Search

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15,529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.

Streptococcal protease precursor, secreted by the human pathogen Streptococcus pyogenes, becomes activated to a cysteine protease. The precursor and the mature enzyme appear to contribute to S. pyogenes virulence. The precursor protein was crystallized in the form of very thin flexible flakes. X-ray diffraction data were collected to 3.15 A resolution at 100 K using synchrotron radiation. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 41.6, b = 136.0, c = 156.7 A, beta = 95.7 degrees, and contain four copies of the protein in the asymmetric unit.

The mitogenic factor (MF) of group A streptococci has been reported to be a superantigen stimulating human T cells carrying Vbeta2, 4 and 8 and has been designated streptococcal pyrogenic exotoxin F (SPEF). MF was also shown to possess DNase activity. Here we have purified MF from culture supernatants of different Streptococcus pyogenes strains. Surprisingly, the MF preparations from different strains showed different Vbeta specificities depending on the expression of SPEC or SMEZ3 by the producing strain. Their mitogenic activity decreased upon further purification. In addition, the mitogenic activity could be only neutralized by antibodies against the basic streptococcal superantigens SPEC or SPEX (SMEZ3) but not by antibodies against MF itself although the latter were able to neutralize completely the DNase activity of MF. We found that streptodornase type B (SDB) was expressed in two molecular forms (SDBI and SDBII), differing only by one additional N-terminal arginine at SDBI. MF was found identical to the enzyme SDBII but is devoid of superantigenic properties and should no longer be called a superantigen or a pyrogenic exotoxin.

In this study we have investigated whether streptolysin O contributes to the virulence of group A streptococci. For this purpose we generated M-negative and SLO-negative mutants by insertion mutagenesis into the chromosome of an M type 1 strain. The inactivation of M1 protein expression was achieved by the construction of the integrative plasmid pSFABS, which contains the internal fragment abs of the emm1 gene. Integration of pSFABS by homologous recombination into the chromosome of strain 38 541 resulted in the generation of mutant EMM1. Inactivation of slo with plasmid pFWSLOD resulted in two different mutant forms. The homologous recombination with plasmid pFWSLOD carrying the two slo fragments slo1 (899 base pairs in the 5' region) and slo2 (709 base pairs in the downstream part) resulted in mutants SLO3, SLO4 and SLO17. In SLO17 a double crossover event took place with insertion of the spectinomycin resistance gene aad9 between the slo fragments 1 and 2. In mutants SLO3 and SLO4 the homologous recombination with the same plasmid led to the integration of the whole plasmid construct into the chromosome of strain 38 541. Both forms of mutation failed to express SLO. In mutant SLO4 additionally M1 protein expression was significantly decreased. The mutants EMM1 (M − , SLO + ) and SLO4 (M decreased, SLO − ) showed a reduced binding to collagen-coated surfaces. In contrast the mutants SLO3 and SLO17 (both M + , SLO − ) and the wild-type strain 38 541 (M + , SLO + ) showed an affinity to collagen similar to purified M1 protein. All mutants were less virulent for chicken embryos compared to the wild-type strain after infection by intravenous injection as well as by application onto the chorioallantoic membrane. The results show that besides M protein SLO can also influence virulence of group A streptococci. Moreover, it became obvious that streptococci need more than one tool to fully develop their infectious potential.

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.

The gene for NAD-glycohydrolase (nga) of group A streptococci (Streptococcus pyogenes) was identified and shown to be located immediately adjacent to the gene for streptolysin O (slo). The nga gene contains 1341 base pairs and encodes a protein of 447 amino acids, including an N-terminal signal peptide. Results from analysis with the polymerase chain reaction indicated that the nga gene is present in all of the strains tested. Functional extracellular NAD-glycohydrolase, also known as NADase, was detected among a wide variety of clinical isolates and known laboratory strains and shown to be present in 72% of 100 strains examined. In contrast, 92% of strains isolated from patients with invasive streptococcal infections were positive for NADase production.

Highly purified extracellular superoxide dismutase was obtained from Streptococcus pyogenes strain 12,714 (type 12) by adsorption of culture supernatant on phenyl-Sepharose following preparative isoelectric focusing of eluates and a final gel filtration purification on Superdex 200. The purified superoxide dismutase of S. pyogenes was found to be a homodimer. The monomeric protein had a molecular mass of 22,442 Da and an isoelectric point of 4.0. The enzymatic activity was strongly manganese-dependent. The N-terminal sequence of the purified mature protein was AIILPELPYAYDALEPQUFDA and corresponded to the first amino acids following the methionine initiation codon with no evidence of a leader sequence for the mature protein. The DNA sequence of the superoxide dismutase gene of strain 12,714 was found to be almost identical to the corresponding sequences reported in the gene bank data from other S. pyogenes serotypes and showed strong homology to superoxide dismutases from other Gram-positive bacteria.

In various cells including monocytes the cytokine interferon-gamma as well as lipopolysaccharide induce indoleamine 2,3-dioxygenase which degrades tryptophan to form L-kynurenine. We addressed the question of whether the exposure of human peripheral mononuclear cells to superantigens derived from streptococci is associated with tryptophan degradation in vitro.Peripheral blood mononuclear cells were exposed to streptococcal erythrogenic toxins A and B and a streptococcal-derived mitogen named BX. In addition, the myelomonocytic cell line THP-1 was treated with these toxin preparations.In peripheral blood mononuclear cells all three toxins induced tryptophan degradation. In parallel, production of interferon-gamma was found, and the tryptophan degradation could be blocked by antihuman interferon-gamma antibodies. Tryptophan degradation was not induced when the human myelocytoma cell line THP-1 was stimulated with these toxins, but there was a costimulatoty effect to interferon-gamma.In peripheral blood mononuclear cell culture streptococcal erythrogenic toxins are able to stimulate tryptophan degradation in humans via the induction of interferon-gamma production. There seems to be no direct effect on myelomonocytic THP-1 cells. Because some of the degradation products of tryptophan, such as quinolinic acid and kynurenic acid, are toxic, superantigen-driven degradation oftryptophan may play a role for example in the development of the toxic-shock-like syndrome associated with severe group A streptococcal infections.

We tested whether the exposure of human monocytic cells to streptococcal erythrogenic toxins A, B, C and a streptococcal-derived Mitogert BX is associated with synthesis of neopterin in vitro. Neopterin production was not induced when the human myelomono-cytoma cell line THP-1 was stimulated with these toxins, and there was only a slight co-stimulatory effect of streptococcal erythrogenic toxin A together with interferon-gamma stimulation. However, these toxins induced interferon-gamma and further neopterin production in peripheral blood mononuclear cells of three healthy individuals. This neopterin formation could be blocked by anti-human interferon-gamma. From our investigations we conclude that there is no direct effect of streptococcal erythrogenic toxins on neopterin production by monocytic cells. However, the data obtained in peripheral blood mononuclear cell culture imply that these toxins are able to stimulate neopterin production in humans via the induction of huge amounts of interferon-gamma.

NAD-glycohydrolase (NADase) was purified from culture supernatant fluids of group C streptococci by adsorption on silica gel, chromatography on hydroxyapatite and ion exchange on Mono S column. After inactivation of a chymotrypsin-like protease, a homogeneous enzyme was isolated with an N-terminal sequence of VSGKEGKKSDVKYEMTKVMEANATSSKEDKHVMHTLDKVM. According to serological methods, the purified enzyme of group C streptococci was identical to the group A enzyme showing a specific activity of 10 000 000 U mg-1. It did not attack NADH, NADP or NADPH. In addition, a streptodornase was isolated having an N-terminal sequence of KTVSVNQTYGE.

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein.

The streptokinase gene of Streptococcus equisimilis H46 was inactivated by plasmid insertion mutagenesis to study the relationship between elaboration of streptokinase and acquisition of cell-associated plasmin activity after incubation of wild-type and mutant cells in media containing plasminogen or plasmin. The results showed that H46A binds both the zymogen and active enzyme, generates surface-associated plasmin activity in the presence of plasminogen when producing streptokinase, and expresses its plasmin(ogen) receptor(s) independently of a functional streptokinase gene. At least part of the plasmin(ogen) binding capacity may be due to the glyceraldehyde-3-phosphate dehydrogenase type of receptor molecule, as judged by the detection of the corresponding gene.

Hybridoma cell lines producing monoclonal antibodies against streptococcal erythrogenic toxins type A and C were established from fusions of immunized BALB/c mice splenocytes with P3X63-Ag8.653 myeloma cells. Six MAbs recognize ETA and 11 MAbs bind to ETC. Two MAbs (designated ETA-2 and ETC-10) were produced in ascitic fluid and further characterized. ETA-2 (IgG2a) binds to ETA with an affinity constant of 1.8 × 1010 M−1 and ETC-10 (IgG1 binds to ETC with an affinity constant of 3.5 × 109 M−1. The specificities of the MAbs were evaluated by ELISA and immunoblotting. Both MAbs ETA-2 and ETC-10 are useful in developing specific double-sandwich ELISAs, in which the MAbs were used as solid-phase capture antibodies for the quantitative determinations of ETA and ETC.

In an effort to understand the molecular genetic basis of temporal variation in frequency and severity of bacterial disease, genetic relationships among strains of Streptococcus pyogenes that caused scarlet fever epidemics in Canada in the early 1940s and in eastern Germany in the 1960s to 1980s were studied. Application of multilocus enzyme electrophoresis and comparative sequencing of the gene (speA) encoding streptococcal pyrogenic exotoxin A (scarlet fever toxin) revealed that new waves of scarlet fever are associated with an increase in frequency of S. pyogenes clones carrying variant speA alleles. This finding suggests that the occurrence of new scarlet fever epidemics can be predicted by comprehensive monitoring of the frequency of S. pyogenes clones with variant toxin alleles.

Streptococcus pyogenes may cause tonsillitis, scarlet fever and so-called "streptococcal toxic shock-like syndrome" (STSS). These streptococci produce exotoxins which are implicated as superantigens in the pathogenesis of STSS and scarlet fever. Using human peripheral blood-derived mononuclear cells in vitro, such toxins were shown to induce neopterin production and degradation of the amino acid tryptophan to metabolites such as kynurenine by activating indoleamine (2,3)-dioxygenase via interferon-gamma. We investigated the sera of seven patients with streptococcal tonsillitis and of four patients with STSS. Those with STSS showed higher serum neopterin concentrations (median: 152 nmol/l; 95th percentile in healthy controls: 8.7 nmol/l) than those with tonsillitis (median: 12 nmol/l). Similarly, kynurenine to tryptophan ratios were increased in tonsillitis and extremely high in patients with STSS. Highly increased neopterin production and tryptophan degradation in patients with STSS suggest an association between a high degree of T cell activation and the severity of the disease manifestation.

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.

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-d-galactosamine/N-acetyl-d-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS–PAGE and MALDI-TOF analysis. In SDS–PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)2-Man]2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5′- or 3′-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.

Streptococcus pyogenes (GAS) causes about 90% of streptococcal human infections while group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Especially the human pathogenic GCS and GGS, Streptococcus dysgalactiae, subsp. equisimilis, account for 5–8% of the human streptococcal diseases like wound infections, otitis media, purulent pharyngitis and also streptococcal toxic shock syndrome. A defined superantigen so far was not identified in GCS and GGS strains. In the present investigation we screened DNA of GCS and GGS human isolates for the presence of genes for streptococcal pyrogenic exotoxins (spe) by hybridisation with probes that stand for the GAS genes speA, speC, speZ (smeZ), speH, speG, speI, speJ and ssa. In many GCS and GGS strains we found positive reactions with the probes speG, speJ and ssa, but not with the probes for the remaining genes under investigation. PCR amplification with subsequent sequence analysis of the PCR fragments revealed only the presence of the gene speG in GCS and GGS strains, while no DNA fragments specific for speJ and ssa could be amplified. Additionally, the upstream and downstream regions flanking speG in GGS strain 39072 were sequenced. Remarkable differences were found in the neighbourhood of speG between GAS and GGS sequences. Downstream of speG we identified in strain GGS 39072 two new open reading frames encoding proteins with no similarity to protein sequences accessible in the databases so far. In the compared GAS strains SF370 and MGAS8232, this segment, apart from some small fragments, had been deleted. Our analysis suggests that a gene transfer from GGS to GAS has preceded following deletion of the two genes orf1 and orf2 in GAS.

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3–5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.

A potent basic superantigen (designated streptococcal pyrogenic exotoxin X, SPEX/SMEZ3) was purified to homogeneity from culture supernatants of a Streptococcus pyogenes scarlatina strain of type 12 (genotype speA(-), speC(-)) and characterized. Sequence alignments revealed SPEX to be an allele of the streptococcal mitogens type Z (SMEZ). The N-terminal amino acid sequence of SPEX was found with LEVDNNSLLR to be identical to the recently described acidic superantigen SMEZ. Although SPEX/SMEZ genes were present in all of the streptococcal strains tested, a toxin production could only be detected in a small number of strains. The produced toxin concentration in the culture supernatants of positive strains differed between 0 and 20 ng ml(-1). The purified SPEX stimulated human T-lymphocytes with Vbeta8 specificity at extremely low concentrations (lower than 100 pg ml(-1)).

Many group A streptococci (GAS) bind the Fc part of IgG. In the present work, the possible influence of growth time and incubation atmosphere on the expression of the IgG binding activity by GAS of various serotypes was studied. Among 13 GAS reference strains, two categories were distinguished on aerobic incubation at 37 degrees C, one expressing similar IgG binding activity at 6 h and 18 h (types M1, M4, M13, M15), and a second one which showed higher binding at 6 h than at 18 h (M9, M14, M22, M25, M48, M49). Only one strain (M36) bound less IgG at 6 h than at 18 h. Seven of the strains (M5, M6, M22, M25, M36, M48, M49) showed higher binding of IgG when grown in a 5% CO2 atmosphere than in air, whereas one strain (M14) showed a reverse pattern and in the remaining five strains, no influence was found. Protease activity was detected in the growth supernatant of most of the strains. For five selected strains, the time of appearance of supernatant protease activity coincided with a decay of surface IgG binding activity. Purified streptococcal cysteine protease was found to reduce or abolish the binding of IgG by each of three studied strains (M1, M13 and M15) and of type M1 or M15 purified IgG binding material. When tested in the stationary phase, a majority of 62 clinical GAS isolates belonging to 6 different M types showed high protease activity but low binding of IgG. We conclude that streptococcal IgG binding is often better expressed on growth in 5% CO2 atmosphere than in air. Furthermore, due to its sensitivity to streptococcal protease, the IgG binding activity is mostly higher during the logarithmic than during the stationary phase of growth.

A quantitative and kinetic study of the release of the hematopoietic cytokines IL-3, IL-5 and GM-CSF, the immunoregulatory cytokine IL-12 heterodimer (and its p40 subunit) and IL-13 by human peripheral blood mononuclear cells (PBMC) stimulated in vitro with the superantigen streptococcal pyrogenic (erythrogenic) exotoxin A (SPE A) from Streptococcus pyogenes is reported. PBMC were stimulated in parallel with heat-killed group A streptococcal cells, E. coli lipopolysaccharide (LPS) and with concanavalin A (Con A) in certain experiments for comparative purposes. The cytokines were assayed in the supernatant fluids by ELISA. IL-13 expression was also determined by a quantitative competitive PCR. IL-3, IL-5, GM-CSF, IL-12 p40, IL-12 heterodimer and IL-13 expression was induced by SPE A in a time- and dose-dependent manner in rather substantial amounts except the IL-12 heterodimer, which was released in small quantities. In contrast to SPE A, IL-3, IL-5 and IL-13 were not or poorly elicited by streptococcal cells or LPS whereas these two stimulants induced relatively high amounts of GM-CSF. Interestingly, both IL-12 p40 and IL-12 heterodimer were released in much higher amounts by streptococcal cells. Con A induced IL-3, IL-5, GM-CSF and IL-13 production in amounts comparable to those elicited by SPE A. The possible pathophysiological relevance of the elicitation by SPE A and streptococcal cells of these cytokines is discussed.

The cleavage by specific enzymes of nicotinamide adenine dinucleotide (NAD+, synonym diphosphopyridine nucleotide (DPN)) in animal tissues has been described first by HANDLER and KLEIN (3). One class of these enzymes are the ADP-ribosyl transferases of bacterial origin (Cory neb acterium diphtheriae, Vibrio cholerae, Bordetella pertussis, Clostridium ssp., and Pseudomonas ssp.) which catalyze the transfer of ADP-ribosyl moieties to proteins like EF2 (elongation factor 2) and also show a distinct NAD+ glycosidase activity.

The erythrogenic (pyrogenic) toxins ETA and ETC have been characterized as superantigens, which bind as native molecules to MHC class II molecules of antigen processing cells and to T-cell receptors possessing specific Vs-chains. Linking these two receptors results in a high release of cytokines. ETB was recognized as the precursor of the streptococcal proteinase. Mitogenic activity detected in preparations of ETB by some authors was found to be a contamination, recently isolated and described as toxin AX (1). It is accompanied by a second component BX, which differs only by the lack of the amino terminal arginine. The immunological reactions of both proteins are identical, but the mitogenic activity of BX is failed higher than that of AX.

Among bacterial derived proteins, the superantigens possess the unique property to interact polyclonally with a high proportion of T lymphocytes bearing appropriate Vs motifs of the T cell receptor. In the presence of antigen presenting cells expressing MHC class II molecules the superantigens stimulate in a cascade of activation signals the production of a broad array of cytokines. Clinical and experimental observations strongly suggest that the pathophysiological features of streptococcal toxic shock syndrome (STSS) are mainly due to massive release of proinflammatory cytokines. Furthermore, the production of Th1 and Th2 derived cytokines by superantigen stimulated immune cells in vitro strongly indicates that these effectors have the capacity to play an important role in pathogenic disorders other than acute inflammatory processes by interfering with the regulation of Th1 and Th2 cytokine network in direction of either cell mediated, allergic or autoimmune responses. The precise role of bacterial superantigen involvement in the pathogenesis of chronic sequelae remains to be clarified.

Conformation and stability of three Sks from Streptococcus equisimilis strain H46A, Streptococcus pyogenes strain A374, and Streptococcus pyogenes strain AT27 were compared by limited proteolysis, CD, and fluorescence measurements and by DSC. The general similarity of the peptide CD spectra in the spectral region 185 to 260 nm indicates the same type of folding for the three proteins. Fluorescence and aromatic CD spectra are consistent with a predominant surface localization of the aromatic amino acids and a low rigidity of their surroundings. A major difference among the three Sks is shown by deconvolution of their excessive heat capacity functions. Deconvolution reveals two energetic folding units in Sk H46A but three energetic folding units in Sk A374 and Sk AT27. Digestion of the Sks with trypsin indicates a reduced sensitivity of the C-terminal region of Sk A374 and Sk AT27 in comparison to Sk H46A. This suggests that amino acids of the C-terminal region participate in the formation of the third folding unit of Sk A374 and Sk AT27.

A worldwide increase of severe invasive Streptococcus pyogenes infections beginning from the skin or soft tissue infections (cellulitis, fasciitis, myositis) has been reported during the last decade. This so-called Streptococcal Toxic Shock Syndrome (STSS) is considered as a resurgence of the historically known fulminant or toxic scarlet fever. It is characterized by fever, multiorgan failure, skin desquamation and occurs mainly in young/middle-aged, otherwise healthy adults within hours of the onset of symptoms. The onset of STSS seems to be related to the efficacy of bacterial products and host factors (particularly the immune state) of the patients. Bacterial products which are believed to be causally involved in these infections are the M-protein, different enzymes and the various erythrogenic toxins (ET or SPE), the streptococcal mitogenicity factor MF, the streptococcal superantigen (SSA), low molecular weight proteins (LMP) and the cell bound streptococcal membrane protein (CAP) (1). Our interest in this investigation has been focused on the examination of STSS strains isolated in Germany since 1989, when the first case was reported. More than 70 S. pyogenes strains isolated from cases of STSS in Germany and a group of control strains isolated from healthy people or from patients suffering from harmless streptococcal diseases were typed and investigated with regard to content of speA, speB, speC, and MF genes, toxin expression (ETA and ETC) and for mitogenic activity in culture supernatants. The results of this investigation are presented and discussed.

Cultivation of peripheral blood mononuclear cells (PBMC) in the presence of streptococcal pyrogenic exotoxins (SPE) A and C resulted in a significant induction of sister chromatid exchange (SCE)-inducing DNA lesions. Concomitantly, the expression of interleukin-2 receptor alpha chain (IL-2R alpha chain), transferrin receptor (TfR), and major histocompatibility complex class II molecule HLA-DR on the surface of phytohemagglutinin-activated T cells from whole blood culture cells (WBCC) significantly decreased within 72 h, that is at least two cell cycles, whereas unstimulated T cells from WBCC did not express these markers but had lost their CD3 molecules, an effect reported to precede apoptosis as part of a T cell inactivation pathway. However, no apoptotic cells were observed within a cultivation period of 120 h. We observed clearcut differences in the responses towards SPE A in WBCC and isolated lymphocytes, since SPE A-treated lymphocytes showed an increase in the [3H]thymidine incorporation and did express IL-2R alpha chain and TfR on their cell surface. Regardless of the precise underlying mechanism, T cells from WBCC seem to be in a state of functional incompetence. The data presented here are the first to provide strong evidence that streptococcal toxins produce SCE-inducing DNA lesions in PBMC, an effect that might contribute to the process of immune cell lethality in streptococcal toxic shock-like syndrome and could be of pivotal importance in the pathogenesis of severe streptococcal disease.

Superantigens use an elaborate and unique mechanism of T lymphocyte stimulation. Prototype superantigen are the pyrogenic exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes. Many candidate proteins of bacterial, viral and protozoal origin have recently been reported to be superantigens. In most cases the evidence that these proteins are in fact superantigens is highly indirect. In this review the evidence that gram-positive cocci produce superantigens other than the pyrogenic exotoxins is critically discussed. Evidence in described demonstrating that the epidermolytic toxins of Staphylococcus aureus and the pyrogenic exotoxin B and M-proteins of Streptococcus pyrogenes are not superantigens. Criteria are described for acceptance of a candidate as a superantigen.

Summary It has been supposed for many years that group A streptococci may elaborate more than the three well known erythrogenic toxins A, B or C (ETA, ETB, ETC). The analysis of the culture supernatant of streptococcal strain 27297 carrying neither genes for ETA nor ETC revealed mitogenic activity at pH 7.3 in isoelectric focusing. This mitogen of strain 27297 was purified by hydrophobic adsorption to Phenyl-Sepharose following FPLC chromatography on a Mono S column resulting in two proteins with mitogenic activity called AX and BX, respectively. Both differed in only one aminoterminal residue. The mitogenic activity of BX lacking one aminoterminal arginine was found to be about 100 times higher than that of AX. The aminoterminus of BX does not correspond to a predictable cleavage site for signal peptidase. We assume that BX was produced after translation by cleavage of the mature protein or the AX molecule with streptococcal proteinase (ETB) or an arginylaminopeptidase which is detectable on whole cells. The purified proteins BX and AX showed molecular weights of about 27 kDa in SDS electrophoresis and isoelectric points of 8.3 (AX) and 7.3 (BX) in isoelectric focusing, respectively. Both proteins were produced by practically all group A strains tested but not by groups B, C, G or H streptococci. Therefore, AX or BX seem to be proteins characteristic of group A streptococci.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of „superantigens” produced by Staphylococcus aureus and Streptococcus pyogenes and its T lymphocyte stimulating activity is involved into the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have produced and characterized recombinant SPEA molecules in Escherichia coli. These molecules are indistinguishable from natural SPEA in both T cell stimulatory and HLA class II binding activities. Human class II molecules are more efficient than mouse class II molecules in presenting SPEA to T cells. In binding tests to major histocompatibility complex class II-positive cells SPEA competes with staphylococcal enterotoxin B and A but not with toxic shock syndrome toxin-1.

One problem in investigating group A streptococcal infections and virulence is the lack of appropriate in vivo models. In this study we introduce the chicken embryo model for determining virulence of Streptococcus pyogenes. We found that M protein positive strains, if administered intravenously, were highly virulent for 12-day-old chicken embryos. The LD50 of the strains tested could be correlated directly with the amount of cell wall exposed M protein, which has been determined by the capacity of streptococci to bind fibrinogen and by the ability of streptococci to survive in fresh normal human blood. The number of colony forming units (cfu) of M+ strains necessary to kill 50% of embryonated eggs was significantly lower (10(2) cfu) than for M-variants (10(4) cfu). Albumin and/or IgG binding streptococcal cells, which can also take place in proteins of the M protein family which do not bind to fibrinogen, did not show that clear correlation to the virulence in chicken embryos that did fibrinogen binding. Application of anti-streptococcal M protein antisera from chicken and rabbit reduced the lethality of the chicken embryos. In contrast, no correlation was found between lethality of chicken embryos and the in vitro production of erythrogenic toxins by the administered strains. Thus the results indicate that the presence of M-protein with its fibrinogen binding activity on the streptococcal cell surface is necessary for virulence of group A streptococci in the chicken embryo model.

Streptolysin O (SLO) was purified from culture supernatants of group C streptococci. The final product was either the complete, native molecule (SLOn [pI, 6.0]) with the N-terminal sequence (Asp)-Ser-Asn-Lys-Gln-Asn-Thr-Ala-Asn-Thr-Glu-Thr- or a large fragment (SLOf [pI, 7.3]) with the N-terminal sequence Ala-Pro-Lys-Glu-Met-Pro-Leu-Glu-Ser-Ala-Glu-Lys-Glu-Glu-Lys-.

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.

The mode of pathogenic action of the Steptococcus pyogenes superantigen erythrogenic toxin type A (ETA) in causing toxic shock-like syndrome in humans is thought to be mediated by massive release of cytokines by patients immune cells. The cytokine-inducing capacity of ETA as an extracellular protein was compared with that of lipopolysaccharide (LPS), a component of cell wall of gram-negative bacteria. Peritoneal macrophages and splenocytes of BALB/c and C3H/HeJ mice were stimulated by ETA and LPS. Tumor necrosis factor (TNF), interleukin 3 (IL-3) and interleukin 6 (IL-6) activities in the supernatants of stimulated cells were evaluated. In contrast to LPS, ETA induced only low amounts of IL-6 and no detectable TNF activities in peritoneal macrophage supernatants. ETA-triggered BALB/c and C3H/HeJ splenocytes produced great amounts of IL-6. ETA triggered the production of IL-3 by both mice strains splenocytes in a dose dependent manner. The amounts of IL-3 in supernatants were comparable to those induced by concanavalin A. The simultaneous presence of ETA and LPS in macrophage and splenocyte cultures induced a slight enhancement above an additive value after 72-96 h. Challenge of BALB/c mice with ETA 6 h before the harvest of peritoneal macrophages led to an enhanced production of IL-6 upon stimulation with ETA as well as with LPS. Splenocytes of nude BALB/c mice did not produce IL-6 upon stimulation with ETA, whereas LPS-induced IL-6 production was similar in these mice and in their littermates. The pathogenic effect of ETA on host's immune cells could most likely be explained as a consequence of T cell activation. The results confirm also that LPS- and ETA-induced shock is mediated by different cell types.

Summary 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 μg/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.

Hyaluronate lyase of group C strain H 46 A ( Streptococcus equisimilis ) was purified and characterized by isoelectric focusing, sodium-dodecylsulfate-acrylamide electrophoresis, polyacrylamide-gradient-electrophoresis and crossed immunoelectrophoresis. The purification of the hyaluronate lyase was performed successively by adsorption on Florisil, chromatography on DEAE-cellulose, and - for separation of streptokinase - stirring with CPG-pore-glass. The last step was gel filtration on Sepharose 6B. Purified hyaluronate lyase showed a high specific activity. The purified enzyme was found to be antigenically homogeneous. No contaminating streptococcal components could be detected. The molecular size was determined as to be 90000 dalton by gel filtration and 110000 dalton by sodium dodecylsulfate-acrylamide electrophoresis. The amino acid composition was also determined. In the isoelectric focusing, using gels with reducing conditions, one protein band at a pi 4.95 was observed. Under nonreducing conditions two or three diffuse protein bands which showed lower enzymatic activity were found. It might be possible that the hyaluronate lyase exists in two different forms.

A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase (sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human interferon alpha 1 (hIFN alpha 1) and hybrid hIFN alpha 1/2 genes to this sak ESU resulted in secretory expression of the two gene products in both Escherichia coli and Bacillus subtilis. While most of the IFN alpha was exported to the periplasmic space of E. coli, about 99% was secreted to the culture medium by recombinant B. subtilis strains. The total yield in E. coli was 1.2 x 10(5) IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of the sak ESU through insertion of an IS1 element. No such instability was observed with B. subtilis although expression and secretion levels reached even 3 x 10(6) IU/ml. Proteolytic degradation of IFN alpha by extracellular proteases was avoided by a combination of constitutive expression and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFN alpha 1 protein purified from B. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed efficient secretion of hIFN alpha 1 in B. subtilis gave poor yields when introduced into Streptococcus sanguis.

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.

The gene coding for the bacterial plasminogen activator staphylokinase was cloned from the Staphylococcus aureus phage 42D, a serogroup F phage used for lysotyping, onto the standard Escherichia coli plasmid vector pACYC184. The coding and flanking sequences of the sak42D gene were largely identical to those of a sak gene cloned from the serologically different S. aureus phage SøC (Sako and Tsuchida 1983). Subcloning of a 2.5 kb phage 42D DNA fragment onto plasmid pGB3631 allowed the sak42D gene to be introduced into the gram-positive hosts Bacillus subtilis and Streptococcus sanguis. The sak42D gene was expressed and secreted most efficiently by B. subtilis cells (25 micrograms/ml of culture supernatant) reduced in exoprotease production. In this host expression and secretion of Sak was initiated at the early growth phase and continued through the logarithmic phase. Formation of Sak was, however, also observed with the other cloning hosts. The Sak elaborated by the heterologous hosts was serologically identical with authentic Sak derived from S. aureus.

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.

By interaction of streptococcal erythrogenic toxins A and B with chick embryo fibroblasts and human amnion (FL) cells an antiviral interferon-like factor was secreted. It inhibited the replication of vesicular stomatitis, vaccinia and Mengo viruses. The streptococcal toxin type B was 50 times more cytotoxic for both cell cultures in comparison with streptococcal toxin A. The maximum tolerated doses of the two types of streptococcal toxins induced approximately the same antiviral protection effect. The production curve of the antiviral factor showed a maximum at the 12 th hour after incubation at 37°C with graduate decrease up to the 24 th hour using a 6 hours induction time. The interferons induced by the streptococcal erythrogenic toxins A and B were thermostable at 56°C for 30 min and were partially destroyed at pH 2 as tested against Mengo virus in FL cells. The antiviral effect could be reversed by addition of streptococcal erythrogenic toxin A at the maximum tolerated dose simultaneously with the glycosylating inhibitors strepto-virudin and D-glucosamine by 90 and 100 per cent, respectively.

The streptokinase expressed by the cloned gene in Streptococcus sanguis has a molecular weight of about 44 000 [Malke, H., Gerlach, D., Kohler, W., & Ferretti, J.J. (1984) MGG, Mol. Gen. Genet. 196, 360-365] while the molecular weight of the native streptokinase is 47 000. The structural and activity differences of the cloned streptokinase (cSK) as expressed by S. sanguis and the native streptokinase (nSK) were investigated. From a partially purified cSK, two active fractions were obtained by reversed-phase HPLC. The minor fraction cSKL was nearly as active as SK in plasminogen activation. The major fraction cSKs had only about one-fourth of the specific activity. The structures of cSKL and cSKs were studied and compared to the known amino acid sequence of SK [Jackson, K. W., & Tang, J. (1982) Biochemistry 21, 6620-6625]. From the NH2- and COOH-terminal sequences and amino acid composition of the cyanogen bromide (CNBr) fragments, it could be deduced that cSKL and cSKs are without 31 and 32 residues, respectively, from the COOH-terminal end of SK. Since the cloned gene contained the full SK structure, the missing structures must have been due to posttranslational proteolysis. An SK fragment similar in size to cSK was observed from a chymotryptic digest of SK.

Summary A gene coding for the bacterial plasminogen activator staphylokinase (SAK) was cloned from Staphylococcus aureus bacteriophage 42D into an exoprotease reduced mutant strain of Bacillus subtilis (1). Yields of up to 50 mg SAK per litre of culture supernatant were obtained depending on the medium used. SAK purified by ion exchange chromatography and gel filtration had a specific activity of 16 000 units/mg protein. Isoelectric focusing of the purified SAK revealed heterogeneity with respect to the isoelectric points. Four different SAK proteins were identified among which the majority fraction had an IEP of 6.3 and a N-terminal amino acid sequence of NH2-Lys-Gly-Asp… This N-terminus was 10 amino acids downstream of the expected signal peptide cleavage site beyond AA 27. It resulted most likely from a postsecretory proteolytic modification of the transiently appearing and correct processing product. In contrast to other plasminogen activators SAK was found to be resistant to proteolytic inactivation by plasmin.

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).