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Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica
Karthikayalu, S;Rama, V;Venkatesan, R;
Journal of Venomous Animals and Toxins including Tropical Diseases , 2010, DOI: 10.1590/S1678-91992010000300019
Abstract: pore-forming cytolysins of 19 kda from sea anemones present a remarkable cytolytic property. in the present work, a purified 19-kda cytolysin was obtained from the sea anemone heteractis magnifica. the purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mm sodium phosphate buffer (ph 7.4), followed by anion exchange chromatography in deae-sepharose? column (ge healthcare, sweden) and gel filtration chromatography using sephadex? g-50 matrix (ge healthcare, sweden). the active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. the final active fraction showed a prominent protein band of molecular mass of 19 kda when analyzed by sds-page.
Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity  [PDF]
Christina F?rtsch,Sabrina Hupp,Jiangtao Ma,Timothy J. Mitchell,Elke Maier,Roland Benz,Asparouh I. Iliev
Toxins , 2011, DOI: 10.3390/toxins3010043
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.
Structure, Function, and Biology of the Enterococcus faecalis Cytolysin  [PDF]
Daria Van Tyne,Melissa J. Martin,Michael S. Gilmore
Toxins , 2013, DOI: 10.3390/toxins5050895
Abstract: Enterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. The current understanding of cytolysin biosynthesis, structure/function relationships, and contribution to the biology of E. faecalis are reviewed, and opportunities for using emerging technologies to advance this understanding are discussed.
The Pore-Forming Toxin β hemolysin/cytolysin Triggers p38 MAPK-Dependent IL-10 Production in Macrophages and Inhibits Innate Immunity  [PDF]
Magali Bebien,Mary E. Hensler,Suzel Davanture,Li-Chung Hsu,Michael Karin,Jin Mo Park,Lena Alexopoulou,George Y. Liu,Victor Nizet,Toby Lawrence
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002812
Abstract: Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns and immune-compromised adults. The pore-forming toxin (PFT) β hemolysin/cytolysin (βh/c) is a major virulence factor for GBS, which is generally attributed to its cytolytic functions. Here we show βh/c has immunomodulatory properties on macrophages at sub-lytic concentrations. βh/c-mediated activation of p38 MAPK drives expression of the anti-inflammatory and immunosuppressive cytokine IL-10, and inhibits both IL-12 and NOS2 expression in GBS-infected macrophages, which are critical factors in host defense. Isogenic mutant bacteria lacking βh/c fail to activate p38-mediated IL-10 production in macrophages and promote increased IL-12 and NOS2 expression. Furthermore, targeted deletion of p38 in macrophages increases resistance to invasive GBS infection in mice, associated with impaired IL-10 induction and increased IL-12 production in vivo. These data suggest p38 MAPK activation by βh/c contributes to evasion of host defense through induction of IL-10 expression and inhibition of macrophage activation, a new mechanism of action for a PFT and a novel anti-inflammatory role for p38 in the pathogenesis of invasive bacterial infection. Our studies suggest p38 MAPK may represent a new therapeutic target to blunt virulence and improve clinical outcome of invasive GBS infection.
Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage  [PDF]
Carolin Wippel,Jana Maurer,Christina F?rtsch,Sabrina Hupp,Alexandra Bohl,Jiangtao Ma,Timothy J. Mitchell,Stephanie Bunkowski,Wolfgang Brück,Roland Nau,Asparouh I. Iliev
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003380
Abstract: Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.
Novel Allosteric Sites on Ras for Lead Generation  [PDF]
Barry J. Grant, Suryani Lukman, Harrison J. Hocker, Jaqueline Sayyah, Joan Heller Brown, J. Andrew McCammon, Alemayehu A. Gorfe
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025711
Abstract: Aberrant Ras activity is a hallmark of diverse cancers and developmental diseases. Unfortunately, conventional efforts to develop effective small molecule Ras inhibitors have met with limited success. We have developed a novel multi-level computational approach to discover potential inhibitors of previously uncharacterized allosteric sites. Our approach couples bioinformatics analysis, advanced molecular simulations, ensemble docking and initial experimental testing of potential inhibitors. Molecular dynamics simulation highlighted conserved allosteric coupling of the nucleotide-binding switch region with distal regions, including loop 7 and helix 5. Bioinformatics methods identified novel transient small molecule binding pockets close to these regions and in the vicinity of the conformationally responsive switch region. Candidate binders for these pockets were selected through ensemble docking of ZINC and NCI compound libraries. Finally, cell-based assays confirmed our hypothesis that the chosen binders can inhibit the downstream signaling activity of Ras. We thus propose that the predicted allosteric sites are viable targets for the development and optimization of new drugs.
Group B Streptococcal β-Hemolysin/Cytolysin Directly Impairs Cardiomyocyte Viability and Function  [PDF]
Mary E. Hensler, Shigeki Miyamoto, Victor Nizet
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002446
Abstract: Background Group B Streptococcus (GBS) is a leading cause of neonatal sepsis where myocardial dysfunction is an important contributor to poor outcome. Here we study the effects of the GBS pore-forming β-hemolysin/cytolysin (Bh/c) exotoxin on cardiomyocyte viability, contractility, and calcium transients. Methodology/Principal Findings HL-1 cardiomyocytes exposed to intact wild-type (WT) or isogenic Δβh/c mutant GBS, or to cell-free extracts from either strain, were assessed for viability by trypan blue exclusion and for apoptosis by TUNEL staining. Functionality of exposed cardiomyocytes was analyzed by visual quantitation of the rate and extent of contractility. Mitochondrial membrane polarization was measured in TMRE-loaded cells exposed to GBS βh/c. Effects of GBS βh/c on calcium transients were studied in fura-2AM-loaded primary rat ventricular cardiomyocytes. Exposure of HL-1 cardiomyocytes to either WT GBS or βh/c extracts significantly reduced both rate and extent of contractility and later induced necrotic and apoptotic cell death. No effects on cardiomyocyte viability or function were observed after treatment with Δβh/c mutant bacteria or extracts. The βh/c toxin was associated with complete and rapid loss of detectable calcium transients in primary neonatal rat ventricular cardiomyocytes and induced a loss of mitochondrial membrane polarization. These effects on viability and function were abrogated by the βh/c inhibitor, dipalmitoyl phosphatidylcholine (DPPC). Conclusions/Significance Our data show a rapid loss of cardiomyocyte viability and function induced by GBS βh/c, and these deleterious effects are inhibited by DPPC, a normal constituent of human pulmonary surfactant.. These findings have clinical implications for the cardiac dysfunction observed in neonatal GBS infections.
Allosteric Modulation of Muscarinic Acetylcholine Receptors  [PDF]
Jan Jakubík,Esam E. El-Fakahany
Pharmaceuticals , 2010, DOI: 10.3390/ph3092838
Abstract: An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.
The Structural Basis of ATP as an Allosteric Modulator  [PDF]
Shaoyong Lu ,Wenkang Huang ,Qi Wang,Qiancheng Shen,Shuai Li,Ruth Nussinov ,Jian Zhang
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003831
Abstract: Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.
Change in Allosteric Network Affects Binding Affinities of PDZ Domains: Analysis through Perturbation Response Scanning  [PDF]
Z. Nevin Gerek,S. Banu Ozkan
PLOS Computational Biology , 2011, DOI: 10.1371/journal.pcbi.1002154
Abstract: The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation.
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