Crystal structure of the superantigen enterotoxin C2 from Staphylococcus aureus reveals a zinc-binding site.

BACKGROUND: Staphylococcus aureus enterotoxin C2 (SEC2) belongs to a family of proteins, termed 'superantigens', that form complexes with class II MHC molecules enabling them to activate a substantial number of T cells. Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties. Comparison of the structure of SEC2 with those of two other superantigens--staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)--may provide insight into their mode of action. RESULTS: The crystal structure of SEC2 has been determined at 2.0 A resolution. The overall topology of the molecule resembles that of SEB and TSST-1, and the regions corresponding to the MHC class II and T-cell receptor binding sites on SEB are quite similar in SEC2. A unique feature of SEC2 is the presence of a zinc ion located in a solvent-exposed region at the interface between the two domains of the molecule. The zinc ion is coordinated to Asp83, His118, His122 and Asp9* (from the neighbouring molecule in the crystal lattice). Atomic absorption spectrometry demonstrates that zinc is also bound to SEC2 in solution. CONCLUSIONS: SEC2 appears to be capable of binding to MHC class II molecules in much the same manner as SEB. However, structure-function studies have suggested an alternative binding mode that involves a different site on the toxin. The zinc ion of SEC2 lies within this region and thus may be important for complex formation, for example by acting as a bridge between the two molecules. Other possible roles for the metal cation, including a catalytic one, are also considered.

Crystal structure of microbial superantigen staphylococcal enterotoxin B at 1.5 A resolution: implications for superantigen recognition by MHC class II molecules and T-cell receptors.

Staphylococcal enterotoxin B is a member of a family of toxins known as superantigens that activate a large number of T-cells (up to 20%) by cross-linking MHC class II molecules with T-cell receptors in a Vbeta-restricted fashion. The crystal structure of staphylococcal enterotoxin B presented here has been determined at 1.5 A resolution, the highest resolution so far for a superantigen. The final model contains 1948 protein atoms and 177 water molecules and has excellent geometry with root-mean-square (rms) deviation of 0.007 A and 1.73 degrees in bond lengths and bond angles, respectively. The overall fold is similar to that of other microbial superantigens, but as it lacks the zinc-binding site found in other members of this family, such as staphylococcal enterotoxin A, C2 and D, this enterotoxin possesses only one MHC class II binding site. Comparison of the crystal structure of free SEB and in complex with an MHC class II molecule revealed no major changes in the MHC-binding site upon complex formation. However, a number of water molecules found in the free SEB may be displaced in the complex or contribute further to its stability. Detailed analysis of the TcR-binding site of SEB, SEA and SEC2 shows significant differences which may account for the ability of each superantigen to bind specific Vbeta sequences.

The refined crystal structure of toxic shock syndrome toxin-1 at 2.07 A resolution.

The pyrogenic toxin toxic shock syndrome toxin-1 from Staphylococcus aureus is a causative agent of the toxic shock syndrome disease. It belongs to a family of proteins known as superantigens that cross-link major histocompatibility class II molecules and T-cell receptors leading to the activation of a substantial number of T cells. The crystal structure of this protein has been refined to 2.07 A with an Rcryst value of 20.4% for 51,240 reflections. The final model contains three molecules in the asymmetric unit with good stereochemistry and a root-mean-square deviation of 0.009 A and 1.63 from ideality for bond lengths and bond angles, respectively. The overall fold is considerably similar to that of other known microbial superantigens (staphylococcal enterotoxins). However, a detailed structural analysis shows that toxic shock syndrome toxin-1 lacks several structural features that affect its specificity for V beta elements of the T-cell receptor and also its recognition by major histocompatibility class II molecules.

Purification, crystallization and preliminary X-ray analysis of toxic shock syndrome toxin-1 from Staphylococcus aureus.

High yields of toxic shock syndrome toxin-1, from Staphylococcus aureus, have been purified (> 99%) using a novel, simple, two-step procedure involving dye ligand chromatography. Crystals suitable for X-ray diffraction work were obtained by vapour diffusion using ammonium sulphate and polyethylene glycol as precipitants. They belong to the orthorhombic space group C222(1), with unit cell dimensions a = 108.6 A, b = 177.6 A and c = 97.5 A, with three molecules per asymmetric unit. The crystals diffract to at least 2.5 A resolution and are suitable for three-dimensional X-ray structural analysis.

Crystallization and preliminary X-ray analysis of a microbial superantigen staphylococcal enterotoxin C2.

High yields of staphylococcal enterotoxin C2, from Staphylococcus aureus, have been purified using dye ligand chromatography. Crystals suitable for X-ray diffraction work were obtained by vapour diffusion using ammonium sulphate and polyethylene glycol as precipitants. They belong to the tetragonal space group P4(1)22 with unit cell dimensions a = b = 43.2 A and c = 290.9 A with one molecule per asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for three-dimensional X-ray structural analysis.

Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin-1 at 2.5 A resolution.

Toxic shock syndrome toxin-1 (TSST-1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST-1 with a significant proportion of the T-cell repertoire. TSST-1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of V beta chains of the T-cell receptor (TCR). The three-dimensional structure of a mutant (His-135-Ala) TSST-1 was compared with the structure of the native (wild-type) TSST-1 at 2.5 A resolution. The replacement of His 135 of TSST-1 with an Ala residue results in the loss of T-cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin-TCR interactions, is located on the major helix alpha 2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix alpha 2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix alpha 1 by disrupting helix-helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the alpha 1 helix.

Comparative phenotypic characteristics of Staphylococcus aureus isolates from line and non-line associated septicaemia, CAPD peritonitis, bone/joint infections and healthy nasal carriers.

This study compared specific phenotypic and potential virulence characteristics of Staphylococcus aureus isolates from invasive infections and nasal carriers. Three hundred and sixty isolates were studied; 154 from septicaemia (69 line associated, 85 non-line), 79 from continuous ambulatory peritoneal dialysis (CAPD) peritonitis, 64 from bone/joint infections and 64 from healthy nasal carriers. The isolates were tested for production of enterotoxins (SE) A, B, C or E, toxic shock syndrome toxin-1 (TSST-1) protein A, and also for lipolytic, proteolytic, fibrinolytic and haemolytic activities. In addition phage typing, crystal violet reaction, urease and galactose breakdown were studied. Seventy-one percent of isolates were enterotoxigenic. Production of SEA was significantly lower amongst the bone/joint isolates. Production of SEB, was lower among the control group compared with CAPD, bone/joint, and non-line septicaemia isolates. SEE production was higher among the bone/joint isolates compared with the CAPD and non-line septicaemias and production of TSST-1 was significantly higher among nasal isolates compared with isolates causing infection. Almost all of the isolates were lipolytic, with highest activity amongst nasal and bone/joint isolates. Fibrinolytic activity was similar in the five groups of isolates. Proteolytic activity ranged from 35 to 62% of isolates with the lowest frequency among septicaemia isolates. In all, 80-90% of isolates were haemolytic, although CAPD isolates were less likely to be haemolytic. Isolates from the control and CAPD group more frequently belonged to phage group I. TSST-1 does not appear to be an important requirement for invasive infections, but SEB may be. Proteolysis and intensity of lipolysis appear to be less important in septicaemia, and haemolysis may not be important in CAPD peritonitis.

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid.

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vbeta type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21-32, 93-107 and 202-217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.

Stimulation of rat spleen cells by staphylococcal enterotoxins.

There is much interest in staphylococcal enterotoxins as T cell mitogens in humans, mice and rabbits. Rat spleen cells were shown to proliferate in response to staphylococcal enterotoxins A and B and toxic shock syndrome toxin-1 at concentrations (5 to 500 ng ml-1) which also stimulate mouse spleen cells. The proliferative response to all these enterotoxins was inhibited by cyclosporin A, indicating the response to be predominantly that of T cells. These results indicate that the rat provides another convenient model for the analysis of T cell responses to enterotoxins.

The effects of irradiation and temperature on the immunological activity of staphylococcal enterotoxin A.

The effects of irradiation and temperature on purified staphylococcal enterotoxin A were investigated using sensitive ELISA systems. Thermal inactivation of staphylococcal enterotoxin A in phosphate-buffered saline was considerably faster at temperatures of 60, 70, 115 and 121 degrees C than at 90 and 100 degrees C. In gelatin phosphate buffer, staphylococcal enterotoxin A was completely inactivated by irradiation at 8.0 kGy; in a 15% mince slurry, however, 27-37% of staphylococcal enterotoxin A remained at this level of irradiation. Even at a dose of 23.7 kGy, 16-26% residual staphylococcal enterotoxin A could still be detected. Generally, increasing the mince concentration increased the protection against the effect of irradiation on staphylococcal enterotoxin A. However, the protective effect of mince at a concentration of 50% was less than at a mince concentration of 30%. Both irradiation and heat processing of food should only be used in conjunction with good manufacturing practices to prevent proliferation of microorganisms and toxin productions.

The influence of incubation temperature and pH on the antimicrobial properties of hen egg albumen.

Gram positive bacteria, including lysozyme-resistant strains, and yeasts were killed in hen egg albumen with or without iron at 30 of 39.5 degrees C. The albumen was more toxic at 39.5 degrees C than at 30 degrees C for Gram negative bacteria. With the exceptions of Pseudomonas fluorescens, Acinetobacter sp. and Proteus vulgaris, iron caused the growth of Gram negative bacteria or protected them from being killed in hen albumen at 39.5 degrees C. At this temperature, however, maximal growth of and glucose utilization by Escherichia coli C20 only occurred in albumen supplemented with growth factors, trace metals, additional nitrogen and sufficient iron to quench ovotransferrin. The bactericidal properties of albumen could be negated by changing its pH from 9.0 or above to 7.5 or below. At 39.5 degrees C, enterochelin allowed growth of E. coli in albumen at pH 7.9, but not at 9.4, whereas iron allowed growth at both pH values.

Purification of staphylococcal enterotoxin E.

Staphylococcal enterotoxin E has been purified by a combination of Red-A ligand chromatography and Mono-S (cation-exchange) chromatography. Commencing with 30 litres of fermenter-grown culture the method has been used to purify 17 mg of toxin, giving a purification yield of around 40%. The purity of the toxin was over 98% when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, but demonstrated three isoelectric forms by isoelectric focusing, with pl values of 7.6, 7.4, and 7.0. This method adds to the versatility of the Red-A matrix system which can also be used for the purification of staphylococcal enterotoxins A, B, and C2.

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.

Scaled-up production and purification of Clostridium perfringens type A enterotoxin.

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38.8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119.3 micrograms/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty micrograms of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.

The effect of the olive phenolic compound, oleuropein, on growth and enterotoxin B production by Staphylococcus aureus.

The presence of low concentrations (0.1% w/v) of oleuropein, a phenolic compound extracted from olives, delayed the growth of Staphylococcus aureus in NZ amine A and brain heart infusion media modified by the addition of growth factors and glucose (NZA+ and BHI+), as indicated by changes in conductance, whilst higher concentrations (0.4-0.6% w/v) inhibited growth completely. Intermediate concentrations of oleuropein (0.2%) prevented growth in BHI+ but allowed growth to occur in NZA+ despite an extended lag phase (30 h). Concentrations of oleuropein > 0.2% inhibited growth and production of enterotoxin B in both types of media. Lower levels (0.1%) did not affect the final viable count and production of toxin in BHI+ but decreased the number of viable organisms and reduced the toxin production in NZA+ by eightfold. An increase in the concentration of oleuropein resulted in a decrease in the amount of glucose assimilated and consequently the amount of lactate produced. In addition, oleuropein prevented the secretion of a number of exoproteins. Addition of oleuropein during the exponential phase appeared to have no effect on the growth of Staph. aureus in NZA+.

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.

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation.

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose. Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.

Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus.

The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.

Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes.

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.