Werner Köhler

A New Source of Antibody‐Like Substances Having Anti‐Blood Group Specificity: A Discussion on the Specificity of Helix Agglutinins

Summary. A new source of agglutinins has been found in snails (albumin gland) and in many sorts of fish roe. These agglutinins do not only agglutinate red cells of various origin before and (or) after treatment with several enzymes (pronase, neuraminidase), but also different strains of bacterial microorganism. These reactions are not unspecific, but follow the rule of strong chemospecificity as could be demonstrated in several cases by haemagglutination inhibition and immunodiffusion tests, especially with the agglutinins from Helix snails, where artificially conjugated antigens were included, as well as a discussion of the nomenclature and topo‐chemical arrangement of the receptors.

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.

Expression of a streptokinase gene from Streptococcus equisimilis in Streptococcus sanguis

SummaryUsing recombinant DNA techniques, we introduced a previously cloned streptokinase gene from Streptococcus equisimilis into the Challis strain of S. sanguis (group H). The gene was expressed in the new host under the control of its own promoter and the gene product had biological properties identical to authentic streptokinase. However, the molecular weight of cloned streptokinase (42 K) as expressed by S. sanguis was substantially lower than that of authentic streptokinase (47 K). Since the cloned streptokinase gene encoded a 47 K mature protein, the lowered molecular weight of S. sanguis streptokinase may reflect posttranslational proteolytic cleavage, which leaves the biological activity of the gene product and its serological reactivity unimpaired.

Routine Identification of Group-C Streptococci by Means of an Agglutinin (Protectin) from the Albumen Gland of the Edible Snail, Helix Pomatia

Summary A crude extract of the albumen gland of the edible snail, Helix pomatia, was used for the routine identification of group-C streptococci. The reaction is specific; all of the 338 group-C strains reacted with the extract but none of either 3560 group-A or 262 group-G strains. The test may be performed by slide-agglutination with trypsinised cells, or by capillary precipitation or agar-gel immunodiffusion tests with formamide extracts of streptococci.

Isolierung und Charakterisierung von erythrogenen Toxinen I. Untersuchung des von Streptococcus pyogenes, Stamm NY-5 gebildeten Toxins A

Abstract 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 characterization of two new low molecular weight proteins (LMPs) from Streptococcus pyogenes.

Two novel extracellular mitogenic substances were isolated from Streptococcus pyogenes strain NY-5 and characterized. The purification steps involved an initial enrichment of the proteins from culture supernatant by silica gel adsorption, followed by ion exchange chromatography and gel filtration. The purified materials were homogeneous in SDS-PAGE, showed estimated molecular weights of 12 kD and isoelectric points of 4.7 and 4.3, respectively. Both proteins (LMP-12k-4.3pI and LMP-12k-4.7pI) demonstrated lymphocyte transformation activity at a concentration of 0.1 microgram/ml. The LMP-12k-4.7pI showed a 69.2% homology of the amino acid sequence with that of a phosphocarrier protein of Staphylococcus aureus and with a total identity in the active centre. The same protein was also isolated from streptococcal group C strain H46A with an N-terminal amino acid sequence being identical. The LMP-12k-4.7pI demonstrated biochemical properties identical with those of the earlier described streptococcal pyrogenic exotoxin type D. The LMP-12k-4.3pI did not show such a clear relation to other functional proteins.

Haptoglobin: An immunoregulatory role in tuberculosis?

A significant correlation was found between levels of haptoglobin in sera from Indonesian patients with tuberculosis and the extent to which these sera suppressed mitogen-driven activation of normal lymphocytes in vitro. As in a previous study we showed that there was an inverse relationship between haptoglobin levels and the circulating lymphocyte count in the same group of patients, it is now suggested that this protein has an immunoregulatory role in tuberculosis. Similar observations have been made by other workers on patients with cancer. There was no difference in the distribution of the 3 allotypes of haptoglobin in patients and healthy subjects nor was there any association between such allotypes and the lymphocyte count, the dermal reactivity to tuberculin or the degree of suppression.

Purification and characterization of streptococcus pyogenes erythrogenic toxin type a produced by a cloned gene in streptococcus sanguis

The gene of Streptococcus pyogenes erythrogenic toxin type A (speA) 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 speA gene specified an ET A which had a molecular weight identical to that of ET A from S. pyogenes previously reported from this laboratory. NH2-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 speA gene according to Weeks and Ferretti and different from the sequence published by Johnson et al.

Scarlet Fever and Types of Erythrogenic Toxins Produced by the Infecting Streptococcal Strains

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.

Tissue Cages for Study of Experimental Streptococcal Infection in Rabbits: I. Production of Erythrogenic Toxins in vivo

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 SF130, while ET type C was produced by strain T18. Differences between production of toxins in vitro and in vivo might be of significance for the understanding of the pathogenetic mechanisms in streptococcal infection.