Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle.

Synthesis of enterotoxins C1 and C2 and thermonuclease throughout the growth cycle was investigated with Staphylococcus aureus type strains FRI137 and FRI361 and S. aureus isolates M5 (C1) and L2 (C2) of animal origin. Both enterotoxins were produced during the exponential growth phase or at the beginning of the stationary phase. The minimal incubation time (7 to 12 h) and the lowest population (10(7) to 2 x 10(9) CFU/ml) associated with detectable enterotoxin (1 to 6.5 ng/ml) were related to the total amount of toxin produced after 24 h. Thermonuclease was detected in all samples whenever enterotoxins were detected. Furthermore, strain FRI137 produced thermonuclease earlier and at lower cell populations than it did enterotoxin C1. Patterns of enterotoxin and thermonuclease synthesis did not correlate. The concentration of toxins increased throughout the growth cycle, while the concentration of thermonuclease remained constant during the last hours of the growth cycle.     

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.     

Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy

Secondary and tertiary structural parameters of two functionally and serologically related proteins, staphylococcal enterotoxins B and C1, have been determined by using circular dichroism and fluorescence spectroscopy. The secondary structures derived from the respective far-UV circular dichroic spectra were 9.5% alpha-helix, 55.0% beta-pleated sheets, 16.5% beta-turns, and 19.0% random coils for enterotoxin B and 15.0% alpha-helix, 38.0% beta-pleated sheets, 25.5% beta-turns, and 21.5% random coils for staphylococcal enterotoxin C1. The values matched well with the secondary structures derived from the amino acid sequences (Chou and Fasman method). Seven antigenic sites have been predicted for both staphylococcal enterotoxins B and C1 by using the hydrophilicity and the secondary structure information. Three of these antigenic sites appear similar. Fluorescence quantum yield of the single tryptophan residue (Trp-197) of both the enterotoxins showed the tryptophan residue in staphylococcal enterotoxin B to be approximately 46% more fluorescent than in staphylococcal enterotoxin C1. Tryptophan fluorescence quenching by the surface quencher I- and the neutral quencher acrylamide revealed that the single tryptophan residue in each of the enterotoxins is buried in the protein matrix and is not accessible to the surface quencher I-. The tryptophan residue in staphylococcal enterotoxin C1 is 14% less accessible to acrylamide than in staphylococcal enterotoxin B. The data, in general, reflect several similarities and significant differences between the two related enterotoxins.     

Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor

The goal of this study was to investigate the molecular interaction between superantigens and the T-cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T-cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing Vβ3, Vβ12, Vβ13.2, Vβ14, Vβ15, Vβ17 and Vβ20. SEC2 and SEC3 have the additional ability to expand T cells bearing Vβ13.1, and their expansion of Vβ3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate Vβ3 and Vβ13.1 was determined by the residue in position 26, located in the base of the SEC α3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a Vβ expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co-ordinates of a recently reported murine TCR β-chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T-cell receptor. In this model complex, the complementarity-determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the Vβ element contact SEC3 predominantly through residues in the α3 cavity of the toxin. CDR3 of the β chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of Vβs 3 and 13.1 by the SEC superantigens.     

Staphylococcal growth and enterotoxins (A—D) and thermonuclease synthesis in the presence of dehydrated garlic

E. GONZÁLEZ-FANDOS, M.L. GARCÍA-LÓPEZ, M.L. SIERRA AND A. OTERO. 1994. The inhibition of Staphylococcus aureus growth and enterotoxin and thermonuclease production by various concentrations of garlic ( Allium sativum ) was studied in BHI broth. The growth of Staph. aureus was inhibited by dehydrated garlic at levels of 1.5% (w/v) and over. Enterotoxins A, B and C₁ were only detectable in broth containing < 1% of garlic while enterotoxin D was produced at a level of 2%. Garlic also inhibited thermonuclease (TNAse) production, complete inhibition being observed at levels ≥ 1.5%. TNAse was not always detected when enterotoxin was present.     

Nucleotide sequence of the staphylococcal enterotoxin C3 gene sequence comparison of all three type C staphylococcal enterotoxins

Summary The structural gene entC3, which encodes staphylococcal enterotoxin C3 was cloned from the genome of Staphylococcus aureus FRI-913 and sequenced. The primary amino acid sequence of the toxin was deduced from the nucleotide sequence data. entC3 contains 801 by and encodes a precursor protein of 266 amino acids. Glutamic acid was found to be the N-terminus of mature enterotoxin C3. Thus, the first 27 residues of the toxin precursor comprise the signal peptide, and the mature toxin contains 239 amino acids with a molecular weight of 27 563 daltons. Enterotoxin C3 differs from enterotoxin C2 by four amino acids and from enterotoxin C1 by nine residues. The 167 C-terminal residues of the three toxins are identical, except for one conservative amino acid substitution in enterotoxin C3. The degree of immunological relatedness among the three Type C enterotoxins is proportional to their molecular relatedness. This study also provides evidence that the N-termini of Type C enterotoxins determine subtype-specific antigenic epitopes, while more conserved C-terminal regions determine biological properties and cross-reactive antigenic epitopes shared with other pyrogenic toxins.     

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.     

The secondary structure of staphylococcal enterotoxins A,B and C

The circular dichroism (CD) of staphylococcal enterotoxins A, B and C was measured. The CD of enterotoxins B and C were almost identical from 250 to 320 nm, but differed from the CD of enterotoxin A. The spectrum of enterotoxin A in this wavelength region contained the same bands with respect to both location and sign, but with significant differences in intensity. The CD spectra of enterotoxins B and C were also much more alike from 190 to 250 nm. Although all three enterotoxins had a major negative extremum at 215–218 nm, its magnitude was equal in enterotoxins B and C, but was substantially decreased in enterotoxin A. The secondary structure of the enterotoxins contained little α-helix as analyzed with CD models. A secondary structure of enterotoxin B computed from a scheme based on a joint prediction histogram of five separate methods, placed 29 residues in α-helices, 71 in β-pleated sheets, 88 in β-turns and 55 in aperiodic conformation.     

Cross-reactions between tryptic polypeptides of staphylococcal enterotoxins B and C.

The strong cross-reactions demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by measurement of antigen-binding capacity were reflected in well defined polypeptides obtained by limited tryptic digestion from SEB and SEC1. Two antigenic determinants on each enterotoxin were capable of reacting with heterologous antibody, one on the first 57 amino acids and one on the last 150 residues of the polypeptide backbone. The larger, carboxyl terminal polypeptides bound efficiently to homologous antiserum but about two orders of magnitude less efficiently to heterologous antibody. The amino terminal peptides showed only weak homologous binding but nearly comparable heterologous binding. It is proposed that the determinant on the amino terminal polypeptides is largely responsible for the strong reciprocal binding of the intact enterotoxins and that their low antigen-binding capacity is due to a random or a structurally distorted conformation in solution.     

Production of a monovalent staphylococcal enterotoxic antiserum type A and its use for typing staphylococcal enterotoxins

A monovalent specific staphylococcal antiserum, type A, was obtained by means of the isolated and purified preparation of type A staphylococcal enterotoxin. This antiserum was proved to be identical to antiserum of the same type, manufactured by Serva Feinbiochemica GmbH & Co. (West Germany). The titer of the newly obtained antiserum in the precipitation test was 1 : 16, and its use allowed one to detect enterotoxin of the above-mentioned type at a concentration of 0.004 mg/ml. The study of 320 staphylococcal strains with the use of this antiserum revealed that 25 strains (7.8%) produced type A enterotoxin.     

On the cross-reactivity of staphylococcal enterotoxins A, B, and C.

Strong cross-reactions were demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by antigen-binding capacity and by competitive binding ability. Both SEB and SEC1 combined completely with the heterologous antibody although requiring four times as much antiserum as the homologous enterotoxin and both displaced about one-third of the other enterotoxin from a heterologous antigen-antibody system. It is proposed that one of the three major antigenic determinants of these enterotoxins possesses a significant similarity but probably not an identity of structure. SEB and SEC1 did not combine with antiserum to enterotoxin A nor inhibit the reaction of SEA with anti-SEA. SEA had no intrinsic binding capacity for anti-SEB or anti SEC1 nor did it inhibit the binding of either enterotoxin to its own antibody. Affinity chromatography was employed to demonstrate that a small apparent binding of SEA to anti-SEB was due to antibody to SEA in the anti-SEB serum and that an almost complete displacement of SEC1 binding to anti-SEC1 was caused by contaminating SEC (about 0.01%) in preparations of enterotoxin A.     

Production and characterization of murine monoclonal antibodies against staphylococcal enterotoxins A and E

Six murine monoclonal antibodies (MAbs) against staphylococcal enterotoxin A (SEA) and enterotoxin E (SEE) were prepared by fusion of myeloma cells with mouse spleen cells immunized with SEA and SEE. Of five MAbs to SEA tested, two MAbs were reactive with only SEA, whereas three were specific for both SEA and SEE. On the other hand, one MAb to SEE was found to be specific for only SEE. To study specificities of the combining sites of these MAbs, competitive binding assays with either SEA or SEE and horseradish peroxidase conjugated MAbs were performed using unconjugated MAbs as inhibitors. The results obtained in the assays suggest that different epitopes may be located on SEA and that some of them may be cross-reacting epitopes between SEA and SEE.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

Superantigen staphylococcal enterotoxin C1 inhibits the growth of bladder cancer

Superantigens can induce cell-mediated cytotoxicity preferentially against MHC II-positive target cells with large amounts of inflammatory cytokines releasing. In this study, superantigen staphylococcal enterotoxin C (SEC) 1 was investigated to evaluate its potential in bladder cancer immunotherapy in vitro and in vivo. Our results revealed that SEC1 could stimulate the proliferation of human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner, accompanied with the release of interleukin-2, interferon-γ, and tumor necrosis factor-α, and increased the population of CD4⁺ T cells and CD8⁺ T cells. PBMCs stimulated by SEC1 could initiate significant cytotoxicity towards human bladder cancer cells in vitro. The results of in vivo antitumor experiment indicated that SEC1 could decrease the rate of tumor formation and prolong the survival time of tumor-bearing mice. Our study demonstrated that SEC1 inhibited the growth of bladder cancer. And it is also suggested that SEC1 may become a candidate for bladder cancer immunotherapy.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.