A simple method of purifying staphylococcal alpha-toxin and a study of its properties]

The simple method is proposed for isolation and purification of staphylococcal alpha-toxin that permits one to obtain the homogeneous toxic protein with high activity. The time necessary for maximal toxin production at cultivation has been defined. The thermostability and interferonogenic characteristics of the obtained alpha-toxin were studied.     

Identification of serine138 residue in the 4-residue segment K135K1361137S138 of LukS-I component of Staphylococcus intermedius leukocidin crucial for the LukS-I-specific function of staphylococcal leukocidin

Luk-I produced by Staphylococcus intermedius was found to be a new member of the staphylococcal bi-component pore-forming toxin family, in which staphylococcal leukocidin, Panton-Valentine leukocidin, and gamma-hemolysin are included. Luk-I consists of LukS-I and LukF-I. From the deduced amino acid sequence of LukS-I, a 4-residue sequence, K135K1361137S138, at the root of the stem region was found to be identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. A mutant of LukS-I (MLSI-SA), in which the Ser138 residue was replaced by an alanine residue, was created, purified, and assayed for its leukocytolytic and pore-forming activities with LukF-I. Both LukS-I and MLSI-SA formed a ring-shaped complex with LukF-I on rabbit erythrocytes and human polymorphonuclear leukocytes (HPMNLs) membrane. However, MLSI-SA showed no leukocytolytic activity with LukF-I. LukS-I was phosphorylated by protein kinase A in the presence of [gamma-32P] ATP in a cell-free system, but MLSI-SA was not phosphorylated significantly. A potent and selective inhibitor of protein kinase A (N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89)) showed 50% inhibition of the Luk-I-induced cell lysis at 0.5 nM. Thus, it is concluded that the phosphorylation of the Ser138 residue in the 4-residue segment K135K1361137S138 of LukS-I is important for the leukocytolysis of HPMNLs.     

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.     

Anaphylactogenic, antigeni and avid properties of the antibotulin serum from the blood of various animal species]

It was shown that purified and concentrated by the "Diaferm" method antibotulin sera from horse and cattle blood failed to differ by anaphylactogenic properties; at the same time in sensitization of the organism to protein of one animal species the use of the sera of another species provided a lesser reactogenicity of the preparation. The antigenic activity of the purified and concentrated sera from the blood or horses and cows in testing on rabbits was identical, but in response to cow alpha-globulin the animals responsed by a more intensive production of precipitins. The activity of cow and horse antibotulin serum (determined by the rate and stability of their association with the corresponding toxin) proved to be identical.     

Interaction of Staphylococcal Alpha Toxin and the Estrogenic Hormone Diethylstilbestrol

Previous work in this laboratory showed that diethylstilbestrol was capable of suppressing induced furunculosis in rabbits. The present study indicates that the synthetic estrogenic hormone diethylstilbestrol which is used for acne, estrogen deficiency, cancer, and other disorders, can reduce the cytolytic action of staphylococcal alpha toxin. The cytotoxic action of purified alpha toxin for tissue cultures was evaluated by use of such parameters as total and viable cell counts, glucose, and protein determination, and cytopathic effects (CPE) in the presence and absence of steroids. To 3-day-old primary rabbit baby kidney tissue cultures, 1 to 5 μg of diethylstilbestrol per ml was added; growth of tissue cultures in Eagles medium was continued till the 6th day, and then one tissue cytopathic dose per milliliter of alpha toxin was added, and the subsequent fate of tissue cultures was assayed. Such cultures yielded higher total and viable cell counts, utilized more glucose, and contained more protein than the control cultures. In control cultures, CPE was observed on the 3rd hr after the addition of alpha toxin, and it was complete in 24 hr, whereas in tissue cultures treated with diethylstilbestrol, the CPE was significantly reduced. The data presented in this study made possible the availability of a suppressor of the cytolytic action of alpha toxin and might be useful in assaying the action of alpha toxin in an in vitro inexpensive test system.     

Structural basis for the recognition of superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules and T-cell receptors.

Streptococcal pyrogenic exotoxin A (SpeA) is a superantigen produced by Streptococcus pyogenes and is associated with severe infections characterized by rash, hypotension, multiorgan failure and a high mortality rate. In this study, an allelic form of this toxin, SpeA1, was crystallized with four molecules in the crystallographic asymmetric unit and its crystal structure was determined at 2.6 A resolution. The crystallographic R-factor was 19.4% (33 497 reflections) for 7031 protein atoms and 88 water molecules. The overall structure of SpeA1 is considerably similar to that of other prototype microbial superantigens, either of staphylococcal or streptococcal origin, but has greatest similarity to staphylococcal enterotoxin C (SEC). Based on structural and mutagenesis data, we have mapped several important residues on the toxin molecule, which are involved in the recognition of major histocompatibility complex (MHC) class II molecules and T-cell receptors. Also, the toxin appears to possess a potential zinc-binding site which may have implications in binding to particular MHC class II molecules. Finally, we propose models for SpeA1-MHC class II and SpeA1-T-cell receptor association and the relevance of this phenomenon to the superantigenic action of this toxin is considered.     

Staphylococcal toxic shock exotoxin (its isolation and characteristics)

The method for obtaining the preparation of toxic shock exotoxin (TSE) has been developed. This method comprises the following operations: the sorption of the toxin from the culture fluid on Amberlite CG-50, elution, dialysis, gel chromatography in a column with biogel P-2, isoelectric focusing, and gel chromatography in a column with Sephadex G-75. TSE is a relatively thermostable protein with a molecular weight of 24,000. Its isoelectric point is 7.2. Monospecific antiserum to TSE with precipitating antibody titer equal to 1:16, identical to the reference serum (M. S. Bergdoll), has been prepared. This antiserum has shown no cross reactions with the homogeneous preparations of staphylococcal enterotoxins.     

Effect of Staphylococcal Enterotoxin B on the Electroencephalogram of Monkeys

A highly purified preparation of staphylococcal enterotoxin B was administered intravenously, 1 mg/kg, to rhesus monkeys. Electroencephalograms (EEG) were recorded from electrodes attached to the skin or implanted on the dura. The dose of toxin employed consistently produced a sequence of vascular collapse followed by death; in control studies, animals were bled periodically to produce a similar pattern of shock. Regardless of the time to death following administration of the enterotoxin, there were essentially no changes from base line EEG patterns until shortly before death. With the development of preterminal severe shock, there was a marked decrease in EEG wave frequency and an initial increase in amplitude. The latter diminished progressively to produce an isoelectric tracing immediately prior to death. This could be reversed for a brief period by epinephrine. An identical sequence of EEG changes was observed during the terminal period of hemorrhagic shock. It is postulated that cerebral anoxia, caused by inadequate blood flow, is the primary cause of the altered EEG patterns that accompany enterotoxin toxicity. In this respect, staphylococcal enterotoxin B produces changes apparently similar to bacterial endotoxin but distinctly different from the EEG effects reported after botulinum toxin, anthrax toxin, or rattlesnake and cobra venom.     

Transport and processing of staphylococcal alpha-toxin

Two larger precursors to staphylococcal alpha-toxin were identified and partially characterized. Both precursor proteins were present on the cell membrane at very low levels and appeared to be rapidly processed to the mature form. Dinitrophenol inhibited processing such that the two precursors accumulated in the membranes, whereas little extracellular (mature) alpha-toxin is formed. The peptide maps of the 35S-labeled peptides from extracellular alpha-toxin and the two precursors were almost identical. The larger precursor protein contained four additional peptides and the smaller precursor protein contained three additional peptides not found in the extracellular toxin.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Abstract Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli β-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

A simple purification method for enterotoxin F produced by Staphylococcus aureus and some properties of the toxin

Staphyloccoccus aureus enterotoxin F (SEF), which is associated with S. aureus strains isolated from toxic-shock-syndrome patients, was purified by successive chromatography on CM sephadex C-25 and gelfiltration on sephadex G-75. When tested by disc-polyacrylamide gel-electrophoresis the toxin migrated as a homogeneous protein. In SDS-polyacrylamide gel-electrophoresis three protein bands were observed. The main component had a mol wt of 23000 and the two minor components had a mol wt<13 000. By iso-electric focussing a main protein band with an iso-electric point of 7.2 was obtained. The LD₅₀ for rabbits (3–3.5 kg) by subcutaneous and intravenous application of SEF was 6 g and 180 g, respectively. Antibodies to SEF prepared in a sheep did not react with other staphylococcal enterotoxins (A to E).     

A simple and rapid method for purifying staphylococcal exfoliative toxin A

A rapid and efficient method of purification of staphylococcal exfoliative toxin A, the causative agent of staphylococcal scalded skin syndrome (SSSS), has been developed. It is based on ammonium sulfate precipitation of the culture supernatant and hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B. This procedure results in 87-fold purification of this toxin, which appears as a single band in sodium dodecyl sulfate-polyacrylamide gels.     

Nucleotide sequence of the epidermolytic toxin A gene of Staphylococcus aureus.

The nucleotide sequence of the eta gene, which codes for the epidermolytic toxin serotype A of Staphylococcus aureus TC16, is reported. The coding sequence of 840 nucleotides specifies a protein which, when secreted, has a predicted molecular weight of 26,950. The sequence of eta and the deduced amino acid sequence of the toxin have been compared with those of epidermolytic toxin serotype B. The coding sequences have 52% identical residues, and the polypeptides have 40% identical residues. Amino acid residues have been conserved in the areas of the proteins which correspond to major hydrophobic domains, whereas the regions likely to specify antigenic determinants occur in hydrophilic sequences that have diverged. The level of expression of epidermolytic toxin A in S. aureus 8325-4 was shown to be dependent on the integrity of a regulatory gene called agr.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli beta-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Ultracentrifugal Analysis of Staphylococcal Alpha Toxin

Ultracentrifugal examination of staphylococcal alpha toxin at different stages of purification showed the presence of a major component having a sedimentation coefficient of 2.8S, present to the extent of more than 90% of the sample, and identifiable with active toxin. Several minor components having S(20,w) values of 11.5S, 8.5S, and 2.0S were detected. The 11.5S component presumably is identical with a toxin aggregate studied earlier and designated 12S; the 8.5S component appears to be delta toxin. A sedimentation equilibrium study of more highly purified material gave 32,700 as the best estimate of molecular weight of alpha toxin. Lowering the pH of the partially purified alpha toxin from 10.2 to 5.3 resulted in a small increase in S(20,w) of the 11.5S component and in the disappearance of the 8.5S component, whereas the S(20,w), molecular weight, and hemolytic activity of the toxin remained constant. Exposure of toxin to pH 3.5 irreversibly reduced the S(20,w) to 2.0S, the molecular weight to about 16,000, and caused irreversible inactivation. Raising the pH of acid-inactivated toxin and adding sodium dodecyl sulfate to 1% increased the S(20,w) to near its normal value (2.7S) but did not restore activity.     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

Using Common Spatial Distributions of Atoms to Relate Functionally Divergent Influenza Virus N10 and N11 Protein Structures to Functionally Characterized Neuraminidase Structures,Toxin Cell Entry Domains,and Non-Influenza Virus Cell Entry Domains

The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell entry has been supplemented or replaced by sialidase-independent receptor binding to an expanded cell population that may include neurons and T-cells.     

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.