Jörg-Hermann Ozegowski

Isolation and characterization of erythrogenic toxins. V. Communication: identity of erythrogenic toxin type B and streptococcal proteinase precursor.

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.

Purification and characterization of hyaluronidase from streptococcus agalactiae

Hyaluronidase from two different strains of Streptococcus agalactiae was purified and characterized. The purification was performed successively by chromatography and rechromatography on phenylsepharose, gel filtration with FPLC on Superdex G 200 and isoelectric focusing. The purified hyaluronidase had an isoelectric point of 8.75 and a molecular weight of approximately 116,000 D. It showed maximal enzyme activity at pH 6.30 and 40 degrees C. The Michaelis constant was estimated to be 8.17 x 10(-2) mg/ml. Hyaluronidase was stimulated only by Mg++ and inhibited by Zn++, Al , Cu++ and Fe++ at a final concentration of 10 mmol/l, respectively. The enzyme splitted hyaluronic acid and in low amounts dermatan sulphate and chondroitin sulphate A. Additionally, synthetic polyanions (like polymers of gentisic acid with formaldehyde and hydroxy sulphonic acid with formaldehyde) turned out to be also potent inhibitors of the enzyme.

Occurrence of extracellular hyaluronic acid and hyaluronatlyase in streptococci of groups A, B, C, and G.

Streptococci of serological groups A (GAS), B (GBS), C (GCS) and G (GGS) were examined in vitro using an optimized medium in respect of their ability to produce hyaluronic acid (HA) and hyaluronatlyase (HY). In this study, 614 GAS (including 123 streptococcal toxic shock syndrome strains, STSS), 247 GBS, 225 GCS and 143 GGS were investigated in qualitative and quantitative tests. Only 4% of GAS and 2.7% of GCS were able to express HA. In contrast to GAS, isolates of GCS showed a highly specific HA formation (to 1 g HA/g dry biomass). In all strains of GBS and GGS, not even a single isolate was positive for HA. HY expression was detectable in all four serological groups. In GAS, only 12.5% of strains were positive; the most common types being 22 and 4, whereas in GBS, GCS and GGS, 72.1%, 84% and 85.3% of isolates, respectively, could be reported as positive. The data suggest that the HA capsule only plays a secondary role in infections caused by GAS strains pathogenic for humans.

Tissue cages for study of experimental streptococcal infection in rabbits III. Influence of immunization with erythrogenic toxin type A (ET A) and its toxoid on subsequent infection with an ET A producing strain

Purified erythrogenic toxin type A (ET A) and the corresponding toxoid, prepared by formalin treatment, were used for the immunization of rabbits (200 micrograms per rabbit). The impact of anti-erythrogenic toxin and toxoid immunity was investigated under the conditions of experimental infection with the ET A-producing Streptococcus pyogenes strain SF 130 (type 1). Whereas non of the immunized rabbits (n = 14) died after infection, 40% of nonimmunized animals did not survive (Table 1). The increase of the spleen weight after infection was significantly smaller in the immunized groups (Table 2). The immunized rabbits responded after infection with a significantly lower increase of fever which did not exceed 0.8 degree C (2 degrees C in infected non-immunized animals). Humoral antibodies to ET A were detected after immunization by means of ELISA. The challenge infection acted as a booster leading to a further increase of antibodies. The antibodies were found to be neutralizing the nonspecific mitogenicity of ET A in vitro in relation to the antibody titer. Cell-mediated immunity was tested in the lymphocyte transformation reaction with peripheral lymphocytes. The nonspecific mitogenicity of ET A, ET B, ET C and Con A was pronounced after immunization, whereas the nonimmunized rabbits responded to these antigens to a lower degree. The toxoid was found to be nonmitogenic. The altogether higher lymphocyte stimulation was also observed using spleen lymphocytes of immunized animals after infection.

Isolierung und Charakterisierung erythrogener Toxine von Streptococcus pyogenes: 3. Mitteilung: Vergleichende Untersuchungen erythrogener Toxine vom Typ A

Abstract 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 27000 and 28000; i.p. 5.2). They were serologically identical by immunodiffusion (Oucbterlony), and no spur formation was seen with crude concentrates of the toxins produced by the remainder of the strains.

Isolierung und Charakterisierung von erythrogenen Toxinen VIL Untersuchung des von Streptococcus pyogenes gebildeten erythrogenen Toxins Typ C

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.

Einfluß physikalischer parameter auf die bildung extrazellulärer streptokokkenproteine in pH-stabilisierten kulturen II. Mitteilung: Der einfluß der temperatur auf die bildung von extrazellulären streptokokkenprodukten

Summary The influence of the cultivation temperature on growth and production of extracellular proteins was examined for the group C streptococcus S. equisimilis , strain H 46 A. The temperature range studied was from 28°C to 43°C. The analytical methods used included the determination of enzyme activities, isoelectric focusing and crossed Immunoelectrophoresis. The strain was able to grow in the entire temperature range with maximum growth at 28°C. The amounts of extracellular proteins elaborated depended on both the cultivation temperature and the biomass production. The specific temperature for optimal production of certain enzymes were determined. The majority of these was produced at optimal rate at a temperature of 28°C. Besides isoelectric focusing and crossed Immunoelectrophoresis showed that additional proteins exist, the production of which was reduced at low temperatures. Isoelectric focusing revealed that in a temperature range from 28°C to 37°C approximately 28 different extracellular proteins were produced. In the same temperature range the presence of about 30 antigens was demonstrated by crossed immunoelectrophoresis.

Kinetics and Regulation of Erythrogenic Toxins Type A and C During Growth of Streptococcus Pyogenes

The production of erythrogenic toxins type A (ETA) and C (ETC) is described as a function of growth kinetics. Group A streptococcal strains C 203 S and NY 5 were cultivated in yeast-peptone extract, Todd-Hewitt medium and a synthetic medium. Two main growth phases occurred during growth: a first logarithmic phase and a second linear phase. These phases were separated by a short stationary interphase caused by limitation of the amino acids L-serine and L-leucine. Maximum production of ETC was observed during the logarithmic phase, it was correlated to a high level of viable cells. ETA was produced mainly during the short stationary interphase. The production of ETC is regulated by L-isoleucine. A stagnation or reduction of the concentration of viable cells was observed during the interphase. The phosphate limitation caused during streptococcal growth induced expression of the extracellular protein phosphatase and surprisingly, of a serine proteinase activity. The association between these results and the pathogenicity of streptococci is discussed.

Kinetics of growth and product formation in cultures from streptococci of groups a and c

During growth of streptococci of Lancefield groups A and C in a culture medium containing glucose, yeast extract and peptone, two main growth phases occur: growth phase I and growth phase II (diauxic growth). They are separated by a short stationary phase (1st stationary phase). The diauxic growth is caused by transient limitations as well as the availability of new sources of the amino acids L-serine and L-arginine. Growth phase I consists of an exponential and a nearly linear part. These growth kinetics are reflected by the kinetics of gas metabolism as well as by product formation. Hyaluronic acid is formed during the nearly linear phase whereas the enzyme alkaline phosphatase, is exclusively excreted in the 1st stationary phase. Also carbon dioxide and L-lactate are mainly produced in a growth phase-dependent mode. In the late stationary phase (2nd stationary phase) more oxygen is consumed whereas the demand for oxygen in the 1st stationary phase is nearly zero.

Isolierung und charakterisierung von erythrogenen toxinen VIII. Die Reinigung eines biologisch aktiven proteins vom molekulargewicht 10000 (LMP-10k) aus kulturfiltraten des streptococcus pyogenes, Stamm NY-5. Beziehung zum erythrogenen toxin typ A

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.