Phage conversion of exfoliative toxin A production in Staphylococcus aureus

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (phiETA) that encodes ETA and determined the complete nucleotide sequence of the phiETA genome. phiETA has a head with a hexagonal outline and a non-contractile and flexible tail. The genome of phiETA is a circularly permuted linear double-stranded DNA, and the genome size is 43 081 bp. Sixty-six open reading frames (ORFs) were identified on the phiETA genome, including eta, which was found to be located very close to a putative attachment site (attP). phiETA converted ETA non-producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that phiETA or related phages are responsible for the acquisition of eta genes in S. aureus.     

Sequence of the exfoliative toxin B gene of Staphylococcus aureus.

We sequenced the Staphylococcus aureus exfoliative toxin B gene contained on a 1.7-kilobase HindIII fragment of plasmid pRW001. The gene was located by comparison of the amino acid sequences of open reading frames with the amino-terminal sequence of exfoliative toxin B and the total amino acid composition of the protein (A.D. Johnson, L. Spero, J.S. Cades, and B.T. De Cicco, Infect. Immun. 24:679-684, 1979). The primary translation product consists of 274 amino acids and contains a 31-amino-acid N-terminal peptide presumably necessary for transport.     

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.     

Isolation of extrachromosomal deoxyribonucleic acid for exfoliative toxin production from phage group II Staphylococcus aureus.

The ability of phage group II staphylococcal strain UT 0101 to produce exfoliative toxin and bacteriocin could be eliminated at a high frequency after growth at high temperatures or in the presence of ethidium bromide or sodium dodecyl sulfate. Extrachromosomal deoxyribonucleic acid, associated with the genes for exfoliative toxin and bacteriocin production, was isolated from strain UT 0101 but was absent from an ethidium bromide-cured substrain. The molecular weight of the exfoliative toxin plasmid, determined by co-sedimentation with the penicillinase plasmid, PI258, was 3.3 times 10-7. The 56S covalently closed circular form of the exfoliative toxin plasmid converted to a 38S open circular form after storage or exposure to sodium dodecyl sulfate. Plasmid deoxyribonucleic acid associated with penicillin resistance could not be identified in the penicillin-resistance Tox+ strains, UT 0007 and UT 0001.     

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.     

Molecular cloning and genetic analysis of the determinant for gamma-lysin, a two-component toxin of Staphylococcus aureus

The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.     

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.     

Translocation of the chloramphenicol-resistance determinant in Staphylococcus aureus

pC658: a plasmid determining resistance to chloramphenicol in the hospital strain of Staphylococcus aureus JM658 was transduced after irradiation of phage lysate with high doses of UV. The localization of determinants causing resistance to chloramphenicol in the obtained transductants was investigated by modified Arber's method and variants resistant to chloramphenicol, but suspected of absence of chloramphenicolase plasmid were selected. Additionally the absence of plasmid DNA was demonstrated in the selected strains. The possibility of plasmid and chromosomal localization of the same gene indicates its translocable nature. The obtained results suggests transposomal character of the genes determining resistance to chloramphenicol in the pC658 plasmid, occurring in the hospital strain of S. aureus.     

Transduction of penicillinase production in Staphylococcus epidermidis and nature of the genetic determinant.

Four strains of Staphylococcus epidermidis from clinical sources were capable of serving as donors for the transduction of either penicillinase production, ethidium bromide resistance, or tetracycline resistance. Three typing phages served as transducing phages and, depending upon the combination of transducing phage, donor strain, and recipient strain, the rates of transduction ranged between 10(-5) and 10(-9). In one strain, cotransduction of penicillinase production and ethidium bromide resistance was observed. Although ultraviolet irradiation kinetics indicated that both the tetracycline resistance and the penicillin resistance determinants were located on plasmids, only resistance to tetracycline could be eliminated by growth in the presence of curing agents or at elevated temperature. However, evidence was obtained by agarose gel electrophoretic studies that both the tetracycline resistance and the penicillin resistance determinants are located on separate plasmids in this organism.     

Chromosomal and extrachromosomal synthesis of exfoliative toxin from Staphylococcus hyicus

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P(-)) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P(-) substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P(-)) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.     

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.     

Crystallization of the delta toxin of Staphylococcus aureus

Delta toxin is a small cytolytic polypeptide produced and secreted by the organism Staphylococcus aureus and belongs to a family of surface-active toxins that exhibit pronounced effects on a wide variety of cellular membranes. Although this class of proteins has been much studied by a wide variety of physical techniques, no consensus has been reached on their mode of action. Therefore, in order to investigate their role in causing membrane damage, a structural analysis of the delta toxin has been initiated. Crystals of this protein have been grown by dialysis against mixtures of 2-methylpentan-2,4-diol and water. These crystals are relatively insensitive to radiation damage and diffract to high resolution. The results of this study should provide a valuable insight into the cytolytic properties of these molecules.     

Detection and toxin production of Staphylococcus aureus in sudden infant death cases in Hungary

The potential role of microbial agents was investigated in 13 cases of Sudden Infant Death Syndrome and in 9 non-SIDS cases in Budapest between September 1996 and May 1998. Autopsy, histological examination and microbiological tests were performed on samples of blood, cerebrospinal fluid, pharyngeal samples and lung tissue from infants under one year died suddenly, without previous diseases. The multifactorial pathomechanism of SIDS was suggested by the isolation of toxin producing Staphylococcus aureus-, Enterobacteriaceae and Candida albicans strains in large number and by the detection of Parainfluenza Type 2 virus antigen. S. aureus proved the predominant bacteria in the SIDS cases. Nasopharyngeal microbial flora and S. aureus carrier of 100 age matched healthy infants were tested during the same period. S. aureus was isolated from 54% of SIDS cases and 37% from healthy infants /OR = 1.986 (95% Confidence interval = 0.55-7.33), p = 0243/. The enterotoxin and TSST-1 toxin producing activity of S. aureus showed the characteristic difference. The toxigenic S. aureus was detected in 46% of SIDS cases and 16% of healthy infants /OR = 4.5 (95% CI = 1.15-17.72), p = 0.010/. The distribution of toxigenic and nontoxigenic isolates was 86% in SIDS cases and 43% in healthy infants /OR = 7.875 (CI = 0.78-191.89), p = 0.041/.     

Genetic behavior of the methicillin resistance determinant in Staphylococcus aureus.

The cotransformation frequency of mecC5 with pur-102 using Staphylococcus aureus C5 deoxyribonucleic acid was found to be approximately 45%. However, in cotransduction studies, there was a 15% cotransduction of purine prototrophy and methicillin sensitivity but, in the reciprocal cross, no purine-prototrophic plus Mecr cotransductants were obtained (frequency less than 0.06%). The data support the hypothesis that the mec determinant resides on an inserted deoxyribonucleic acid sequence in S. aureus and that there is no allelic equivalent in sensitive cells.     

Staphylococcus aureus mobile genetic elements

Among the bacteria groups, most of them are known to be beneficial to human being whereas only a minority is being recognized as harmful. The pathogenicity of bacteria is due, in part, to their rapid adaptation in the presence of selective pressures exerted by the human host. In addition, through their genomes, bacteria are subject to mutations, various rearrangements or horizontal gene transfer among and/or within bacterial species. Bacteria’s essential metabolic functions are generally encoding by the core genes. Apart of the core genes, there are several number of mobile genetic elements (MGE) acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. These MGE namely bacteriophages, transposons, plasmids, and pathogenicity islands represent about 15 % Staphylococcus aureus genomes. The acquisition of most of the MGE is made by horizontal genomic islands (GEI), recognized as discrete DNA segments between closely related strains, transfer. The GEI contributes to the wide spread of microorganisms with an important effect on their genome plasticity and evolution. The GEI are also involve in the antibiotics resistance and virulence genes dissemination. In this review, we summarize the mobile genetic elements of S. aureus.     

Multiplex PCR for detection of three exfoliative toxin serotype genes inStaphylococcus aureus

Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control.