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J L Brunton

Publications and source records attributed to J L Brunton.

At least 19 recordsLinked to original sources
PubMed1992

Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli.

The Shiga toxin family, a group of cytotoxins associated with diarrhoeal diseases and the haemolytic uraemic syndrome, includes Shiga toxin from Shigella dysenteriae type 1 and verotoxins produced by enteropathogenic Escherichia coli. The family belongs to the A-B class of bacterial toxins, which includes the cholera toxin family, pertussis and diphtheria toxins. These toxins all have bipartite structures consisting of an enzymatic A subunit associated with a B oligomer which binds to specific cell-surface receptors, but their amino-acid sequences and pathogenic mechanisms differ. We have determined the crystal structure of the B oligomer of verotoxin-1 from E. coli. The structure unexpectedly resembles that of the B oligomer of the cholera toxin-like heat-labile enterotoxin from E. coli, despite the absence of detectable sequence similarity between these two proteins. This result implies a distant evolutionary relationship between the Shiga toxin and cholera toxin families. We suggest that the cell surface receptor-binding site lies in a cleft between adjacent subunits of the B pentamer, providing a potential target for drugs and vaccines to prevent toxin binding and effect.

Bacterial Toxins↗
PubMed1992

Alteration of the carbohydrate binding specificity of verotoxins from Gal alpha 1-4Gal to GalNAc beta 1-3Gal alpha 1-4Gal and vice versa by site-directed mutagenesis of the binding subunit.

Verotoxin 1 (VT-1) and Shiga-like toxin II (SLT-II) bind to the glycosphingolipid (GSL), globotriaosylceramide (Gb3), whereas pig edema disease toxin (VTE) binds to globotetraosylceramide (Gb4) and to a lesser degree Gb3. Amino acids important in the GSL binding specificity of VT-1 and VTE have been identified by site-directed mutagenesis. One mutation, Asp-18----Asn, in VT-1 resulted in binding to Gb4 in addition to Gb3 in a manner similar to VTE. Several mutations in VTE resulted in the complete loss of GSL binding; however, one mutation resulted in a change in the GSL binding specificity of the VTE B subunit. The double mutation Gln-64----Glu and Lys-66----Gln (designated GT3) caused a selective loss of Gb4 binding, effectively changing the binding phenotype from VTE to VT-1. Both wild-type VTE and GT3 were purified to homogeneity and binding kinetics in vitro were determined with purified GSLs from human kidney. The cell cytotoxicity spectrum of the mutant toxin was also found to be altered in comparison with VTE. These changes were consistent with the GSL content of the target cells.

Amino Acid Sequence↗
PubMed1991

Development of verotoxin 2- and verotoxin 2 variant (VT2v)-specific oligonucleotide probes on the basis of the nucleotide sequence of the B cistron of VT2v from Escherichia coli E32511 and B2F1.

We and others have noted that there are serological differences between verotoxin 2 (VT2) (also known as Shiga-like toxin II) produced by Escherichia coli C600(933W) and the VT2 variant (VT2v) produced by strain E32511. Recent reports have described nucleotide sequence differences between the VT2v B subunit cistron of E32511 and B2F1 and that of VT2. We have confirmed the sequence differences and have used them to design oligonucleotide probes which differentiate the B subunit cistron of VT2v from that of VT2. Isolates of VT-producing E. coli obtained from human as well as food and veterinary sources were classified according to the toxin phenotype by using a toxin neutralization assay with VT2-specific monoclonal antibody and VT2v-specific polyclonal antisera. Using the oligonucleotide probes in colony hybridization, we detected 35 of 35 VT2 producers and 16 of 16 VT2v producers. One VT2 producer was falsely identified as containing the VT2v gene. The E32511 strain in our collection hybridized only with the VT2-specific probe. Southern hybridization of radiolabeled oligonucleotide probes showed that strains carried zero to one copy of the VT2 gene and zero to two copies of the VT2v gene. We conclude that colony hybridization with the VT2- and VT2-specific probes is highly predictive of the toxin phenotypes in the clinical isolates described in this study.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of a new replication region in the Neisseria gonorrhoeae beta-lactamase plasmid pFA3.

The 7.1-kilobase-pair (kbp) plasmid pFA3 specifies TEM beta-lactamase production in Neisseria gonorrhoeae. We studied the minimal region required for replication of this plasmid in Escherichia coli by constructing a set of nested deletions of the 3.4-kbp PstI-HindIII fragment. The smallest fragment capable of maintenance in E. coli when ligated to a streptomycin-spectinomycin resistance cassette was 2.0 kbp in size and was different from another autonomously replicating fragment of pFA3 reported by K. H. Yeung and J. Dillon (Plasmid 20:232-240, 1988). The fragment contained single BamHI and XbaI sites and specified a 39-K protein. Fragments subcloned from the minimal region or constructed by deletion from the 3' or 5' ends were not capable of autonomous replication. Mutants constructed by end filling and religating DNA cleaved at the BamHI or XbaI sites were not capable of autonomous replication and no longer produced the 39K protein. These results suggest that replication is dependent on the 39K protein. DNA sequence analysis of the region showed an A-T-rich region followed by four 22-bp direct repeats followed by an open reading frame encoding a 39K basic protein.

Amino Acid Sequence↗
PubMed1989

Plasmid mediated antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae.

The genetic basis of antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae was studied. Two Ontario isolates of A. pleuropneumoniae were found to be resistant to sulfonamides (Su), streptomycin (Sm) and ampicillin (Amp). Resistance to Su and Sm was specified by a 2.3 megadalton (Mdal) plasmid which appeared to be identical to pVM104, which has been described in isolates of A. pleuropneumoniae from South Dakota. Southern hybridization showed that the 2.3 Mdal Su Sm plasmid was highly related to those Hinc II fragments of RSF1010 known to carry the Su Sm genes, but was unrelated to the remainder of this Salmonella resistance plasmid. Resistance to Su and Amp was specified by a 3.5 Mdal plasmid and appeared identical to pVM105 previously reported. The beta-lactamase enzyme had an isoelectric point of approximately 9.0. Southern hybridization showed no relationship to the TEM beta-lactamase. A third isolate of A. pleuropneumoniae was found to be resistant to chloramphenicol (Cm), Su and Sm by virtue of a 3.0 Mdal plasmid which specified a chloramphenicol acetyl transferase. We conclude that resistance to Su, Sm, Amp and Cm is mediated by small plasmids in A. pleuropneumoniae. Although the Su and Sm resistance determinants are highly related to those found in Enterobacteriaceae, the plasmids themselves and the beta-lactamase determinant are different.

Actinobacillus↗
PubMed1988

Cloning and nucleotide sequence analysis of the genes determining verocytotoxin production in a porcine edema disease isolate of Escherichia coli.

The structural genes determining the edema disease principle were cloned from the total cellular DNA of Escherichia coli strain 412 (O139:K82) isolated from a case of porcine edema disease. An assay for cytotoxicity in Vero cells was used to detect the edema disease principle. A 7.5 kb EcoRI-SalI fragment specifying cytotoxin production was subcloned in pUC18. Sequences which specified production of cytotoxin were localized to a 0.9 kb region by transposon Tn5 mutagenesis. A 2.4 kb EcoRI-BglII fragment encompassing this region was subcloned into pUC18. Using nucleotide sequence analysis, two open reading frames separated by 12 bp were identified. They encoded proteins of 319 (A subunit) and 87 (B subunit) amino acids which both had N-terminal sequences typical of E. coli signal peptides. Comparison of these with the published sequence for the Shiga-like toxin II (SLT-II) showed 91% overall nucleotide sequence similarity. The nucleotide sequence similarity extended to 200 base pairs upstream of the putative A subunit translational start site suggesting a common regulatory mechanism. The deduced amino acid sequences of the processed A and B subunits had 94% and 84% similarity, respectively. These findings confirm the close genetic relationship between SLT-II and edema disease principle.

Amino Acid Sequence↗
PubMed1987

Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro.

Escherichia coli verotoxin (also known as Shiga-like toxin) has been implicated in the aetiology of the hemolytic uremic syndrome and hemorrhagic colitis. The glycolipid binding specificity of verotoxin purified from E. coli H30 and verotoxin cloned from bacteriophage H19B has been examined. Verotoxin from both sources binds specifically to globotriosyl ceramide containing the carbohydrate sequence galactose alpha 1-4galactose beta 1-4glucose-ceramide. Removal of the terminal galactose or substitution with N-acetylgalactosamine in beta 1-3 linkage deletes toxin binding activity. A ceramide trihexoside species, consistent with a globotriosyl ceramide structure was shown to be the major verotoxin-binding glycolipid of cultured vero cells which are routinely used to measure the cytotoxicity of toxin samples.

Bacterial Toxins↗
PubMed1987

Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.

Bacterial Toxins↗
PubMed1986

Cloning and expression of the genes specifying Shiga-like toxin production in Escherichia coli H19.

Some strains of Escherichia coli produce a protein which is cytotoxic for Vero cell and HeLa cell monolayers. This toxin is very similar to the toxin of Shigella dysenteriae 1 and has been named verotoxin or E. coli Shiga-like toxin. It has been shown that toxin conversion is due to a group of bacteriophages, one of which has been designated H-19B. In this study we report hybridization experiments showing that part of the H-19B genome is homologous to phage lambda. We have cloned a 1.7-kilobase BalI-BglII fragment from the genome of H-19B into pUC18. The recombinant plasmid confers the ability to produce high levels of Shiga-like toxin on transformed E. coli cells. We demonstrate using an in vitro transcription/translation system that the cloned fragment specifies the two verotoxin subunit peptides which have masses of 31 and 5.5 kilodaltons. The identity of peptides was confirmed by immunoprecipitation with verotoxin antiserum and protein A-Sepharose beads.

Animals↗
PubMed1986

In vitro studies of plasmid-mediated penicillinase from Streptococcus faecalis suggest a staphylococcal origin.

A strain of Streptococcus faecalis with plasmid-mediated penicillinase production was studied further. Partially purified penicillinase from the S. faecalis strain hydrolyzed penicillin, ampicillin, and ureido-penicillins but not penicillinase-resistant semisynthetic penicillins, cephalosporins, or imipenem; hydrolysis was inhibited by clavulanic acid. Hydrolysis of a given antibiotic correlated with a marked increase in the minimal inhibitory concentration (MIC) of that drug when a high inoculum was used. As with most enterococci, the MICs of cephalosporins and penicillinase-resistant semisynthetic penicillins were too high for clinical usefulness, although these agents did not show an inoculum effect. Based upon hybridization under stringent conditions of plasmid DNA from the S. faecalis strain to cloned penicillinase genes from Staphylococcus aureus, it appears that these resistance determinants are highly homologous and suggests that this enzyme was introduced into streptococci from staphylococci.

Anti-Bacterial Agents↗
PubMed1984

Q fever: hazard from sheep used in research.

The recent recognition that Q fever is endemic in Ontario and the known occupational risk of Q fever to research personnel working with sheep prompted a study to determine the prevalence of antibodies to the causative organism, Coxiella burnetti, in animals and staff at a Toronto animal research institute. Of 37 sheep 34 (92%) were found to be seropositive--that is, to have a titre of complement-fixing antibody to the phase II antigen of 1:8 or greater. Of 331 staff members tested, 18% were found to be seropositive, compared with 0.6% of a random sample of Toronto blood donors. The highest rate of seropositivity, 68%, was in the 28 animal attendants tested. Seropositivity was associated with working with sheep or fetal lamb tissue (p less than 0.0001) and with visiting the animal facility (p less than 0.001). Of the 59 seropositive staff members 63% had had no direct contact with sheep. There were 12 clinically apparent cases of Q fever, 2 of which required admission to hospital. Q fever remains a serious occupational hazard to staff working in research laboratories using sheep, even to those with indirect exposure to infected animals.

Adult↗
PubMed1983

Evolution of antibiotic resistance plasmids in Neisseria gonorrhoeae and Haemophilus species.

The emergence of beta-lactamase producing strains of Haemophilus influenzae and Neisseria gonorrhoeae has required fundamental changes in the antimicrobial therapy of disease caused by these organisms. Ampicillin resistance in both organisms is caused by plasmid mediated production of TEM beta-lactamase. This enzyme is specified by a sequence of mol. wt 3.2 X 10(6) which is capable of inserting itself at multiple sites in DNA replicons without the requirement for significant base sequence homology between donor and recipient replicon. Further, it does so without requirement for conventional recombination enzymes. Analysis of beta-lactamase specifying plasmids of H. influenzae show that they generally have a molecular mass in the order of 30 X 10(6) and contain the complete TnA sequence. They are conjugative but are incapable of mobilizing smaller beta-lactamase plasmids. Previous studies have presented evidence suggesting that these plasmids may have evolved by insertion of the TnA sequence (perhaps introduced from enteric bacteria) into a phenotypically cryptic plasmid of mol. wt 27 X 10(6) resident in rare strains of H. influenzae. In this study, we review data showing a high degree of homology between the small (3--7 X 10(6) mol. wt), nonconjugative beta-lactamase specifying plasmids of N. gonorrhoeae, H. parainfluenzae and H. ducreyl and present new evidence that cryptic plasmids highly homologous to the beta-lactamase plasmids are present in many strains of H. parainfluenzae. This suggests that the small beta-lactamase specifying plasmids of H. parainfluenzae, H. ducreyi and N. gonorrhoeae may have arisen by insertion of TnA into phenotypically cryptic plasmids present in H. parainfluenzae.

Ampicillin↗
PubMed1982

Primary and secondary healing in infected wounds. An experimental study.

The beneficial effects of delayed closure of contaminated wounds are recognized, but not well defined. Rats with abdominal incisions were infected with Staphylococcus aureus or Escherichia coli and then subgrouped for primary or secondary healing. Noninfected rats served as controls. Between seven and 119 days later, the wounds were subjected to breaking-strength determination, hydroxyproline analysis, and light and scanning electron microscopy. In the controls, secondary closure gave stronger wounds than primary closure. In those infected with S aureus the wounds were stronger than in the primary-closure control group, regardless of the closure method. Of the E coli-infected wounds those primarily closed were weaker than those secondarily closed. Secondary closure gave infected wounds fewer complications. Biochemical and microscopic examination did not explain these findings.

Abdominal Muscles↗
PubMed1982

Plasmid-mediated sulfonamide resistance in Haemophilus ducreyi.

Clinical isolates of Haemophilus ducreyi from patients with chancroid were shown to have one or more 4.9- to 7.0-megadalton non-self-transferable plasmids and to have in vitro resistance to sulfonamides. Transformation of Escherichia coli to sulfonamide resistance was associated with the acquisition of a 4.9-megadalton plasmid, which did not confer linked resistance to streptomycin. The guanine-plus-cytosine content of this plasmid was found to be 57%. Filter-blot hybridization and restriction endonuclease digestion studies suggested a relationship of this plasmid to RSF1010. Electron microscope heteroduplex analysis confirmed this relationship. The identification in H. ducreyi of a plasmid closely related to plasmids found in enteric species, rather than transposition of a resistance determinant to an indigenous plasmid, suggests that further dissemination of the enteric plasmid pool to this genus is possible since plasmid transfer between certain Haemophilus species is readily demonstrated.

Endonucleases↗
PubMed1982

Haemophilus influenzae: comparison of respiratory tract isolates with genitourinary tract isolates.

Haemophilus influenzae isolates recovered from the genitourinary (GU) tract were shown to have a significantly different biotype distribution compared with respiratory tract isolates. Biotype IV strains were recovered more commonly from the GU tract, and most strains were non-serotypable. Antibiotic-susceptible strains isolated from the GU tract more frequently harbored plasmids of less than 10 megadaltons than did antibiotic-susceptible respiratory tract strains. One 2.8-megadalton plasmid resident in a GU tract isolate and one 1.8-megadalton plasmid resident in a respiratory tract isolate were shown to be related to the small ampicillin resistance plasmids previously described in H. influenzae, Haemophilus parainfluenzae, Haemophilus ducreyi, and Neisseria gonorrhoeae. This supports the suggestion that these ampicillin resistance plasmids originated by transposition or recombination of the ampicillin transposon (TnA) with cryptic endogenous Haemophilus plasmids.

Anti-Bacterial Agents↗