Biomedical subjects

J C Paton

Publications and source records attributed to J C Paton.

At least 109 records · Page 6Linked to original sources

PubMed1989

Contribution of autolysin to virulence of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct an autolysin-negative derivative of Streptococcus pneumoniae. This derivative was obtained by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 375-base-pair TaqI DNA fragment from the middle of the autolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, autolysin-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 transformants produced autolysin. Southern blot analysis confirmed that the autolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The autolysin-negative mutants showed markedly reduced virulence for mice compared with that of strain D39; intranasal and intraperitoneal 50% lethal doses were increased 10(2)- and 10(5)-fold, respectively. Autolysin production was reinstated in one of the mutants by back-transformation with the cloned autolysin gene, with the concomitant loss of erythromycin resistance; the virulence of this isolate for mice was indistinguishable from that of D39. The importance of autolysin in pathogenesis was confirmed by immunization-challenge studies. Mice immunized with purified autolysin survived significantly longer than did control mice after intranasal challenge with strain D39. This study provides direct evidence that the pneumococcal autolysin contributes to virulence and identifies it as a potential vaccine antigen.

Amidohydrolases↗

PubMed1988

Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli.

A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.

Blotting, Southern↗

PubMed1988

Comparative efficacy of pneumococcal neuraminidase and pneumolysin as immunogens protective against Streptococcus pneumoniae.

Neuraminidase and pneumolysin were purified from cultures of Streptococcus pneumoniae and used, either singly or in combination, to immunize juvenile mice which were subsequently challenged intranasally with virulent S. pneumoniae. In each of two independent trials, a small but significant (P less than 0.05) increase in survival time (compared with that of non-immunized mice) was observed in groups which had been immunized with neuraminidase, but only if the enzyme had been pre-treated with 3.4% (v/v) formaldehyde. The median extension in survival time was significantly less (P less than 0.01) than that of mice which had been immunized with pneumolysin alone. The median survival time for mice which had received both formaldehyde-treated neuraminidase and pneumolysin was not significantly different from that of mice which had received pneumolysin alone. While these findings provide direct evidence that neuraminidase contributes to the pathogenicity of the pneumococcus in mice, they suggest that this protein may be of less value than pneumolysin as a vaccine component in the present experimental model.

Animals↗

PubMed1988

Purification and immunological characterization of neuraminidase produced by Streptococcus pneumoniae.

Previous workers have suggested that Streptococcus pneumoniae, the pneumococcus, produces multiple forms of the enzyme neuraminidase. By serial chromatography on DEAE-cellulose, Sephacryl S-200, Amicon Red-A gel and hydroxylapatite we have purified to electrophoretic homogeneity a pneumococcal neuraminidase with an apparent molecular weight of 86,000 (as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis). Mouse antiserum raised against the purified material reacted with a single species with molecular weight 107,000 (107K form) in crude pneumococcal cell lysate. During the purification procedure this species was progressively degraded to the molecular weight 86,000 (86K) form whilst retaining enzyme activity. Degradation of neuraminidase was inhibited by phenylmethylsulphonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA). Purification of the enzyme in the presence of these protease inhibitors permitted the isolation of the 107K species substantially undegraded and greater than 98% pure. Our findings on the degradation of neuraminidase during its purification account for previous reports of multiple neuraminidase isoenzymes in Streptococcus pneumoniae.

Antigens, Bacterial↗

PubMed1987

Efficacy of enzyme-linked immunosorbent assay for rapid diagnosis of Bordetella pertussis infection.

We examined the diagnostic efficacy of an enzyme-linked immunosorbent assay (ELISA) for class-specific antibodies to Bordetella pertussis in acute-phase sera collected from 1,240 patients with suspected pertussis. A total of 833 serum specimens (67%) yielded positive results. The proportion of positive results increased to 77% if a second (convalescent-phase) serum was also tested. By comparison, a bacterial agglutination test for B. pertussis antibodies was positive in only 21% of acute-phase specimens and 50% of paired specimens. The high proportion of acute-phase sera which were ELISA positive indicates that a measurable serologic response has usually occurred by the time the diagnosis is suspected. Thus, the ELISA is potentially the most rapid means of laboratory confirmation of B. pertussis infection.

Adolescent↗

PubMed1986

Antibody response to pneumococcal vaccine in children aged 5 to 15 years.

The type-specific antibody response of 99 healthy children, aged 5 to 15 years, to a 14-valent pneumococcal vaccine was determined and analyzed according to age at the time of vaccination. The response to the majority of the serotypes, including the important "pediatric" types 6A, 18C, and 19F, appeared to plateau in the 8- to 10-year-old age group, whereas for types 14 and 23F, which are also important in children, the response continued to improve with age to ages 13 to 15 years. The antibody response of a group of 23 children, aged 5 to 15 years, with various hematologic disorders (12 of whom had undergone splenectomy) was also determined and compared with that of the healthy children. For all serotypes the antibody response was similar in the two groups.

Adolescent↗

PubMed1986

Pneumococcal carriage and type-specific antibody. Failure of a 14-valent vaccine to reduce carriage in healthy children.

No consistent effect on nasal carriage rates of Streptococcus pneumoniae belonging to vaccine types was observed during a randomized, controlled trial of a 14-valent pneumococcal vaccine, which included 1273 Australian children aged 6 to 54 months. Nasal carriage of S pneumoniae was associated with a significantly elevated homotypic serum antibody concentration for types 18C, 19F, and 23F, but not for types 6A and 14 (these five types are the most important causes of pneumococcal infections in children). Upper respiratory tract carriage seems to play an important role in natural acquisition of antibody to some but not all pneumococcal serotypes. These findings help to explain why pneumococcal vaccine fails to protect young children from acute otitis media.

Antibodies, Bacterial↗

PubMed1986

Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin.

A gene bank of Sau3A1-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. Clones expressing the pneumolysin determinant were selected by testing for hemolytic activity which could be inhibited by antibody to purified pneumolysin and by cholesterol. Restriction analysis of pneumolysin-positive recombinant cosmid DNA indicated that the coding sequence for the toxin was located within a 2.9-kilobase-pair (kbp) ClaI DNA fragment. This fragment, which included 0.35 kbp of vector pHC79 DNA, was subcloned into the plasmid pBR322. E. coli cells harboring this recombinant plasmid (designated pJCP20) produced approximately one-third of the amount of pneumolysin found in the donor S. pneumoniae strain. Plasmid pJCP20 was stably maintained in E. coli and resulted in the accumulation of active pneumolysin in the cytoplasm. Western blot analysis showed that E. coli harboring pJCP20 produced two forms of the toxin with molecular weights of 54,000 and 52,000. The lower-molecular-weight form was indistinguishable from native pneumolysin. Subcloning the 2.9-kbp DNA fragment into the expression vector pEV31 allowed the determination of the direction of transcription of the pneumolysin gene. The pneumolysin-coding sequence (approximately 1.5 kbp) has been localized to within a 1.75-kbp segment of pneumococcal DNA.

Bacterial Proteins↗

PubMed1986

Inhibition of human monocyte respiratory burst, degranulation, phospholipid methylation and bactericidal activity by pneumolysin.

The interaction between the pneumococcal toxin pneumolysin and human monocytes was examined. At non-cytotoxic concentrations (0.5-2.5 HU/10(6) cells) pneumolysin depressed the oxygen-dependent respiratory burst in monocytes, induced by opsonized zymosan or phorbol myristate acetate (PMA). This included depressed hexose-monophosphate shunt activity and hydrogen peroxide production. The toxin also depressed the ability of monocytes to degranulate (measured by release of lysozyme) in response to the above stimuli. Phospholipid transmethylation was also markedly decreased by pretreating monocytes with pneumolysin. These effects on monocyte functions were accompanied by a decreased ability of pneumolysin-treated monocytes to kill Streptococcus pneumoniae, the organism that produces the toxin. Cholesterol, which inhibits the haemolytic activity of the toxin, was shown to abrogate the effects of pneumolysin on monocytes.

Bacterial Proteins↗

PubMed1984

Activation of human complement by the pneumococcal toxin pneumolysin.

Highly purified pneumolysin (at a concentration of 10 micrograms/ml) caused significant activation of human complement, as measured by conversion of C3. Complement activation in the presence of pneumolysin was not observed in sera chelated with a combination of Mg2+ and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid, and activation was only slight in C2-deficient sera. This suggests that the toxin is capable of activating the classical complement pathway. Treatment of normal human serum with pneumolysin also significantly reduced its opsonic activity for Streptococcus pneumoniae.

Bacterial Proteins↗

PubMed1984

Inhibition of in vitro human lymphocyte response by the pneumococcal toxin pneumolysin.

The effects of pneumolysin, a sulfhydryl-activated cytolytic toxin produced by Streptococcus pneumoniae, on the in vitro human lymphocyte response was examined. The toxin, at concentrations of one to five hemolytic units per ml, caused marked inhibition of the response of lymphocytes to concanavalin A, phytohemagglutinin, pokeweed mitogen, and protein A. The response was assessed by measuring both [3H]thymidine incorporation and the ability of lymphocytes to produce immunoglobulins and lymphokine activity. The effects of pneumolysin were irreversible, could be prevented by pretreatment of the toxin with cholesterol, and were not related to a direct cytotoxic effect on the lymphocytes. Pneumolysin appeared to act at the initiation phase of the immune response and had no effect on lymphocytes committed to DNA synthesis or to the synthesis and secretion of immunoglobulins. Furthermore, pneumolysin-mediated inhibition of the lymphocyte response was not due to the inhibition of binding of mitogens to leukocytes and is likely to be related to effects on membrane-mediated signals essential for lymphocyte triggering. This may be one means by which pneumolysin plays a role in the pathogenesis of pneumococcal infections.

Antibody Formation↗

PubMed1983

Antibody response to pneumococcal vaccination in children younger than five years of age.

Sera were analyzed by radioimmunoassay for type-specific pneumococcal antibody in 249 children aged six to 54 months, who were participating in a controlled trial of a 14-valent pneumococcal vaccine. Levels of serum antibody to all serotypes increased after immunization in all age groups tested. For all serotypes, the antibody response increased progressively with age whether response was viewed as antibody doubling, relative increase in geometric mean, or final antibody level. Responses were poor up to the age of five years for the important pediatric serotypes 6A, 14, 19F, and 23F. Seventeen children under the age of two years at the time of primary immunization received booster doses of vaccine six months later. There was no significant increase in antibody to any serotype, and the geometric mean antibody levels fell for most types. Immune response to the pediatric serotypes was poor until the age of 4.5 years.

Aging↗

PubMed1983

Effect of immunization with pneumolysin on survival time of mice challenged with Streptococcus pneumoniae.

The role of the cytolytic toxin pneumolysin in the pathogenicity of Streptococcus pneumoniae was investigated. Pneumolysin was purified to homogeneity and used to immunize mice. When these mice were subsequently challenged via the nasal route with virulent S. pneumoniae, they survived significantly longer than control mice. The mean survival times were 5.52 and 2.48 days for immunized and control mice, respectively. This work provides direct evidence for the involvement of pneumolysin in pneumococcal pathogenicity.

Animals↗

PubMed1983

Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin.

The in vitro effects of pneumolysin, a sulfhydryl-activated toxin produced by Streptococcus pneumoniae, on various functions of human polymorphonuclear leukocytes (PMNLs) was investigated. Treatment of PMNLs with highly purified toxin significantly inhibited respiratory burst (in response to stimulation), ability to kill opsonized pneumococci, chemotaxis, and random migration. These inhibitions were observed at very low toxin doses (less than or equal to 1 hemolytic unit (2 ng) per 10(6) PMNLs), which had no effect on PMNL viability. These results suggest that pneumolysin could function in pathogenicity by interfering with the ability of PMNLs to migrate toward and kill pneumococci.

Bacterial Proteins↗

PubMed1983

Quantities of Clostridium botulinum organisms and toxin in feces and presence of Clostridium botulinum toxin in the serum of an infant with botulism.

A 7-week-old boy presented with symptoms and signs characteristic of infant botulism, and the diagnosis was confirmed by the detection of Clostridium botulinum type A organisms and toxin in the feces. The levels of organisms and toxin in the feces were measured throughout the 81-day period in hospital. The maximum levels detected were 2.46 x 10(8) C. botulinum type A colony-forming units and 64,000 mouse 100% lethal doses of type A toxin per g (wet weight) of feces. C. botulinum toxin was also detected in two samples of the patient's serum, collected 3 and 10 days after admission. Improvement in the patient's clinical condition occurred before the levels of organisms and toxin in the feces reached their maxima. A slight improvement may also have occurred while toxin was still present in the serum.

Botulinum Toxins↗

PubMed1982

Quantitation of Clostridium botulinum organisms and toxin in the feces of an infant with botulism.

A 4-month-old boy presented with symptoms and signs characteristic of infant botulism. Examination of feces revealed Clostridium botulinum type B and type B toxin. The numbers of C. botulinum and the amount of toxin in feces were measured throughout the 4-week period in hospital. The maximum numbers and amounts were detected in a fecal specimen collected 16 days after admission: this contained 8.4 X 10(6) C. botulinum type B colony-forming units and 61,440 mouse 100% lethal doses of type B toxin per g (wet weight) of feces. This latter figure is the highest fecal toxin titer reported yet for a case of infant botulism. By day 16, however, substantial improvement in the patient's clinical condition had occurred. This suggests that initiation of recovery from infant botulism is not necessarily preceded by a reduction in the numbers of C. botulinum organisms and the quantity of toxin in the gut.

Action Potentials↗

PubMed1980

Infant botulism.

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Botulism↗

PubMed1978

Membrane phospholipid asymmetry in Bacillus amyloliquefaciens.

The phospholipid distribution in the membrane of Bacillus amyloliquefaciens was studied by using phospholipase C (B. cereus), phospholipase A2 (Crotalus), and the nonpenetrating chemical probe trinitrobenzenesulfonic acid. After treatment of intact protoplasts of B. amyloliquefaciens with either phospholipase, about 70% of total membrane phospholipid was hydrolyzed; specifically, about 90, 90, and 30% of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold-shock treatment were incubated with either of the phospholipases, up to 80% of cardiolipin was hydrolyzed and phosphatidylglycerol and phosphatidylethanolamine were hydrolyzed virtually to completion. In intact cells, 92% of the phosphatidylethanolamine could be labeled with trinitrobenzenesulfonic acid under conditions in which the reagent did not penetrate the membrane to any significant extent. These results indicate that 70% of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation of this is required.

Bacillus↗