The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform

Timothy J Mitchell - One of the best experts on this subject based on the ideXlab platform.

  • Pneumolysin is responsible for differential gene expression and modifications in the epigenetic landscape of primary monocyte derived macrophages.
    2020
    Co-Authors: Joby Cole, Timothy J Mitchell, Adrienn Angyal, Richard D. Emes, Mark J. Dickman, David H Dockrell
    Abstract:

    Abstract Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of Pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether Pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a Pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed Pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that Pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF production, reprising the pattern observed with Pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by Pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse Pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by Pneumolysin plays a role in dampening the innate responses to pneumococci. Author summary Pneumolysin is a toxin that contributes to how Streptococcus pneumoniae, the leading cause of pneumonia, causes disease. In this study, the toxin alters gene expression in immune cells called macrophages, one of the first lines of defence against bacteria at sites of infection. Modulation involved multiple immune responses, including generation of chemical signals coordinating responses in immune cells termed cytokines. In addition, changes were observed in histone proteins that are involved in controlling gene expression in the cell. Pneumolysin reduced early production of the cytokine TNF-α and a medicine vorinostat that modifies these ‘epigenetic’ histone modifications had a similar affect, suggesting epigenetic mechanisms contribute to the ability of Pneumolysin to reduce immune responses.

  • Missing elimination via membrane vesicle shedding contributes to the diminished calcium sensitivity of listeriolysin O
    Nature Publishing Group, 2018
    Co-Authors: Jana Maurer, Timothy J Mitchell, Sabrina Hupp, Helena Pillich, Trinad Chakraborty, Asparouh I Iliev
    Abstract:

    Abstract The lytic capacity of cholesterol-dependent cytolysins is enhanced in the extracellular calcium-free environment through a combination of limited membrane repair and diminished membrane toxin removal. For a typical neurotoxin of the group, Pneumolysin, this effect has already been observed at reduced (1 mM) calcium conditions, which are pathophysiologically relevant. Here, we tested another neurotoxin of the group, listeriolysin O from L. monocytogenes, active in the primary vacuole after bacterium phagocytosis in host cells. Reduced calcium did not increase the lytic capacity of listeriolysin (in contrast to Pneumolysin), while calcium-free conditions elevated it 2.5 times compared to 10 times for Pneumolysin (at equivalent hemolytic capacities). To clarify these differences, we analyzed membrane vesicle shedding, known to be a calcium-dependent process for toxin removal from eukaryotic cell membranes. Both Pneumolysin and listeriolysin initiated vesicle shedding, which was completely blocked by the lack of extracellular calcium. Lack of calcium, however, elevated the toxin load per a cell only for Pneumolysin and not for listeriolysin. This result indicates that vesicle shedding does not play a role in the membrane removal of listeriolysin and outlines a major difference between it and other members of the CDC group. Furthermore, it provides new tools for studying membrane vesicle shedding

  • Chapter 14 – Pneumolysin
    Streptococcus Pneumoniae, 2015
    Co-Authors: Daniel R. Neill, Timothy J Mitchell, Aras Kadioglu
    Abstract:

    Pneumolysin is a cholesterol-dependent cytolytic toxin that plays a crucial role in pneumococcal pathogenesis as well as acting as a major instigator of both inflammatory and protective immune responses in the host. This dichotomy between stimulating damaging pro-inflammatory responses and immune regulatory pathways during infection makes Pneumolysin a fascinating toxin to study and a key target for novel therapeutics and vaccines. In this chapter we present the past and present of our knowledge of Pneumolysin.

  • Effects of cigarette smoke condensate on pneumococcal biofilm formation and Pneumolysin
    The European respiratory journal, 2012
    Co-Authors: N. Daphney Mutepe, Timothy J Mitchell, Charles Feldman, Riana Cockeran, Annette J. Theron, Helen C. Steel, Ronald Anderson
    Abstract:

    Although the well-recognised predisposition of cigarette smokers to the development of severe pneumococcal disease may be attributable to impairment of local host defences, less is known about the direct effects of smoke exposure on airway pathogens, or their virulence factors. In the current study, we have investigated the effects of cigarette smoke condensate (CSC) on biofilm formation by Streptococcus pneumoniae , and on the pore-forming activity of its major toxin, Pneumolysin. Biofilm formation following exposure of the pneumococcus to CSC (20–160 μg·mL−1) was measured using a crystal violet-based spectrophotometric procedure, while the pore-forming activity of recombinant Pneumolysin was determined by a fura-2/acetoxymethyl ester-based spectrofluorimetric procedure to monitor the uptake of extracellular Ca2+ by isolated human neutrophils. Exposure of the pneumococcus or Pneumolysin to CSC resulted in significant dose-related augmentation of biofilm formation (p≤0.05 at 80 and 160 μg·mL−1) and substantial attenuation of the pore-forming interactions of Pneumolysin, respectively. Augmentation of biofilm formation and inactivation of Pneumolysin as a consequence of smoking are likely to favour microbial colonisation and persistence, both being essential precursors of pneumococcal disease.

  • changes in astrocyte shape induced by sublytic concentrations of the cholesterol dependent cytolysin Pneumolysin still require pore forming capacity
    Toxins, 2011
    Co-Authors: Christina Fortsch, Timothy J Mitchell, Sabrina Hupp, Elke Maier, Roland Benz, Asparouh I Iliev
    Abstract:

    Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, Pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of Pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of Pneumolysin's pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of Pneumolysin treatment, the toxin's lytic capacity remained critical for the initiation of cell shape changes. The non-lytic Pneumolysin mutants W433F-Pneumolysin and delta6-Pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the Pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of Pneumolysin and the actin cytoskeleton reorganization is suggested.

Peter W. Andrew - One of the best experts on this subject based on the ideXlab platform.

  • Pneumolysin causes cyotoxicity and ciliary stasis in human respiratory epithelial cells
    European Respiratory Journal, 2014
    Co-Authors: Khodor S. Hazime, Peter W. Andrew, Christopher O'callaghan, Gwyneth Williams, Robert A. Hirst
    Abstract:

    Background: Pneumolysin is the cholesterol dependent pore forming toxin that is released from Streptococcus pneumoniae during respiratory infection. The release of Pneumolysin is thought to be a sudden event following activation of the bacterial autolysin enzyme. Here we tested the dose response relationship of acute exposure of Pneumolysin on human respiratory epithelial cells grown at an air-liquid interface. Aims: To determine the concentration of Pneumolysin required to adversely affect human respiratory ciliary function. Methods: Normal human respiratory epithelial cells were grown to a confluent layer and re-growth of cilia from the basal epithelial cells was stimulated by use of an air-liquid interface culture. Light microscopy and high speed video recordings at 1000x magnification were used to determine cilia function and morphological cytotoxicity. Results: Pneumolysin displayed a dose dependent inhibition of ciliary beat frequency with an IC 50 of 43.3± 6.9 HU . The common cytotoxic events that were quantified included ciliary dyskinesia, immotility and cytoplasmic extrusion. Conclusion: Pneumolysin caused dose dependent, rapid ciliary inhibition and cytotoxicity, indicating an important role of this toxin in pneumococcal infection. The comparative toxicity with un-ciliated basal cells will be presented.

  • human lung mast cells mediate pneumococcal cell death in response to activation by Pneumolysin
    Journal of Immunology, 2010
    Co-Authors: Glenn Cruse, Peter W. Andrew, Vitor E Fernandes, Jose De Salort, Depesh Pankhania, Marta S Marinas, Hannah Brewin, Peter Bradding, Aras Kadioglu
    Abstract:

    Mast cells are emerging as contributors to innate immunity. Mouse mast cells have a pivotal role in protection against bacterial infection, and human cord blood-derived mast cells reduce bacterial viability in culture. The objectives of this study were to determine whether human lung mast cells (HLMCs) might be protective against pneumococcal lung infection through direct antimicrobial activity. Tissue-derived HLMCs and the human mast cell lines HMC-1 and LAD2 were cocultured with wild-type and mutant pneumococci, and viability and functional assays were performed. Mast cells were also stimulated with purified Pneumolysin. HLMCs killed wild-type serotype-2 (D39) pneumococci in coculture but had no effect on an isogenic Pneumolysin-deficient (PLN-A) pneumococcus. D39 wild-type, but not PLN-A pneumococci, induced the release of leukotriene C4 from human mast cells in a dose-dependent manner, which was not accompanied by histamine release. Stimulation of mast cells with sublytic concentrations of purified Pneumolysin replicated this effect. Furthermore, Pneumolysin induced the release of the cathelicidin LL-37 from HLMCs, purified LL-37 reduced pneumococcal viability, and neutralizing Ab to LL-37 attenuated mast cell-dependent pneumococcal killing. In addition, at high concentrations, all pneumococcal strains tested reduced HLMC viability through a combination of Pneumolysin and H2O2-dependent mechanisms. HLMCs exhibit direct antimicrobial activity to pneumococci through their activation by Pneumolysin. This antimicrobial activity is mediated, in part, by the release of LL-37 from HLMCs. This suggests that mast cells provide an early warning system and potentially limit pneumococcal dissemination early in the course of invasive pulmonary pneumococcal disease.

  • Oligomerisation of Pneumolysin on cholesterol crystals: Similarities to the behaviour of polyene antibiotics
    Toxicon : official journal of the International Society on Toxinology, 2008
    Co-Authors: Andreas F.-p. Sonnen, Peter W. Andrew, Arthur J. Rowe, Robert J. C. Gilbert
    Abstract:

    Pneumolysin is a cytolytic toxin of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. The prepore and pore states of Pneumolysin have recently been investigated by cryo-electron microscopy and atomic force microscopy, confirming the existence of arc-shaped as well as ring-form oligomers. Here we provide further insights into the Pneumolysin oligomer by studying the interaction of Pneumolysin with cholesterol crystals, comparing the results to those obtained for polyene antibiotics, which also bind cholesterol.

  • pneumococcal behavior and host responses during bronchopneumonia are affected differently by the cytolytic and complement activating activities of Pneumolysin
    Infection and Immunity, 2003
    Co-Authors: Rania Jounblat, Timothy J Mitchell, Aras Kadioglu, Peter W. Andrew
    Abstract:

    Pneumolysin, a multifunctional toxin produced by all clinical isolates of Streptococcus pneumoniae, is strongly implicated in the pathogenesis of pneumococcal bronchopneumonia and septicemia. Using isogenic mutant strains, we examined the effect of deletion of the cytotoxic activity or complement-activating activity of Pneumolysin on bacterial growth in lungs and blood, histological changes in infected lung tissue, and the pattern of inflammatory cell recruitment. Both of the activities of Pneumolysin contributed to the pathology in the lungs, as well as the timing of the onset of bacteremia. Histological changes in the lungs were delayed after infection with either mutant compared to the changes seen after infection with the wild-type pneumococcus. The complement-activating activity of Pneumolysin affected the accumulation of T cells, whereas the toxin's cytolytic activity influenced neutrophil recruitment into lung tissue.

  • Sensitivities of Human Monocytes and Epithelial Cells to Pneumolysin Are Different
    Infection and immunity, 2002
    Co-Authors: Robert A. Hirst, Timothy J Mitchell, Hasan Yesilkaya, Edwin Clitheroe, Andrew Rutman, Nicola Dufty, Christopher O'callaghan, Peter W. Andrew
    Abstract:

    The Streptococcus pneumoniae pore-forming toxin, Pneumolysin, is an important virulence factor in pneumococcal pneumonia. The effect of Pneumolysin on human lung epithelial and monocyte cell viability was compared. Pneumolysin caused a dose-dependent loss of viability of human lung epithelial (A549 and L132) and monocyte (U937 and THP-1) cell lines. Analysis of the dose-response curves revealed similar log 50% inhibitory concentration (pIC(50)) values for A549, L132, and THP-1 of 0.12+/- 0.1, 0.02+/- 0.04, and 0.12+/- 0.13 hemolytic units (HU), respectively, but U937 cells showed a significantly greater pIC(50) of 0.42+/- 0.12 HU. Differentiation of A549 and L132 with phorbol ester or THP-1 with gamma interferon had no effect on their sensitivity to Pneumolysin. However, a significant decrease in the potency of Pneumolysin against U937 cells followed gamma interferon treatment. The Hill slopes of the inhibition curves were greater than unity, indicating that Pneumolysin may act with positive cooperativity. Analysis of Pneumolysin-treated THP-1 cells by electron microscopy revealed membrane lesions of between 100 and 200 nm in diameter.

James C. Paton - One of the best experts on this subject based on the ideXlab platform.

  • Toll-like receptor 2 contributes to antibacterial defence against Pneumolysin-deficient pneumococci
    Cellular Microbiology, 2007
    Co-Authors: Mark C. Dessing, James C. Paton, Sandrine Florquin, Tom Van Der Poll
    Abstract:

    © 2008 Blackwell Publishing LtdToll-like receptors (TLRs) are pattern recognition receptors that recognize conserved molecular patterns expressed by pathogens. Pneumolysin, an intracellular toxin found in all Streptococcus pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by TLR4. Although TLR2 is considered the most important receptor for Gram-positive bacteria, our laboratory previously could not demonstrate a decisive role for TLR2 in host defence against pneumonia caused by a serotype 3 S. pneumoniae. Here we tested the hypothesis that in the absence of TLR2, S. pneumoniae can still be sensed by the immune system through an interaction between Pneumolysin and TLR4. C57BL/6 wild-type (WT) and TLR2 knockout (KO) mice were intranasally infected with either WT S. pneumoniae D39 (serotype 2) or the isogenic Pneumolysin-deficient S. pneumoniae strain D39 PLN. TLR2 did not contribute to antibacterial defence against WT S. pneumoniae D39. In contrast, Pneumolysin-deficient S. pneumoniae only grew in lungs of TLR2 KO mice. TLR2 KO mice displayed a strongly reduced early inflammatory response in their lungs during pneumonia caused by both Pneumolysin-producing and Pneumolysin-deficient pneumococci. These data suggest that Pneumolysin-induced TLR4 signalling can compensate for TLR2 deficiency during respiratory tract infection with S. pneumoniae.Mark C. Dessing, Sandrine Florquin, James C. Paton and Tom van der Pol

  • Toll-like receptor 2 contributes to antibacterial defence against Pneumolysin-deficient pneumococci
    Cellular microbiology, 2007
    Co-Authors: Mark C. Dessing, James C. Paton, Sandrine Florquin, Tom Van Der Poll
    Abstract:

    Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved molecular patterns expressed by pathogens. Pneumolysin, an intracellular toxin found in all Streptococcus pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by TLR4. Although TLR2 is considered the most important receptor for Gram-positive bacteria, our laboratory previously could not demonstrate a decisive role for TLR2 in host defence against pneumonia caused by a serotype 3 S. pneumoniae. Here we tested the hypothesis that in the absence of TLR2, S. pneumoniae can still be sensed by the immune system through an interaction between Pneumolysin and TLR4. C57BL/6 wild-type (WT) and TLR2 knockout (KO) mice were intranasally infected with either WT S. pneumoniae D39 (serotype 2) or the isogenic Pneumolysin-deficient S. pneumoniae strain D39 PLN. TLR2 did not contribute to antibacterial defence against WT S. pneumoniae D39. In contrast, Pneumolysin-deficient S. pneumoniae only grew in lungs of TLR2 KO mice. TLR2 KO mice displayed a strongly reduced early inflammatory response in their lungs during pneumonia caused by both Pneumolysin-producing and Pneumolysin-deficient pneumococci. These data suggest that Pneumolysin-induced TLR4 signalling can compensate for TLR2 deficiency during respiratory tract infection with S. pneumoniae.

  • The Apoptotic Response to Pneumolysin Is Toll-Like Receptor 4 Dependent and Protects against Pneumococcal Disease
    Infection and immunity, 2005
    Co-Authors: Amit Srivastava, James C. Paton, Philipp Henneke, Alberto Visintin, Sarah C. Morse, Victoria Martin, Claire Watkins, Michael R. Wessels, Douglas T. Golenbock, Richard Malley
    Abstract:

    Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with Pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that Pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and Pneumolysin is direct and specific; ligand-binding studies demonstrated that Pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in Pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to Pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with Pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced Pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced Pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, Pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of Pneumolysin-induced apoptosis.

  • Recognition of Pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection.
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Richard Malley, James C. Paton, Philipp Henneke, Sarah C. Morse, Michael R. Wessels, Michael J. Cieslewicz, Marc Lipsitch, Claudette M. Thompson, Evelyn A. Kurt-jones, Douglas T. Golenbock
    Abstract:

    Streptococcus pneumoniae is one of the leading causes of invasive bacterial disease worldwide. Fragments of the cell wall and the cytolytic toxin Pneumolysin have been shown to contribute substantially to inflammatory damage, although the interactions between pneumococcal components and host-cell structures have not been elucidated completely. Results of a previous study indicated that cell-wall components of pneumococci are recognized by Toll-like receptor (TLR)2 but suggested that Pneumolysin induces inflammatory events independently of this receptor. In this study we tested the hypothesis that Pneumolysin interacts with surface proteins of the TLR family other than TLR2. We found that Pneumolysin stimulates tumor necrosis factor-α and IL-6 release in wild-type macrophages but not in macrophages from mice with a targeted deletion of the cytoplasmic TLR-adapter molecule myeloid differentiation factor 88, suggesting the involvement of the TLRs in Pneumolysin recognition. Purified Pneumolysin synergistically activated macrophage responses together with preparations of pneumococcal cell walls or staphylococcal peptidoglycan, which are known to activate TLR2. Furthermore, when compared with wild-type macrophages, macrophages from mice that carry a spontaneous mutation in TLR4 (P712H) were hyporesponsive to both Pneumolysin alone and the combination of Pneumolysin with pneumococcal cell walls. Finally, these TLR4-mutant mice were significantly more susceptible to lethal infection after intranasal colonization with Pneumolysin-positive pneumococci than were control mice. We conclude that the interaction of Pneumolysin with TLR4 is critically involved in the innate immune response to pneumococcus.

  • The Autolytic Enzyme LytA of Streptococcus pneumoniae Is Not Responsible for Releasing Pneumolysin
    Journal of bacteriology, 2001
    Co-Authors: Priya Balachandran, James C. Paton, Susan K. Hollingshead, David E. Briles
    Abstract:

    It was previously proposed that autolysin's primary role in the virulence of pneumococci was to release Pneumolysin to an extracellular location. This interpretation came into question when Pneumolysin was observed to be released in significant amounts from some pneumococci during log-phase growth, because autolysis was not believed to occur at this time. We have reexamined this phenomenon in detail for one such strain, WU2. This study found that the extracellular release of Pneumolysin from WU2 was not dependent on autolysin action. A mutant lacking autolysin showed the same pattern of Pneumolysin release as the wild-type strain. Addition of mitomycin C to a growing WU2 culture did not induce lysis, indicating the absence of resident bacteriophages that could potentially harbor lytA-like genes. Furthermore, release of Pneumolysin was unaltered by growth in 2% choline, a condition which is reported to inactivate autolysin, as well as most known pneumococcal phage lysins. Profiles of total proteins in the cytoplasm and in the supernatant media supported the hypothesis that release of Pneumolysin is independent of pneumococcal lysis. Finally, under some infection conditions, mutations in Pneumolysin and autolysin had different effects on virulence.

Anthony Watts - One of the best experts on this subject based on the ideXlab platform.

  • structural analysis of the protein lipid complexes associated with pore formation by the bacterial toxin Pneumolysin
    Journal of Biological Chemistry, 2001
    Co-Authors: Boyan B. Bonev, Peter W. Andrew, Robert J. C. Gilbert, Olwyn Byron, Anthony Watts
    Abstract:

    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.

  • Structural Analysis of the Protein/Lipid Complexes Associated with Pore Formation by the Bacterial Toxin Pneumolysin
    The Journal of biological chemistry, 2000
    Co-Authors: Boyan B. Bonev, Peter W. Andrew, Robert J. C. Gilbert, Olwyn Byron, Anthony Watts
    Abstract:

    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.

  • Structural investigations of Pneumolysin/lipid complexes.
    Molecular membrane biology, 2000
    Co-Authors: Boyan B. Bonev, Robert J. C. Gilbert, Anthony Watts
    Abstract:

    Pneumolysin, a virulence factor from the human pathogen Streptococcus pneumoniae, is a water-soluble protein which forms ring-shaped oligomeric structures upon binding to cholesterol-containing lipid membranes. It induces vesicle aggregation, membrane pore formation and withdrawal of lipid material into non-bilayer proteolipid complexes. Solid-state magic angle spinning and wideline static NMR, together with freeze-fracture electron microscopy, are used to characterize the phase changes in fully hydrated cholesterol-containing lipid membranes induced by the addition ofPneumolysin. A structural model for the proteolipid complexes is proposed where a 30-50-meric Pneumolysin ring lines the inside of a lipid torus. Cholesterol is found to be essential to the fusogenic action of Pneumolysin.

Tom Van Der Poll - One of the best experts on this subject based on the ideXlab platform.

  • Toll-like receptor 2 contributes to antibacterial defence against Pneumolysin-deficient pneumococci
    Cellular microbiology, 2007
    Co-Authors: Mark C. Dessing, James C. Paton, Sandrine Florquin, Tom Van Der Poll
    Abstract:

    Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved molecular patterns expressed by pathogens. Pneumolysin, an intracellular toxin found in all Streptococcus pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by TLR4. Although TLR2 is considered the most important receptor for Gram-positive bacteria, our laboratory previously could not demonstrate a decisive role for TLR2 in host defence against pneumonia caused by a serotype 3 S. pneumoniae. Here we tested the hypothesis that in the absence of TLR2, S. pneumoniae can still be sensed by the immune system through an interaction between Pneumolysin and TLR4. C57BL/6 wild-type (WT) and TLR2 knockout (KO) mice were intranasally infected with either WT S. pneumoniae D39 (serotype 2) or the isogenic Pneumolysin-deficient S. pneumoniae strain D39 PLN. TLR2 did not contribute to antibacterial defence against WT S. pneumoniae D39. In contrast, Pneumolysin-deficient S. pneumoniae only grew in lungs of TLR2 KO mice. TLR2 KO mice displayed a strongly reduced early inflammatory response in their lungs during pneumonia caused by both Pneumolysin-producing and Pneumolysin-deficient pneumococci. These data suggest that Pneumolysin-induced TLR4 signalling can compensate for TLR2 deficiency during respiratory tract infection with S. pneumoniae.

  • Toll-like receptor 2 contributes to antibacterial defence against Pneumolysin-deficient pneumococci
    Cellular Microbiology, 2007
    Co-Authors: Mark C. Dessing, James C. Paton, Sandrine Florquin, Tom Van Der Poll
    Abstract:

    © 2008 Blackwell Publishing LtdToll-like receptors (TLRs) are pattern recognition receptors that recognize conserved molecular patterns expressed by pathogens. Pneumolysin, an intracellular toxin found in all Streptococcus pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by TLR4. Although TLR2 is considered the most important receptor for Gram-positive bacteria, our laboratory previously could not demonstrate a decisive role for TLR2 in host defence against pneumonia caused by a serotype 3 S. pneumoniae. Here we tested the hypothesis that in the absence of TLR2, S. pneumoniae can still be sensed by the immune system through an interaction between Pneumolysin and TLR4. C57BL/6 wild-type (WT) and TLR2 knockout (KO) mice were intranasally infected with either WT S. pneumoniae D39 (serotype 2) or the isogenic Pneumolysin-deficient S. pneumoniae strain D39 PLN. TLR2 did not contribute to antibacterial defence against WT S. pneumoniae D39. In contrast, Pneumolysin-deficient S. pneumoniae only grew in lungs of TLR2 KO mice. TLR2 KO mice displayed a strongly reduced early inflammatory response in their lungs during pneumonia caused by both Pneumolysin-producing and Pneumolysin-deficient pneumococci. These data suggest that Pneumolysin-induced TLR4 signalling can compensate for TLR2 deficiency during respiratory tract infection with S. pneumoniae.Mark C. Dessing, Sandrine Florquin, James C. Paton and Tom van der Pol