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J P Arbuthnott

Publications and source records attributed to J P Arbuthnott.

At least 19 recordsLinked to original sources
PubMed1997

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.

Amino Acid Sequence↗
PubMed1993

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.

Animals↗
PubMed1992

Investigation of microbial growth in vivo: evaluation of a novel in vivo chamber implant system.

An intraperitoneal chamber implant system has been used to investigate the phenotype of Staphylococcus aureus growing in the rat and the effect of the antibiotic flucloxacillin on bacterial growth in vivo. Titanium chambers were implanted in the peritoneum: a period of 3-4 days equilibration allowed diffusion of host proteins into the chamber fluid prior to inoculation with bacteria. S. aureus inoculated into the chamber fluid, grew rapidly over a 72 h period, reaching counts of > 10(9) per ml. Organisms harvested from chambers were analysed by SDS-PAGE and showed significant differences in polypeptide profiles from the same strain grown in nutrient broth in vitro. Analysis of whole cell extracts by Western-blotting revealed that protein A expression was repressed in S. aureus grown in vivo. Following subcutaneous administration, flucloxacillin levels in serum peaked earlier and were higher than those detected in chamber fluid. The inhibitory effect of the antibiotic on the growth of S. aureus in chambers in treated animals could be monitored easily by sequential sampling of the chamber fluid. These results indicate the potential of the chamber implant model for investigation of microbial phenotype in vivo and development of alternative methods for assessment of antimicrobial efficacy in vivo.

Animals↗
PubMed1992

Characterization of staphylococcal gamma-lysin.

gamma-Lysin was purified from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated gamma 1 and gamma 2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by gamma-lysin than human erythrocytes. The molecular mass of gamma 1 was 32 kDa and its pI value was 9.4. gamma 2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with gamma 2 to induce increased haemolysis, no such synergism was seen with gamma 1. Also, protease inhibitors acted to inhibit synergism between gamma 1 and gamma 2. These findings suggest that gamma 1 could be a protease.

Endopeptidases↗
PubMed1992

Cell envelope proteins of Staphylococcus epidermidis grown in vivo in a peritoneal chamber implant.

Staphylococcus epidermidis was grown in vivo in chambers implanted intraperitoneally in rats. The cell wall and cytoplasmic membrane protein profiles of the in vivo-grown organisms were compared with those of S. epidermidis grown in vitro in nutrient broth (NB), in iron-restricted NB, or in pooled human peritoneal dialysate (HPD). Compared with growth in broth and in common with growth in HPD, growth in vivo in chambers resulted in the repression of many S. epidermidis wall proteins, with proteins of 27, 42, 54, and 70 kDa predominating. Growth in vivo also resulted in the induction of two iron-repressible cytoplasmic membrane proteins of 32 and 36 kDa, which were also present in staphylococci grown in HPD and in iron-restricted NB. Immunoblotting experiments revealed that in sera taken 21 days after inoculation of the intraperitoneal chambers, the predominant antibody response to cell envelope proteins was directed against the 32- and 36-kDa iron-repressible membrane proteins.

Animals↗
PubMed1991

A toxic shock syndrome toxin mutant of Staphylococcus aureus isolated by allelic replacement lacks virulence in a rabbit uterine model.

The gene coding for toxic shock syndrome toxin-1 in S. aureus was inactivated by allelic replacement in two TSS-associated strains. One mutant derived from FRI1169 (a non-enterotoxigenic strain) lacked virulence in the rabbit uterine chamber infection model. This suggests that TSST-1 is the only determinant produced by this strain that can induce the symptoms of shock in rabbits. A novel method for allelic replacement involving transduction of plasmid integrants is described.

Alleles↗
PubMed1991

Development and design of a novel in vivo chamber implant for the analysis of microbial virulence and assessment of antimicrobial therapy.

An accurate reflection of the pathogenicity of microorganisms and the therapeutic effects of antimicrobial agents on their growth necessitates testing within an in vivo environment. We have developed a novel diffusion chamber, incorporating two 0.22 microns membrane filters, for the growth of in vivo organisms. The chamber, which is implanted intraperitoneally into the rat, has an external sampling portal. This portal allows multiple and sequential sampling of the microbial inoculum without killing the rat, thus significantly reducing the total number of animals used in such studies. In addition, the chamber is superior to other reported implants since it is well tolerated, reusable, easily constructed and can be used within two days of implantation. Staphylococcus epidermidis and a toxic shock syndrome toxin-1 (TSST-1) producing strain of S. aureus have been successfully grown within in vivo chambers, with 10(8)-10(9) organisms per millilitre being recovered within 48 h. Scanning electron microscopy revealed clusters of staphylococci and fibrous material adhering to the inner surface of the filters, with numerous phagocytic cells attached to the outer side. Western immunoblotting indicated that higher levels of TSST-1 were produced by S. aureus grown in vivo as opposed to cells grown in vitro.

Animals↗
PubMed1989

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion.

A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.

Antimicrobial Cationic Peptides↗
PubMed1989

A new methicillin- and gentamicin-resistant Staphylococcus aureus in Dublin: molecular genetic analysis.

In June 1985 two new strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) were isolated in a Dublin hospital. Of these, one strain spread rapidly, affecting a total of 65 patients during the following 18 months, and subsequently spread to a second Dublin hospital. Detailed laboratory studies, including plasmid screening, plasmid restriction enzyme digest pattern analysis, hybridisation analysis, location of resistance determinants, and bacteriophage typing with a set of experimental S. aureus typing phages, demonstrated that the "new" MGRSA organisms, termed Phenotype III Dublin isolates, were completely distinct from, and unrelated to, the MGRSA strains responsible for serious nosocomial infections in Dublin hospitals during the decade before June 1985. These Phenotype III isolates were very similar to MGRSA organisms isolated in a Baghdad hospital during 1984. Data from plasmid curing, plasmid transfer and hybridisation experiments indicated that 20% of the Phenotype-III isolates expressed chromosomally encoded, high level resistance to ethidium bromide (MIC 120 micrograms/ml), and that this was possibly due to chromosomal integration of a penicillinase-like plasmid.

Autoradiography↗
PubMed1989

Enterotoxin production by Staphylococcus aureus isolates from cases of septicaemia and from healthy carriers.

In a prospective study, 52 Staphylococcus aureus isolates from individual patients with septicaemia and 27 nasal strains from separate, healthy carriers were compared for production of a range of extracellular proteins and toxins. Whereas there was no difference (p greater than 0.05) between septicaemic and nasal isolates with respect to incidence of alpha, beta, gamma and delta haemolysins, toxic shock syndrome toxin-1 or staphylokinase production, the incidence of enterotoxin A, B, and C production was higher among isolates from septicaemia (p less than 0.01). Of the isolates from septicaemia, 33 (63%) produced enterotoxins A, B, C or D alone or in combination. Only three (11%) of the nasal isolates produced a single enterotoxin, enterotoxin D. Of the isolates from septicaemia, 67% were hospital-acquired and greater than 25% of these were endemic, methicillin-resistant (MRSA) strains. All MRSA strains produced either enterotoxin A, or enterotoxin B, or both. These findings suggest a possible role for enterotoxins in the pathogenesis of S. aureus disease other than food poisoning.

Bacterial Toxins↗
PubMed1988

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.

Bacterial Proteins↗
PubMed1988

Induction of tumour necrosis factor by staphylococcal toxic shock toxin 1.

Staphylococcal toxic shock syndrome toxin 1 (TSST1) induced the release of tumour necrosis factor (TNF) from human and rabbit monocytes in vitro. Nanogram amounts of TSST1 were sufficient to induce TNF release. There was considerable variation in response between cells from different rabbits and different donors. Rabbit monocytes were slightly more sensitive to TSST1 than were human monocytes. Release of TNF in vivo could explain many of the symptoms of toxic shock syndrome.

Adult↗