The role of streptococcal toxins in disease.
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Animals↗
Biomedical subjects
T J Mitchell
Publications and source records attributed to T J Mitchell.
Molecular basis of virulence.
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Bacteria↗
Structural and functional characterisation of two proteolytic fragments of the bacterial protein toxin, pneumolysin.
Proteolytic cleavage of the bacterial protein toxin pneumolysin with protease K creates two fragments of 37 and 15 kDa. This paper describes the purification of these two fragments and their subsequent physical and biological characterisation. The larger fragment is directly involved in the cytolytic mechanism of this pore-forming protein, via membrane binding and self-association. The smaller fragment lacks ordered structure or discernible activity.
Bacterial Proteins↗
The chemistry of pseudomonic acid. 18. Heterocyclic replacement of the alpha,beta-unsaturated ester: synthesis, molecular modeling, and antibacterial activity.
The electronic requirements around the C1-C3 region of pseudomonic acid analogues were investigated. Synthetic routes were developed to access a range of compounds where the alpha, beta-unsaturated ester moiety had been replaced by a 5-membered ring heterocycle. The inhibition of isoleucyl tRNA synthetase from Staphylococcus aureus NCTC 6571 was determined as was the minimum inhibitory concentration (MIC) of the test compounds against that organism. Compounds possessing a region of electrostatic potential corresponding to that of the carbonyl group in the alpha, beta-unsaturated ester, and a low-energy unoccupied molecular orbital in the region corresponding to the double bond, were found to have IC50 values of 0.7-5.3 ng mL-1. However the MIC values of these compounds were in the range 2.0-8.0 micrograms mL-1, reflecting their poorer penetration into the bacterial cell.
Anti-Bacterial Agents↗
Streptococcus pneumoniae produces a second haemolysin that is distinct from pneumolysin.
Pneumococci are described as alpha-haemolytic but under certain circumstances they produce zones of beta-haemolysis on blood-containing medium. This observation was investigated using wild type strains and a genetically-modified strain unable to produce the haemolytic toxin, pneumolysin. beta-haemolysis was produced by all pneumococci tested. It was not inhibited by anti-pneumolysin antibody but could be inactivated by cholesterol. These data confirm that pneumococci elaborate a second haemolysin, distinct from pneumolysin.
Antibodies, Bacterial↗
Relationship of structure to function in pneumolysin.
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Amino Acid Sequence↗
Biological properties of pneumolysin.
Pneumolysin is a thiol-activated membrane-damaging toxin produced by Streptococcus pneumoniae. The toxin plays a role in virulence of the pneumococcus in animal models of infection. Pneumolysin has a range of biological activity including the ability to lyse eukaryotic cells and to interfere with the function of cells and soluble molecules of the immune system. The use of purified native and mutant toxin and of isogenic mutants of the pneumococcus expressing altered versions of the toxin has allowed the contribution of the various activities of this multifunctional toxin to virulence to be defined.
Animals↗
Structural and functional analogy between pneumolysin and proaerolysin.
Pneumolysin and proaerolysin are bacterial toxins that form pores in host cells by oligomerization. We propose that they may have similar structures despite a poor sequence identity. The crystal structure of proaerolysin reveals a protein composed of four domains, arranged in the shape of an elongated comma. Electron microscopy of the pneumolysin monomer shows a similar arrangement of domains. The sequence of pneumolysin recognizes the template of proaerolysin from a library of protein folds. A three-dimensional model of pneumolysin has been constructed by the comparative approach using the structure of proaerolysin. This model, together with results on the activity of site-specific mutants and the positions of antigenic sites, has been used to propose functional roles of individual domains.
Amino Acid Sequence↗
A role for pneumolysin but not neuraminidase in the hearing loss and cochlear damage induced by experimental pneumococcal meningitis in guinea pigs.
We investigated the roles of pneumolysin and neuraminidase in the pathogenesis of deafness and cochlear damage during experimental pneumococcal meningitis. Anesthetized guinea pigs were inoculated intracranially with 7.5 log10 CFU of either (i) wild-type Streptococcus pneumoniae D39 (n = 8), (ii) PLN-A, a defined isogenic derivative of D39 deficient in pneumolysin (n = 5), or (iii) deltaNA1, a new derivative of D39 deficient in neuraminidase constructed by insertion-duplication mutagenesis of the nanA gene (n = 5). To quantify hearing loss, the auditory nerve compound action potential evoked by a tone pulse was recorded from the round window membrane of the cochlea every 3 h for 12 h. The organ of Corti was intravitally fixed for subsequent examination by high-resolution scanning and transmission electron microscopy. All animals sustained similar meningeal inflammatory responses. PLN-A induced significantly less hearing loss than D39 over the frequency range of 3 to 10 kHz. Levels of mean hearing loss at 10 kHz 12 h postinoculation were as follows: D39, 50 dB; deltaNA1, 52 dB (P = 0.76 versus D39), and PLN-A, 12 dB (P < 0.0001 versus D39). The mean rates of hearing loss at 10 kHz were 4.4 dB/h for D39, 4.3 dB/h for deltaNA1, and just 1.0 dB/h for PLN-A (P < 0.0001 versus D39). Suppurative labyrinthitis was universal. PLN-A induced the accumulation of less protein in the cerebrospinal fluid (P = 0.04 versus D39). Infection with D39 and deltaNA1 induced significant damage to the reticular lamina, the sensory hair cells, and supporting cells of the organ of Corti. By contrast, after infection with PLN-A, the organ of Corti appeared virtually intact. Pneumolysin seems to be the principal cause of cochlear damage in this model of meningogenic deafness. No clear pathogenic role was demonstrated for neuraminidase.
Animals↗
A method for estimating occupational radiation dose to individuals, using weekly dosimetry data.
Statistical analyses of data from epidemiological studies of workers exposed to radiation have been based on recorded annual radiation doses. It is usually assumed that the annual doses are known exactly, although it is generally recognized that the data contain uncertainty due to measurement error and bias. We propose the use of a probability distribution to describe an individual's dose during a specific period and develop statistical methods for estimating this distribution. The methods take into account the "measurement error" that is produced by the dosimetry system and the bias that was introduced by policies of recording doses below a threshold as zero. The method is applied to a sample of dose histories over the period 1945 to 1955 obtained from hard-copy dosimetry records at Oak Ridge National Laboratory (ORNL). The result of this evaluation raises serious questions about the validity of the historical personnel dosimetry data that are currently being used in studies of the effects of low doses in nuclear industry workers. In particular, it appears that there was a systematic underestimation of doses for ORNL workers. This may result in biased estimates of dose-response coefficients and their standard errors.
Bayes Theorem↗
Molecular analysis of virulence factors of Streptococcus pneumoniae.
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Bacterial Vaccines↗
Synthesis and protein kinase C inhibitory activities of acyclic balanol analogs that are highly selective for protein kinase C over protein kinase A.
A series of balanol analogs in which the perhydroazepine ring and the p-hydroxybenzamide moiety were combined into an acyclic linked unit have been prepared and evaluated for their inhibitory properties against the serine/threonine kinase PKC. Several low-micromolar to low-nanomolar inhibitors of the alpha, beta I, beta II, gamma, delta, epsilon and eta PKC isozymes were prepared. In general, these acyclic balanol analogs were found to be highly selective for PKC over the serine/threonine kinase PKA. The type and number of atoms linking the benzophenone ester to the p-hydroxyphenyl group necessary for optimal PKC inhibition were investigated. The most potent compounds contained a three-carbon linker in which the carboxamide moiety of balanol had been replaced by a methylene group. The effect of placing substituents on the three-carbon chain was also investigated. The preferred compounds contained either a 2-benzenesulfonamido (6b) or a 1-methyl (21b) substituent. The preferred compounds 6b and 21b were tested against a panel of serine/threonine kinases and found to be highly selective for PKC. The more active enantiomer of 6b, (S)-12b, was 3-10-fold more active than the R-enantiomer against the PKC isozymes. The effect of making the analogs more rigid by making the three-carbon chain part of a five-membered ring, but with retention of the methylene replacement for the carboxamide moiety, led to potent PKC inhibitors including anti-substituted pyrrolidine analog 35b and the most potent PKC inhibitor in the series, anti-substituted cyclopentane analog 29b. The anti cyclopentane analog 29b, was a low-micromolar inhibitor of the PMA-induced superoxide burst in neutrophils, and its carboxylic ester was a high-nanomolar inhibitor of neutrophils. Finally esterification of 21b, (S)-12b, and 35b turned these potent PKC inhibitors into low-micromolar inhibitors of neutrophils.
Animals↗
IFN-gamma up-regulates expression of the complement components C3 and C4 by stabilization of mRNA.
We investigated the mechanisms underlying the regulation of complement genes C3 and C4 by IFN-gamma. IFN-gamma (500 U/ml, 24 h incubation) increased steady state mRNA levels for both C3 and C4 in three different cell types (Hep G2, U937, and primary fibroblasts). The response to IFN-gamma in Hep G2 cells was time and dose dependent. At all doses of IFN-gamma and at all incubation times, the transcription rate for these two genes, determined by nuclear run-on assays, was reduced (0.3 +/- 0.1; unstimulated rate = 1.00). The t1/2 of mRNA for C3 and C4 in unstimulated cells was 1.8 +/- 0.3 and 2.2 +/- 0.2 h, respectively. After high-dose IFN-gamma stimulation, both C3 and C4 mRNA levels remained at 100% with respect to baseline at 5 h, but after 12 h, levels fell to 13 +/- 2% (C3) and 8 +/- 3% (C4) of baseline values, giving a half-life for these mRNA species of between 5 and 12 h. IFN-gamma stimulation increased C3 and C4 protein synthesis measured at 24 h. We suggest that it is the increase in mRNA stability that is the major effector mechanism by which IFN-gamma regulates C3 and C4 gene expression.
Cell Line↗
Functional analysis of pneumolysin by use of monoclonal antibodies.
We have produced a panel of monoclonal antibodies to pneumolysin, the membrane-damaging toxin from Streptococcus pneumoniae. We have used these antibodies to identify three regions of the toxin sequence that are involved in the lytic mechanism of this toxin. Two of these sites probably form the cell binding site of this toxin. Antibodies to the third site inhibit the lytic action of this toxin but not the binding of this toxin to cells. This site is engaged in the oligomerization process involved in the formation of pores in cell membranes. Two of these epitopes are also present in the related toxin perfringolysin O.
Animals↗
Distinct roles for pneumolysin's cytotoxic and complement activities in the pathogenesis of pneumococcal pneumonia.
Pneumolysin, the major Streptococcus pneumoniae cytotoxin, contributes to the early pathogenesis of invasive pneumococcal pneumonia by facilitating intrapulmonary bacterial growth and invasion into the blood. Pneumolysin is a multifunctional toxin, with distinct cytolytic ("hemolytic") and complement-activation ("complement") activities that have been mapped to several regions of the molecule. To characterize the specific contributions of pneumolysin's hemolytic and complement properties to the pathogenesis of pneumococcal pneumonia, we compared the in vivo effects of type 2 S. pneumoniae mutant strains, which produce pneumolysins deficient in these activities. The absence of either pneumolysin's hemolytic or complement activities rendered mutant strains less virulent than the wild-type strain during pulmonary infection. Pneumolysin's hemolytic activity correlated with acute lung injury and bacterial growth at 3 and 6 h after endotracheal instillation. In contrast, pneumolysin's complement activity correlated with bacterial growth and bacteremia at 24 h after pulmonary infection. Pneumolysin's complement activity was not associated with the degree of alveolar-capillary injury or recruitment of leukocytes during initial pulmonary infection. However, pneumolysin's complement activity inhibited killing of mutant bacteria in an in vitro complement-dependent neutrophil killing assay. Thus, both pneumolysin's hemolytic and complement activities made specific contributions to the early pathogenesis of pneumococcal pneumonia at different stages of infection and by different mechanisms.
Animals↗
Subunit organisation and symmetry of pore-forming, oligomeric pneumolysin.
We present a detailed analysis of the oligomeric subunit organisation of pneumolysin by the use of negative stain electron microscopy and image processing to produce a projection density map. Analysis of the rotational symmetry has revealed a large and variable subunit number, between 40-50. The projected subunit density by rotational averaging shows at least two distinct subunit domains at different radial positions. Side views of the rings reveal further details concerning the dimensions of the oligomer in the membrane. On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4-domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer.
Bacterial Proteins↗
The role of pneumolysin and autolysin in the pathology of pneumonia and septicemia in mice infected with a type 2 pneumococcus.
Mice were infected intranasally with a serotype 2 pneumococcus, a pneumolysin-negative derivative (PLN-A), or an autolysin-negative derivative (AL-2). Numbers of wild type pneumococci were seen in the lung from approximately 12 h after infection and were first detected in the blood around this time. Immunofluorescent staining of lung sections showed that pneumolysin was produced in vivo. Pneumococcal infection resulted in alteration of the composition of the blood but not the bone marrow. Some of the hematologic changes did not occur after PLN-A. PLN-A had a slower growth rate in the lung and bacteremia was delayed. AL-2 was rapidly cleared from the lungs and was not detected in the blood. These events paralleled the pattern of histology in the lung, with the severity of inflammation reduced with PLN-A and no inflammation or hematologic changes with AL-2.
Analysis of Variance↗
Interaction of pneumolysin-sufficient and -deficient isogenic variants of Streptococcus pneumoniae with human respiratory mucosa.
Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, and pneumolysin, a hemolytic toxin, is thought to be an important virulence factor. We have studied the interaction of a pneumolysin-sufficient type II S. pneumoniae strain (PL+) and an otherwise identical pneumolysin-deficient derivative (PL-) with human respiratory mucosa in an organ culture with an air interface for up to 48 h. Ciliary beat frequency (CBF) was measured by a photometric technique, and adherence to and invasion of the epithelium were assessed by scanning and transmission electron microscopy. PL+ and PL- caused a progressive fall in CBF compared with the control which became significant (P < 0.01) at 24 h for PL+ and at 48 h for PL-. At 24 h, there was a significant increase in the percentage of the mucosa of the organ culture that was damaged for PL+ compared with the control (P < 0.01) and PL- (P < 0.02). At 48 h, there was a significant increase in mucosal damage for both PL+ (P < 0.005) and PL- (P < 0.05) compared with the control. At 24 and 48 h, PL+ and PL- adhered predominantly to mucus and damaged cells. PL+ infection alone caused separation of tight junctions between epithelial cells, and at 48 h PL+ cells were adherent to the separated edges of otherwise healthy unciliated cells. PL+ and PL- both caused damage to the epithelial cell ultrastructure. S. pneumoniae infection caused patchy damage to the respiratory mucosa and a lowered CBF. These changes were more severe and occurred earlier with the pneumolysin-sufficient variant.
Adenoids↗