Biomedical subjects

P W Andrew

Publications and source records attributed to P W Andrew.

At least 19 recordsLinked to original sources

PubMed2005

The role of surfactant protein D in the colonisation of the respiratory tract and onset of bacteraemia during pneumococcal pneumonia.

We have shown previously that surfactant protein D (SP-D) binds and agglutinates Streptococcus pneumoniae in vitro. In this study, the role of SP-D in innate immunity against S. pneumoniae was investigated in vivo, by comparing the outcome of intranasal infection in surfactant protein D deficient (SP-D-/-) to wildtype mice (SP-D+/+). Deficiency of SP-D was associated with enhanced colonisation and infection of the upper and lower respiratory tract and earlier onset and longer persistence of bacteraemia. Recruitment of neutrophils to inflammatory sites in the lung was similar in both strains mice in the first 24 hrs post-infection, but different by 48 hrs. T cell influx was greatly enhanced in SP-D-/- mice as compared to SP-D+/+ mice. Our data provides evidence that SP-D has a significant role to play in the clearance of pneumococci during the early stages of infection in both pulmonary sites and blood.

Animals↗

PubMed2004

The role of pneumolysin in pneumococcal pneumonia and meningitis.

Diseases caused by Streptococcus pneumoniae include pneumonia, septicaemia and meningitis. All these are associated with high morbidity and mortality. The pneumococcus can colonize the nasopharynx, and this can be a prelude to bronchopneumonia and invasion of the vasculature space. Proliferation in the blood can result in a breach of the blood-brain barrier and entry into the cerebrospinal fluid (CSF) where the bacteria cause inflammation of the meningeal membranes resulting in meningitis. The infected host may develop septicaemia and/or meningitis secondary to bronchopneumonia. Also septicaemia is a common precursor of meningitis. The mechanisms surrounding the sequence of infection are unknown, but will be dependent on the properties of both the host and bacterium. Treatment of these diseases with antibiotics leads to clearance of the bacteria from the infected tissues, but the bacteriolytic nature of antibiotics leads to an acute release of bacterial toxins and thus after antibiotic therapy the patients can be left with organ-specific deficits. One of the main toxins released from pneumococci is the membrane pore forming toxin pneumolysin. Here we review the extensive studies on the role of pneumolysin in the pathogenesis of pneumococcal diseases.

Bacteremia↗

PubMed2003

Stress response of Listeria monocytogenes isolated from cheese and other foods.

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.

Adaptation, Physiological↗

PubMed2001

Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho-epithelial cells in vivo.

The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.

Animals↗

PubMed2001

Listeria monocytogenes adheres to many materials found in food-processing environments.

AIMS: To investigate the adhesion of Listeria monocytogenes 10403S to 17 different, food-use approved materials representing metals, rubbers and polymers. METHODS AND RESULTS: Adhesion assays were conducted by placing 'coupons' of the materials in planktonic cultures at 30 degrees C, and then immediately withdrawing them ('short contact') or leaving them submerged in the cultures for 2 h. Adherent cells were recovered by sonication. In the short contact experiments, the logarithm of the mean viable counts ranged from 3.67 +/- 0.43 to 4.78 +/- 0.38. After 2 h contact time, the numbers of adherent cells had increased significantly for all materials with the exception of polypropylene. The highest count (6.33 +/- 0.31) recorded was for stainless steel 405. CONCLUSION: Adhesion to a wide range of materials was time-dependent and characterized by reversible and irreversible stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhesion test protocols must account for cell carry-over and cells which are only weakly bound. Material selection may only have a limited role in reducing food contamination by listeria.

Bacterial Adhesion↗

PubMed2001

Role of genetic resistance in invasive pneumococcal infection: identification and study of susceptibility and resistance in inbred mouse strains.

From a panel of nine inbred mice strains intranasally infected with Streptococcus pneumoniae type 2 strain, BALB/c mice were resistant and CBA/Ca and SJL mice were susceptible to infection. Further investigation revealed that BALB/c mice were able to prevent proliferation of pneumococci in the lungs and blood, whereas CBA/Ca mice showed no bacterial clearance. Rapidly increasing numbers of bacteria in the blood was a feature of CBA/Ca but not BALB/c mice. In the lungs, BALB/c mice recruited significantly more neutrophils than CBA/Ca mice at 12 and 24 h postinfection. Inflammatory lesions in BALB/c mice were visible much earlier than in CBA/Ca mice, and there was a greater cellular infiltration into the lung tissue of BALB/c mice at the earlier time points. Our data suggest that resistance or susceptibility to intranasal pneumococci may have an association with recruitment and/or function of neutrophils.

Animals↗

PubMed2000

Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin.

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.

Bacterial Proteins↗

PubMed2000

Structural basis of pore formation by cholesterol-binding toxins.

In this paper we describe reconstructions by electron cryo-microscopy of two oligomeric states of the pore-forming toxin pneumolysin. The results are interpreted by the fitting of atomic models of separated domains to the 3-dimensional electron density maps, revealing two steps in the mechanism of pore formation by the family of cholesterol-binding toxins. We briefly describe the observation of the toxin pore in model membranes and contrast the apparent mechanism of pneumolysin with that of other pore-forming toxins.

Bacterial Proteins↗

PubMed2000

Rapid methods for testing the efficacy of sterilization-grade filter membranes.

The validation of sterilization-grade membranes is integral to ensuring the efficient and safe use of microfiltration systems. Here validation refers to the production of sterile filtrate for sterilizing-grade membranes under challenge test conditions. Current validation methods require 48 h of culture for results to become available, which creates time delays within the manufacturing process and quality control (QC) backlogs. This work compares four methods for the production of filter challenge test data, to the desired test sensitivity, within 24 h using bioluminescent and fluorescent recombinant strains of the test organism Brevundimonas diminuta. These methods should provide a way to implement more rapid QC test regimens for filters.

Alphaproteobacteria↗

PubMed2000

Host cellular immune response to pneumococcal lung infection in mice.

Although there is substantial evidence that pneumolysin is an important virulence factor in pneumococcal pneumonia, relatively little is known about how it influences cellular infiltration into the lungs. We investigated how the inability of mutant pneumococci to produce pneumolysin altered the pattern of inflammation and cellular infiltration into the lungs. The effect on bacterial growth in the lungs also was assessed. There were three phases of growth of wild-type bacteria in the lungs: a decline followed by a rapid increase and then stasis or decline. The absence of pneumolysin was associated with a more rapid early decline and then a much slower increase in numbers. The pattern of inflammatory-cell accumulation also had distinct stages, and the timing of these stages was influenced by the presence of pneumolysin. Neutrophils began to accumulate about 12 to 16 h after infection with wild-type pneumococci. This accumulation occurred after the early decline in pneumococcal numbers but coincided with the period of rapid growth. Following infection with pneumococci unable to make pneumolysin, neutrophil influx was slower and less intense. Coincident with the third stage of pneumococcal growth was an accumulation of T and B lymphocytes at the sites of inflammation, but the accumulation was not associated with an increase in the total number of lymphocytes in the lungs. Lymphocyte accumulation in the absence of pneumolysin occurred but was delayed.

Animals↗

PubMed2000

Relative roles of pneumolysin and hydrogen peroxide from Streptococcus pneumoniae in inhibition of ependymal ciliary beat frequency.

Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H(2)O(2)) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H(2)O(2) at a concentration as low as 100 microM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H(2)O(2) (10 microM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H(2)O(2) to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H(2)O(2), and in the presence of 10(8) CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H(2)O(2) release from the brain slice. Coincubation with catalase converted the H(2)O(2) produced by the pneumococci to H(2)O. Penicillin-induced lysis of bacteria dramatically reduced H(2)O(2) production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H(2)O(2), which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H(2)O(2) released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H(2)O(2)-induced cell toxicity, indicating a role for H(2)O(2) in the response. There is also a slight additive effect of pneumolysin and H(2)O(2) on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.

Animals↗

PubMed2000

Role of manganese-containing superoxide dismutase in oxidative stress and virulence of Streptococcus pneumoniae.

Streptococcus pneumoniae was shown to contain two types of superoxide dismutase, MnSOD and FeSOD. Levels of MnSOD increased during growth in an aerobic environment. The sodA gene, encoding MnSOD, of virulent S. pneumoniae type 2 strain D39 was inactivated to give mutant D39HY1. Aerobically, D39HY1 had a lower growth rate than the wild type and exhibited susceptibility to the redox-active compound paraquat, but anaerobic growth of D39HY1 was identical to that of the wild type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than that of mice infected with the wild-type pneumococcus. In contrast to the wild type, D39HY1 did not multiply in lungs during the first 24 h but thereafter grew at the same rate as the wild type. Appearance in the bloodstream was also delayed, but growth in the blood was unimpaired by the sodA mutation. The pattern of inflammation in lungs infected with D39HY1 differed from that in wild-type-infected mice. After infection with D39HY1, neutrophils were densely packed around bronchioles, in contrast to the wild-type infection, where neutrophils were more diffusely localized.

Animals↗

PubMed2000

Effect of pneumolysin on rat brain ciliary function: comparison of brain slices with cultured ependymal cells.

This study compares two models for examining ependymal ciliary function: rat brain slices cut from the fourth ventricle and primary ependymal cells in culture. The cilia from both preparations were very reproducible; each preparation had cilia beating at a constant frequency of between 38 and 44 Hz. With the brain slices, ciliary stasis occurred after 5 d in culture. However, ependymal cells had fully functional cilia for up to 48 d in culture. The pneumococcal toxin, pneumolysin, caused a dose-dependent inhibition of cilia beat frequency within 15 min in both models. There were no significant differences in the mean log 50% inhibitory concentration (pIC50) slice = 0.65 +/- 0.05, equivalent to 4.4 hemolytic units (HU)/mL; cells = 0.57 +/- 0.14, equivalent to 3.7 HU/mL. There were also no significant differences in the mean Hill slope factors for the curves (slice = 1.4 +/- 0.05; cells = 1.6 +/- 0.4). These data demonstrate that both models can be used to examine the acute (15-min) effects of pneumolysin on cilia beat frequency. The main advantage of the primary ependymal culture model is that considerably more cultured ependymal cells (approximately 70%) are available, compared with the number of ependymal cells on the brain slices (approximately 2%), thus reducing the number of animals used. A pure ependymal culture was not achieved (approximately 30% of the cells were not ciliated). The increased survival time of the ependymal cells compared with the brain slices make cultured ependymal cells more useful for examining long-term ciliary function, whereas brain slices may be more useful for examining the interactions between ependymal and other nearby cells.

Animals↗

PubMed1999

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation. Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.

Amino Acid Sequence↗

PubMed1999

Two structural transitions in membrane pore formation by pneumolysin, the pore-forming toxin of Streptococcus pneumoniae.

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.

Bacterial Proteins↗

PubMed1999

The effect of the pneumococcal toxin, pneumolysin on brain ependymal cilia.

Densely ciliated ependymal cells cover the ventricular surface of the brain and cerebral aqueducts separating cerebrospinal fluid, which is infected in meningitis, from neuronal tissue. We have established an ex vivo model that allows measurement of ependymal ciliary beat frequency, using high-speed video analysis, during incubation with bacterial toxins. Ciliated ependyma, from Wistar rats, was exposed to the pneumococcal toxin, pneumolysin, and a mutant form with markedly reduced cytotoxic activity (;0.1%). Wild-type pneumolysin (1500 HU/ml and 150 HU/ml: 10 and 1 microg/ml) caused rapid ciliary stasis (30-150 s), sloughing of cilia and cytoplasmic extrusion. Ciliary slowing before stasis was seen at 15 HU/ml (0.1 microg/ml); however, no effect on ciliary beat frequency was seen at lower concentrations (1.5 HU/ml and 0.15 HU/ml: 0.01 and 0.001 microg/ml). Mutant pneumolysin, 99.9% deficient in haemolytic activity, caused rapid ciliary stasis at 10 microg/ml but no effect was seen at lower concentrations (1-0.1 microg/ml). Pneumolysin, at levels which may be produced during severe pneumococcal meningitis, may cause rapid ependymal ciliary stasis.

Animals↗

PubMed1998

Self-interaction of pneumolysin, the pore-forming protein toxin of Streptococcus pneumoniae.

The pathogenically important cholesterol-binding pore-forming bacterial "thiol-activated" toxins (TATs) are commonly believed to be monomeric in solution and to undergo a transition on membrane binding mediated by cholesterol to an oligomeric pore. We present evidence, gained through the application of a number of biochemical and biophysical techniques with associated modelling, that the TAT from Streptococcus pneumoniae, pneumolysin, is in fact able to self-associate in solution to form the same oligomeric structures. The weak interaction leading to solution oligomerization is manifested at low concentrations in a dimeric toxin form. The inhibition of toxin self-interaction by derivatization of the single cysteine residue in pneumolysin with the thiol-active agent dithio (bis)nitrobenzoic acid indicates that self-interaction is mediated by the fourth domain of the protein, which has a fold similar to other proteins known to self-associate. This interaction is thought to have implications for the understanding of mechanisms of pore formation and complement activation by pneumolysin.

Bacterial Proteins↗

PubMed1998

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae.

Pneumolysin, a member of the thiol-activated cytolysin family of toxins, is a virulence factor from the Gram-positive bacterium Streptococcus pneumoniae. The toxin forms large oligomeric pores in cholesterol-containing membranes of eukaryotic cells. A plethora of biochemical and mutagenesis data have been published on pneumolysin, since its initial characterization in the 1930s. Here we present an homology model of the monomeric and oligomeric forms of pneumolysin based on the recently determined crystal structure of perfringolysin O and electron microscopy data. A feature of the model is a striking electronegative surface on parts of pneumolysin that may reflect its cytosolic location in the bacterial cell. The models provide a molecular basis for understanding the effects of published mutagenesis and biochemical modifications on the toxic activity of pneumolysin. In addition, spectroscopic data are presented that shed new light on pneumolysin activity and have guided us to hypothesise a detailed model of membrane insertion. These data show that the environment of some tryptophan residues changes on insertion and/or pore formation. In particular, spectroscopic analysis of a tryptophan mutant, W433F, suggests it is the residue mainly responsible for the observed effects. Furthermore, there is no change in the secondary structure content when the toxin inserts into membranes. Finally, the basis of the very low activity shown by a pneumolysin molecule from another strain of S. pneumoniae may be due to the movements of a key domain-domain interface. The molecular basis of pneumolysin-induced complement activation may be related to the structural similarity of one of the domains of pneumolysin to Fc, rather than the presumed homology of the toxin to C-reactive protein as previously suggested.

Amino Acid Sequence↗