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.     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Detection of Staphylococcus aureus toxins using immuno-PCR

A highly sensitive test system, based on the immuno-PCR method, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was to obtain supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were to be used as DNA-tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not lower than 10 pg/mL.     

A restriction endonuclease from Staphylococcus aureus.

A specific endonuclease, Sau 3AI, has been partially purified from Staphylococcus aureus strain 3A by DEAE-cellulose chromatography. The enzyme cleaves adenovirus type 5 DNA many times, SV40 DNA eight times but does not cleave double-stranded phi X174 DNA. It recognizes the sequence (see article) and cleaves as indicated by the arrows. Evidence is presented that this enzyme plays a role in the biological restriction-modification system of Staphylococcus aureus strain 3A.     

Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus.

Exfoliative toxin type B is produced by bacteriophage group II strains of Staphylococcus aureus and is a causative agent of staphylococcal scalded-skin syndrome. In addition to exfoliative toxin B, most isolates also produce a bacteriocin and are immune to the action of the bacteriocin. These phenotypes, as well as resistance to cadmium, were lost after elimination of a 37.5-kilobase plasmid, pRW001, from S. aureus UT0007. Transduction and transformation showed that pRW001 carries the structural genes for four phenotypic characteristics of S. aureus UT0007: (i) exfoliative toxin B production, (ii) bacteriocin production, (iii) bacteriocin immunity, and (iv) resistance to Cd(NO3)2. The exfoliative toxin B structural gene (etb), which is located on a 1.7-kilobase HindIII fragment of pRW001, was cloned in the plasmid pDH5060 and transformed into phage group III S. aureus RN4220. Transformant clones produced extracellular exfoliative toxin B that was biologically active in the neonatal mouse assay. In the Escherichia coli genetic background, the exfoliative toxin B gene was expressed only after being cloned into the positive selection-expression vector pSCC31. The structural gene for cadmium resistance was also isolated on an HindIII fragment of pRW001 cloned in pDH5060. The loci for the exfoliative toxin B gene and the cadmium resistance gene(s) were identified on a restriction map of plasmid pRW001.     

Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus.

The DNA encoding the exfoliative toxin A gene (eta) of Staphylococcus aureus was cloned into bacteriophage lambda gt11 and subsequently into plasmid pLI50 on a 1,391-base-pair DNA fragment of the chromosome. Exfoliative toxin A is expressed in the Escherichia coli genetic background, is similar in length to the toxin purified from culture medium, and is biologically active in an animal assay. The nucleotide sequence of the DNA fragment containing the gene was determined. The protein deduced from the nucleotide sequence is a polypeptide of 280 amino acids. The mature protein is 242 amino acids. The DNA sequence of the exfoliative toxin B gene was also determined. Corrections indicate that the amino acid sequence of exfoliative toxin B is in accord with chemical sequence data.     

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.     

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.     

Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation

Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. This review, divided into four parts, will focus on the following: (1) the worldwide story of SFP outbreaks, (2) the characteristics and behaviour of S. aureus in food environment, (3) the toxinogenic conditions and characteristics of SEs, and (4) SFP outbreaks including symptomatology, occurrence in the European Union and currently available methods used to characterize staphylococcal outbreaks.     

Preliminary crystallographic data for exfoliative toxin B from Staphylococcus aureus

The serotype B of exfoliative toxin, isolated from Staphylococcus aureus, strain TC 142, has been crystallized. The monoclinic crystals belong to space group P2₁, with $\text{a = 55.9}\text{A}\text{?}\text{, b = 107.9}\text{A}\text{?}\text{, c = 42.8}\text{A}\text{?}\text{, and}\text{β = 90.9 °}$. The asymmetric unit contains two molecules of molecular weight 30,000.     

Understanding the mechanism of action of the exfoliative toxins of Staphylococcus aureus

The exfoliative toxins of Staphylococcus aureus are responsible for the staphylococcal scalded skin syndrome, a blistering skin disorder that particularly affects infants and young children, as well as adults with underlying disease. Their three-dimensional structure is similar to other glutamate-specific trypsin-like serine proteases with two substrate-binding domains and a serine-histidine-aspartate catalytic triad that forms the active site. However, unlike other serine proteases, the exfoliative toxins possess a highly charged N-terminal alpha-helix and a unique orientation of a critical peptide bond, which blocks the active site of the toxins so that, in their native state, they do not possess any significant enzymatic activity. The target for the toxins has recently been identified as desmoglein-1, a desmosomal glycoprotein which plays an important role in maintaining cell-to-cell adhesion in the superficial epidermis. It is speculated that binding of the N-terminal alpha-helix to desmoglein-1 results in a conformation change that opens the active site of the toxin to cleave the extracellular domain of desmoglein-1 between the third and fourth domains, resulting in disruption of intercellular adhesion and formation of superficial blisters. Elucidating the mechanism of action of the toxins and identifying desmoglein-1 as their specific epidermal substrate has not only given us an insight into the pathogenesis of the staphylococcal scalded skin syndrome, but also provided us with useful information on normal skin physiology and the pathogenesis of other toxin-mediated diseases. It is hoped that this knowledge will lead to development of rapid screening and diagnostic tests, and new antitoxin strategies for the treatment and prevention of the staphylococcal scalded skin syndrome in the near future.     

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/ 19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (chi2 = 5.42, P < 0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in > 50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.     

Enterotoxin B formation by fermentation mutants of Staphylococcus aureus.

An appreciable fraction of carbohydrate-negative (car) mutants of Staphylococcus aureus strains ATCC 14458, 778, and S-6 exhibit increased enterotoxin B (SEB) production. In addition, some lac and mtl mutants of these strains also display enhanced SEB formation. All such mutants appear to be point mutations. Mutagen-induced reversions of high SEB producing car, mtl, or lac mutants yield varying amounts of SEB and some clones seem to be restored to the characteristics of the parent type. A few sequentially isolated lac, mtl double mutants of strain 778 elaborate much more or much less SEB than either the lac or the mtl single mutants.