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Biomedical subjects

J C Paton

Publications and source records attributed to J C Paton.

At least 91 records · Page 5Linked to original sources
PubMed1993

Sequence of a variant Shiga-like toxin type-I operon of Escherichia coli O111:H-.

PCR amplification was used to screen faecal isolates of Escherichia coli from a 12-month-old boy with haemolytic uraemic syndrome for the presence of Shiga-like toxin (SLT)-encoding genes. One isolate, belonging to serotype O111:H-, was positive for SLT-I by this method. UV induction indicated that the strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin. Southern hybridization analysis of chromosomal DNA revealed that the SLT-I gene was located on an 8.5-kb EcoRI fragment. SLT-I was further localized to within a 3.0-kb SphI-EcoRI fragment. A separate subclone contained a 3.75-kb HindIII fragment, 1.18 kb of which was common to both. Nucleotide sequence analysis of derivatives of these clones revealed that the SLT-I A subunit gene from E. coli O111:H- differed from the previously published sequences for SLT-I by 5 bp [resulting in two amino acid (aa) changes]. It was more closely related to the gene encoding the A subunit of the Shiga toxin from Shigella dysenteriae type 1, from which it differed by 3 bp (resulting in one aa change). The DNA sequence of the B subunit-encoding gene was identical to that of the other two toxins. The region of DNA upstream from the SLT-I of E. coli O111:H- contained an IS element, as well as a region with strong homology to a portion of the genome of bacteriophage lambda.

Amino Acid Sequence↗
PubMed1993

Immunization of mice with Salmonella typhimurium C5 aroA expressing a genetically toxoided derivative of the pneumococcal toxin pneumolysin.

An attenuated Salmonella strain expressing a genetically toxoided derivative of the pneumococcal toxin pneumolysin was constructed by first transforming a methylation-positive, restriction-negative Salmonella with plasmid pJCP20M, a derivative of pBR322 containing the modified pneumolysin gene. Plasmid DNA was then extracted and transformed into Salmonella typhimurium C5 aroA. The transformant (denoted JM8) was capable of constitutively expressing the modified pneumolysin gene in vitro and stably maintained the recombinant plasmid containing the pneumococcal DNA, even in the absence of antibiotic selection. When JM8, or the parental Salmonella C5 aroA carrying pBR322 (denoted JM6), were administered orally to mice, both strains were capable of at least transient colonization of the Peyer's patches. Sera from JM8 mice (but not those fed JM6) had significant anti-pneumolysin IgG and IgA ELISA titres. Intraperitoneal administration of JM8 resulted in higher anti-pneumolysin IgG titres, but lower specific IgA levels.

Animals↗
PubMed1993

Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction.

A single pair of oligonucleotide primers was used for polymerase chain reaction amplification of a 212- or 215-bp region of Escherichia coli Shiga-like toxin (SLT) genes from crude fecal culture extracts. Genes were typed by hybridization of the polymerase chain reaction products to SLT-I- or SLT-II-specific oligonucleotide probes. The procedure was capable of detecting fewer than 10 SLT-producing E. coli organisms per ml of culture against a background of more than 10(9) other organisms per ml and provides a rapid and sensitive means of screening primary fecal cultures for the presence of such strains. When this procedure was used to test primary cultures from gut contents or feces from various patient groups, including apparently healthy infants, approximately half of all samples yielded positive results for SLT-I and/or SLT-II sequences.

Bacterial Toxins↗
PubMed1993

Molecular analysis of the pathogenicity of Streptococcus pneumoniae: the role of pneumococcal proteins.

For many years the virulence of Streptococcus pneumoniae has largely been attributed to its antiphagocytic polysaccharide capsule. Recent evidence, however, indicates that certain pneumococcal proteins play an important part in the pathogenesis of disease, either as mediators of inflammation or by directly attacking host tissues. Pneumococci carrying defined mutations in the genes encoding any one of at least three pneumococcal proteins (the toxin pneumolysin, the major pneumococcal autolysin, and pneumococcal surface protein A) have significantly reduced virulence. Pneumococcal hydrolytic enzymes, such as neuraminidase, hyaluronidase, and IgA1 protease may also contribute to colonization and/or invasion of the host. Several of these proteins (or their detoxified derivatives) are protective immunogens in animal models and therefore warrant consideration for inclusion in human antipneumococcal vaccine formulations.

Amino Acid Sequence↗
PubMed1992

Cloning and nucleotide sequence of a variant Shiga-like toxin II gene from Escherichia coli OX3:H21 isolated from a case of sudden infant death syndrome.

Escherichia coli OX3:H21 expressing a toxin related to Shiga-like toxin (SLT) was isolated from the small bowel contents of a case of Sudden Infant Death Syndrome (SIDS). This strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin. Southern hybridization analysis of chromosomal DNA revealed that the SLT-related gene was located on a 4.6 kb PstI fragment, which was cloned into E. coli JM109 in both orientations, using the vector pUC19, to generate plasmids pJCP501 and pJCP502. JM109 cells harbouring the recombinant plasmid produced SLT, as judged by cytotoxicity for Vero cells. Nucleotide sequence analysis revealed that the SLT gene was related to, but distinct from, previously reported variants of Shiga-like toxin type II, produced by E. coli from both human and animal sources. The A subunit of the SLT gene from OX3:H21 exhibited 95.9% homology (at both the DNA and derived amino acid sequence level) to the A subunit of the most closely related SLT-II variant. The B subunit was less similar, exhibiting 88.6 and 88.8% homology to the related gene at the DNA and amino acid level, respectively.

Amino Acid Sequence↗
PubMed1992

Effect of insertional inactivation of the genes encoding pneumolysin and autolysin on the virulence of Streptococcus pneumoniae type 3.

Derivatives of Streptococcus pneumoniae type 3 deficient in production of either pneumolysin or autolysin were constructed. This was achieved by transformation of type 3 pneumococci with DNA from derivatives of a rough strain (Rx1), in which the respective genes had been interrupted by insertion-duplication mutagenesis using internal fragments of the cloned genes in the vector pVA891. Southern blot analysis confirmed that the pneumolysin or autolysin genes in the respective transformants had been interrupted by insertion of the plasmid-derived sequences. Both the pneumolysin-negative and the autolysin-negative strains had significantly reduced (P less than 0.0001) virulence in mice, as judged by survival time after intraperitoneal challenge. The median survival time of mice challenged with type 3 pneumococci in which either pneumolysin or autolysin production had been reconstituted by back-transformation of the mutants with an intact copy of the respective cloned gene (with concomitant elimination of plasmid-derived sequences), was indistinguishable from that of mice challenged with the wild-type strain. These results establish the importance of both pneumolysin and autolysin to the virulence of type 3 pneumococci.

Animals↗
PubMed1992

Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae.

Previous studies on Streptococcus pneumoniae have established that the pneumococcal proteins autolysin (N-acetylmuramyl-L-alanine amidase) and pneumolysin both contribute significantly to the virulence of the organism. In the present work, autolysin and a defined toxoid derivative of pneumolysin were tested, individually and in combination, for efficacy in a mouse model as antigens protecting against challenge with virulent, wild-type S. pneumoniae. While each antigen alone provided significant protection, the degree of protection was not increased when the antigens were administered together. In an additional experiment, mice were challenged with a genetically-modified mutant strain of pneumococcus unable to express active pneumolysin. Pre-immunization of such mice with autolysin failed to provide any significant protection against the challenge. The results of this study suggest that the most important contribution made by autolysin to the virulence of S. pneumoniae may be its role in mediating the release of pneumolysin from the pneumococcal cytoplasm during infection.

Animals↗
PubMed1992

Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification.

A rapid method for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates, involving a combination of reverse transcription and polymerase chain reaction amplification (RT-PCR), has been developed. The RT-PCR assay employs oligonucleotide primers specific for the region of the RSV genome which encodes the F1 subunit of the fusion (F) glycoprotein. Other respiratory viruses do not give a positive reaction. The RT-PCR assay was tested on 202 nasopharyngeal aspirates collected from children with clinical signs of respiratory infection, and the results from RT-PCR were compared with those obtained from virus culture and direct detection by enzyme immunoassay (EIA). RT-PCR results were positive in 118 of 125 samples from which RSV was cultured, as well as in 4 of 7 samples which were culture negative but EIA positive. RT-PCR results were negative in 68 of 70 culture-negative, EIA-negative samples, which included 11 samples from which other respiratory viruses were isolated. The speed, sensitivity (94.6%), and specificity (greater than 97%) of the RT-PCR assay suggest that this technique could be useful for rapid detection of RSV in clinical samples.

Base Sequence↗
PubMed1991

Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae.

Pneumolysin is a thiol-activated, membrane-damaging, multifunctional toxin and a known virulence factor of Streptococcus pneumoniae. The toxin can interfere with the functioning of both cellular and soluble components of the human immune system which protects against pneumococcal infection. Different amino acids within the toxin which are important in promoting oligomerization of the toxin in membranes and for the generation of functional lesions have been identified by site-directed mutagenesis. Pneumolysin can also activate the classical pathway of complement, and this appears to involve antibody binding (via Fc) by a region of the toxin homologous to C-reactive protein, a human acute-phase protein also capable of classical pathway activation and implicated in host defence against pneumococcal infection.

Amino Acid Sequence↗
PubMed1991

Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide.

As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

Amino Acid Sequence↗
PubMed1990

Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction.

A tandemly repeated 1,046-base-pair (bp) ClaI DNA fragment from Bordetella pertussis was cloned into Escherichia coli by using the vector pUC19. This fragment, when isolated, hybridized strongly to DNA from all 100 clinical isolates of B. pertussis tested. It was shown to have homology to single-copy sequences in Bordetella bronchiseptica but not Bordetella parapertussis and did not hybridize to lysate blots of a wide range of other bacteria, including members of the closely related genera Pasteurella, Alcaligenes, and Haemophilus. The 1,046-bp fragment was sequenced, and complementary synthetic oligonucleotides flanking a 153-bp region within the repeated element were used as primers for specific amplification of this region using the polymerase chain reaction (PCR). This procedure was then applied to the rapid (5-h) detection of B. pertussis in nasopharyngeal secretions collected from 332 children with suspected pertussis. The test yielded positive results in a total of 98 samples, compared with 66 for culture and 33 for direct immunofluorescence (IF). All of the IF-positive samples were PCR positive, as were 63 of the samples from which B. pertussis was eventually cultured. Two hundred thirty-one specimens which were negative by IF and culture were also negative in the PCR assay. However, 33 culture- and IF-negative specimens were positive by PCR assay. Several of these specimens were collected from close contacts of culture-proven pertussis patients, were follow-up specimens from such patients, or were from patients with serological evidence of pertussis and therefore may be true-rather than false-positives.

Base Sequence↗
PubMed1989

Production of pneumolysin, a pneumococcal toxin, in Bacillus subtilis.

We have expressed the pneumolysin gene of Streptococcus pneumoniae in Bacillus subtilis, both from its own promoter and as a fusion protein. The level of expression of pneumolysin from its own promoter was low. The protein produced was hemolytically active. A higher level of expression (about 10 micrograms/ml of culture) was achieved when either one of two C-terminal fragments (corresponding to amino acids 265-471 and 55-471, respectively) or the entire coding part of the pneumolysin gene were fused to the promoter and signal sequence-coding region of the alpha-amylase gene of Bacillus amyloliquefaciens. The C-terminal fusion peptides reacted with anti-pneumolysin serum, but were not hemolytically active. In both cases most of the peptide remained cell-associated. When the entire pneumolysin gene was fused to the signal sequence, a hemolytically active form of pneumolysin could be detected, and most of the product was found in a processed form in the culture supernatant. The full-length pneumolysin secreted from B. subtilis was partially purified and used as antigen in an enzyme immunoassay with rabbit anti-pneumolysin serum.

Bacillus subtilis↗
PubMed1989

Presence of a small plasmid in clinical isolates of Streptococcus pneumoniae.

We have detected the presence of a small (2.95 kb) plasmid in a clinical isolate of Streptococcus pneumoniae. A restriction map was constructed for this plasmid and for pDP1 (the only previously reported pneumococcal plasmid); no apparent differences were observed and the two plasmids hybridized strongly to each other. Portions of pDP1 were then cloned into Escherichia coli K-12, using the vector pUC19, and the pneumococcal DNA insert was used as a probe to screen 500 clinical isolates of S. pneumoniae for pDP1 sequences. The plasmid was detected in a total of 8 isolates. These were of various serotypes and no correlation could be found between the presence of the plasmid and the geographical location from which it came, the type of infection, or with resistance to antibacterial drugs. Although no function has yet been assigned to pDP1, it may form the basis of a useful vector for cloning in S. pneumoniae, as it contains at least seven unique restriction sites.

Blotting, Southern↗
PubMed1989

Measurement of antibody responses to pneumolysin--a promising method for the presumptive aetiological diagnosis of pneumococcal pneumonia.

An enzymeimmunoassay (EIA) for measuring antibodies to pneumococcal pneumolysin has been developed. The method was used to study the possible pneumococcal aetiology of pneumonia in 159 mostly elderly patients admitted to hospital because of a positive chest X-ray. The results obtained with the assay were compared to those obtained by other diagnostic methods, namely blood culture, detection of pneumococcal antigen in urine and demonstration of an antibody response to pneumococcal C-polysaccharide and capsular polysaccharides. Antibody response to pneumolysin was found in 32 of 39 (82%) patients with pneumococcal pneumonia aetiologically diagnosed presumptively by other methods. In addition, the EIA for pneumolysin antibodies was positive in 31 patients without evidence of pneumococcal aetiology by other methods. The clinical and laboratory investigations of these patients supported the presumption of bacterial infection. We conclude that the EIA we have developed for measuring pneumolysin antibodies is a promising, sensitive method for the presumptive aetiological diagnosis of pneumococcal pneumonia. The assay is simple to perform because only one antigen is needed and measurement of IgG antibodies alone seems to be enough for aetiological diagnosis.

Adolescent↗
PubMed1989

Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.

Animals↗