxin A. Possibly the data given by some authors for the m. w. of erythrogenic toxin type A as 8 000 D are the results of a mix-up with co-purified LMP-10k.

Summary Erythrogenic toxin (ET) type C was purified from culture filtrates of Streptococcus pyogenes strains NY 5, T 18, and AT 13. Methods used included ammonium sulfate precipitation, phosphate precipitation, column chromatography on CM-Sepharose and Sephadex G 100, and isoelectric focusing in Sephadex gel. The molecular weight was determined by SDS gel electrophoresis as 25,500. The preparation reacted only with homologous antiserum (anti-C), but not with antitoxins types A or B in double diffusion tests. The isoelectric point was determined to be 6.8 by analytical isoelectric focusing. Also the amino acid composition was determined. The toxin was found to be mitogenic (as well as pyrogenic and toxic for rabbits). The ET type C is digested by trypsin, pepsin, chymotrypsin and Pronase E, but is rather stable when treated with papain or streptococcal proteinase.

Some in vitro reactions involved in the mitogenic activity of a purified erythrogenic toxin from the group A streptococcus strain NY-5 (ET NY-5) were studied. The optimal dose in the human blood lymphocyte transformation test was 1 to 10(-1) microgram/ml lymphocyte culture, the maximum of 3H-thymidine incorporation was on day 3 or 4. The mitogenic activity showed the signs of nonspecificity, nevertheless, a specific mitogenic effect could not be ruled out either. When Con A and ET NY-5 were added simultaneously in high or low doses at the beginning of lymphocyte cultivation, antagonism or an additive effect was observed, respectively. Incubation of lymphocytes with ET NY-5 resulted in a decrease of mitogenic activity in the supernatants. Erythrocytes had no similar binding activity. ET NY-5 acts as a T-cell mitogen; 98% of ET NY-5-stimulated lymphoblasts formed E rosettes with sheep red blood cells. Thin-layer isoelectric focusing experiments revealed two mitogenic peaks corresponding to toxin types A and C in the ET preparation.

Summary A toxoid of erythrogenic toxin type A (ET A) was prepared by formaldehyde treatment. Already 15 min after exposure to formaldehyde in isoelectric focusing the ET A band at pH 5.2 shifted to a band at pH 4.5. In Ouchterlony double diffusion test ET A and its toxoid were found to be identical, in fused rocket immuno-electrophoresis a reaction of partial identity was seen. Formaldehyde treatment of ET A resulted in an apparent increase of electrophoretic mobility. In contrast to ET A, its toxoid is non-mitogenic, non-pyrogenic and has lost its ability to induce delayed type hypersensitivity. Binding of ET A toxoid to human peripheral lymphocytes is of the same magnitude as binding of gold-labelled ET A.

An erythrogenic toxin was isolated from culture supernatants of Streptococcus pyogenes, strain NY-5 (type 12) grown in yeast extract-pepton-dialysate medium. After concentration by evaporation, a crude material was obtained by precipitation with ethanol. This material was prepurified by ion exchange chromatography using a DEAE-Sepharose Cl-6B column. After precipitation of the active material with (NH4)2SO4 the redissolved precipitate was dialyzed against acetate buffer and rechromatographed on a CM-Sepharose Cl-6B column. The toxin was obtained by stepwise elution with 0.02 M acetate buffer, pH 5.0, and 0.05 M phosphate buffer, pH 6.5, followed by a last purification step on a Sephacryl S-200 column. The purified toxin behaved homogenously in SDS electrophoresis, the molecular weight being about 28000. Amino acid analysis showed only one cysteine residue per molecule. The molecule was insensitive to 2-mercaptoethanol and alkylation. Isoelectric focusing yielded two narrow bands having an isoelectric point of 5.2. The toxin showed a serological reaction of identity with an antiserum against streptococcal pyrogenic exotoxin A (SPE-A) but was shown to have a different molecular weight. The purified material (erythrogenic toxin A) was pyrogenic and toxic for rabbits as well as mitogenic for human lymphocytes. A positive skin reaction in guinea pigs could be obtained by as little as 5 × 10−8 mg.

The separation of a mitogenic substance in M protein preparations of Streptococcus pyogenes, type 1 and type 12, is described. The isolation was achieved by gel chromatography on Biogel A 0.5 m, and by immunochromatography on immobilized type specific antibodies. In the delayed cutaneous hypersensitivity test the immunochromatography purified M 1 protein caused erythema but no unspecific mitogenicity could be found by lymphocyte transformation test. In neutralization experiments the mitogenic activity of M protein preparations was specifically inhibited by anti-erythrogenic toxin antisera.

Tissue cages implanted subcutaneously were used to infect rabbits with erythrogenic toxin (ET) producing streptococci. The in-vivo production of ET was followed during the infection by immunoprecipitation analyses of the tissue cage fluid (TCF). ET types A and C were mainly detected during the first week of infection, but, as late as 4 weeks after the inoculation, ET was occasionally found in TCF. The nonspecific mitogenic activity of ET on human lymphocytes was also used as a biological marker to recognize ET in TCF. Mitogenic activity was detected in 90% of samples during the first week. In order to characterize the mitogenic material released by growing streptococci, TCF was electrofocused in polyacrylamide gel. The eluates of sliced gels were checked for mitogenic activity and compared with a purified ET preparation containing ET types A and C. It could be verified that ET type A was produced under in-vivo conditions by strains NY-5 and SF 130, while ET type C was produced by strain T 18. Differences between production of toxins in vitro and in vivo might be of significance for the understanding of the pathogenetic mechanisms in streptococcal infection.

The gene of Streptococcus pyogenes erythrogenic toxin type A ( spe A) has been previously cloned in Streptococcus sanguis (Challis) and produces extracellular erythrogenic toxin type A (ET A). The ET A produced and secreted by this heterologous host was purified to homogeneity and shown to have properties identical to ET A produced by S. pyogenes strain NY-5; i.e., serological identity in immunodiffusion, migration in SDS-polyacrylamide gel electrophoresis, mitogenic activity, inhibition of mitogenic activity by specific antibody, and precipitation by an international scarlatina antitoxin preparation. The cloned spe A gene specified an ET A which had a molecular weight identical to that of ET A from S. pyogenes previously reported from this laboratory. NH 2 -terminal sequence determination of the purified protein showed the first nine residues to be gln gln asp pro asp pro ser gln leu; this is consistent with predictions made from the nucleotide sequence of the spe A gene according to Weeks and Ferretti (45) and different from the sequence published by Johnson et al. (23).

In a group of 48 children the following streptococcal antigens were tested by means of the lymphocyte transformation test: group A streptococcal M1- and M19-protein, mucopeptide, polysaccharides from group A and group C streptococci, erythrogenic toxins and streptokinase. Specific transformations were obtained only by M-proteins. Erythrogenic toxines induced an unspecific stimulation comparable to the mitogenic activity of phytohaemagglutinins. No reactions could be found with streptococcal mucopeptide, the group-specific carbohydrates of groups A and C-streptococci and streptokinase, resp. A significant difference was seen in the group of young children (mean age 5 years) between the specific transformability by M1-protein (a common type) and M19-protein (a rare type). No difference was seen with both M-proteins in the group of older children (mean age 11 years). In both groups of children no differences exist in the number of positive cutaneous reactions after injection of M1- and M19-proteins, but the intensity of reactions (diameters of erythema) were more pronounced with the M-protein of the common type 1.

Isoelectric focusing (IF) was used to separate erythrogenic toxins (ET) type A, B and C from concentrated culture filtrates of Streptococcus pyogenes strains. The ET's were identified by their mitogenic activity on human lymphocytes in the lymphocyte transformation test: purified ET type A appeared at pH 5.3, ET type C at pH 6.8 and ET type B at pH 7.5 to 8.5; the ET type B was only biologically active when PAGE IF was used. IF on Sephadex G100 failed to yield active B toxin. The application of as little as 0.1 μg ET type A to an isoelectric focusing gel was still sufficient to detect a mitogenic peak in the eluates. ET type A was identified in nine out of 10 culture filtrates, ET type C in 4 out of 10. Detection of ET type B (identical with streptococcal proteinase proenzyme) in culture filtrates after IF proved to be difficult. Here the pH of cultivation media and the auto-catalytic conversion of streptococcal proteinase proenzyme to activated proteinase have to be considered.

Production of erythrogenic toxin type B by Streptococcus pyogenes strain T19 was found to be strongly dependent on the pH of the cultivation medium. Maximum yields (greater than 100 mg of toxin/1) were obtained at pH 6.0. In contrast no toxin production was serologically detectable at pH values above 6.5. Purified B-toxin was shown to consist of two components when assayed by SDS-electrophoresis. The molecular weight of the two components was estimated to be 30 000 and 12 000. Isoelectric focusing revealed a heterogeneity of the preparation with isoelectric points between 8.0 and 9.0. Streptococcal proteinase precursor was isolated from culture supernatants of strains T19 and B220 by ammonium sulfate crystallization and purification on CM-Sepharose CL 6B. The protein obtained was homogeneous by SDS-gel electrophoresis and had a molecular weight of 44 000. After autocatalytic activation with mercaptoethanol two bands appeared corresponding to molecular weights 30 000 and 12 000. Isoelectric focusing of proteinase precursor preparations yielded a double band at pI 8.2-8.3. However, activation of precursor to active proteinase finally resulted in a change of the pI to 9.0. Erythrogenic toxin type B, streptococcal proteinase precursor, its intermediate activation products and the active proteinase itself reacted serologically identical with anti B-toxin antiserum. Streptococcal proteinase precursor provoked a delayed skin reaction and was pyrogenic as well as mitogenic. Its pyrogenic activity could be inhibited by antiserum against scarlet fever toxin (Wellcome Laboratories). We therefore believe erythrogenic toxin type B to be identical with streptococcal proteinase precursor. This helps to understand the heterogeneity of B toxin, its inactivation by trypsin and the different protocols for toxin production described in the literature.