Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties

Mitchell, TJ; Walker, John; Saunders, F. K.; Andrew, Peter W.; Boulnois, Graham J.

doi:10.1016/0167-4781(89)90131-0

Cited by 64 publications

(63 citation statements)

References 12 publications

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“…Wildtype pneumolysin protein was expressed in Escherichia coli JMI01 and purified as described previously [12]. Sample purity was checked by SDS PAGE analysis and haemolytic activity assayed as described previously [12].…”

Section: Pneumolysin Expression and Purificationmentioning

confidence: 99%

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Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

et al. 1995

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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.Key words." Pneumolysin; Pore-forming toxin; Electron microscopy; Image processing copy. This model consists of a single ring of subunits which contains ~30 subunits. The incorporation of pneumolysin oligomers into liposomes rather than sheep erythrocytes and the use of image processing have enabled a more refined analysis of the pneumolysin oligomeric structures. Here we present (i) a symmetry-averaged projected density map of a pneumolysin oligomer and (ii) side views of the oligomers. On the basis of these images we propose a compact 4-domain subunit model for the oligomer. Materials and methods Pneumolysin expression and purificationWildtype pneumolysin protein was expressed in Escherichia coli JMI01 and purified as described previously [12]. Sample purity was checked by SDS PAGE analysis and haemolytic activity assayed as described previously [12].

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Section: Pneumolysin Expression and Purificationmentioning

confidence: 99%

“…Sample purity was checked by SDS PAGE analysis and haemolytic activity assayed as described previously [12].…”

Section: Pneumolysin Expression and Purificationmentioning

confidence: 99%

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

et al. 1995

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“…Pneumolysin was detected using standard Western blotting techniques. Briefly, samples were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, which were transferred to Hybond-C nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, United Kingdom) and blotted for 90 min at 80 V. Blots were incubated in 3% skim milk with polyclonal rabbit antipneumolysin serum (54), washed four times in TrisNaCl (pH 7.4), and then incubated with anti-rabbit horseradish peroxidase (HRP)-linked antibody (Amersham Biosciences) in 3% skim milk, washed four times, and developed with 30 mg 4-chloro-naphthol (Sigma, Haverhill, United Kingdom) dissolved in 10 ml methanol plus 30 l H 2 O 2 (Sigma) in 40 ml Tris-NaCl, pH 7.4. The reaction was stopped with distilled water.…”

Section: Methodsmentioning

confidence: 99%

“…Pneumococcal cell extracts were diluted to 1:500 for the 300 g/ml total protein samples (or 1:2,500 for the 2.35-mg/ml total protein samples) and added in duplicate at 100 l/well. Purified pneumolysin, prepared as described previously (54), was used to provide a standard curve with a range from 2,000 pg/ml to 31.25 pg/ml. One hundred microliters/well rabbit polyclonal antipneumolysin antibody (54) was then added at a 1:2,000 dilution in assay buffer, and plates were shaken for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic Pneumolysin

et al. 2006

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Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing "wild-type" pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.

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“…Pneumococcal DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions. PLY was constructed and purified as previously described (42).…”

Section: Reagentsmentioning

confidence: 99%

Streptococcus pneumoniaeStimulates a STING- and IFN Regulatory Factor 3-Dependent Type I IFN Production in Macrophages, which Regulates RANTES Production in Macrophages, Cocultured Alveolar Epithelial Cells, and Mouse Lungs

Koppe

Högner

Doehn

et al. 2012

The Journal of Immunology

104

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Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. In this study, we examine an innate immune recognition pathway that senses pneumococcal infection, triggers type I IFN production, and regulates RANTES production. We found that human and murine alveolar macrophages as well as murine bone marrow macrophages, but not alveolar epithelial cells, produced type I IFNs upon infection with S. pneumoniae. This response was dependent on the pore-forming toxin pneumolysin and appeared to be mediated by a cytosolic DNA-sensing pathway involving the adapter molecule STING and the transcription factor IFN regulatory factor 3. Indeed, DNA was present in the cytosol during pneumococcal infection as indicated by the activation of the AIM2 inflammasome, which is known to sense microbial DNA. Type I IFNs produced by S. pneumoniae-infected macrophages positively regulated gene expression and RANTES production in macrophages and cocultured alveolar epithelial cells in vitro. Moreover, type I IFNs controlled RANTES production during pneumococcal pneumonia in vivo. In conclusion, we identified an immune sensing pathway detecting S. pneumoniae that triggers a type I IFN response and positively regulates RANTES production.

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Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties

Cited by 64 publications

References 12 publications

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin

Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic Pneumolysin

Streptococcus pneumoniaeStimulates a STING- and IFN Regulatory Factor 3-Dependent Type I IFN Production in Macrophages, which Regulates RANTES Production in Macrophages, Cocultured Alveolar Epithelial Cells, and Mouse Lungs

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