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Rodney K Tweten - One of the best experts on this subject based on the ideXlab platform.
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Intracellular Glycosylphosphatidylinositols Accumulate on Endosomes: Toxicity of Alpha-Toxin to Leishmania major
Eukaryotic cell, 2005Co-Authors: Zhifeng Zheng, Rodney K Tweten, Kojo Mensa-wilmotAbstract:Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur “free” or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using Alpha-Toxin (from Clostridium septicum). Alpha-Toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for Alpha-Toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, Alpha-Toxin killed L. major with a 50% lethal concentration of 0.77 nM.
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clostridium septicum Alpha Toxin uses glycosylphosphatidylinositol anchored protein receptors
Journal of Biological Chemistry, 1999Co-Authors: Valery M Gordon, Rodney K Tweten, Kim L Nelson, Thomas J Buckley, Victoria L Stevens, Patrick C Elwood, Stephen H LepplaAbstract:Abstract The Alpha Toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the Toxin, indicating that they contain Toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to Alpha Toxin. FACS analysis showed that the mutant cells have lost the ability to bind the Toxin, indicating that they lack an Alpha Toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to Alpha Toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lackN-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to Alpha Toxin. On the other hand, the sensitivity of CHO cells to Alpha Toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that Alpha Toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two Toxins bind to different subsets of GPI-anchored proteins.
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expression and properties of an aerolysin clostridium septicum Alpha Toxin hybrid protein
Molecular Microbiology, 1999Co-Authors: Dzung B Diep, Bret R Sellman, Rodney K Tweten, Kim L Nelson, Tracy S Lawrence, Thomas J BuckleyAbstract:Summary Aerolysin is a bilobal channel-forming Toxin secreted by Aeromonas hydrophila. The Alpha Toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas Toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to Alpha Toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than Alpha Toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas Alpha Toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosylphosphatidylinositol-anchored proteins that is located in the large lobe and is also found in Alpha Toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.
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Clostridium septicum Alpha-Toxin is proteolytically activated by furin.
Infection and Immunity, 1997Co-Authors: Valery M Gordon, Stephen H Leppla, Robert Benz, Karen Fujii, Rodney K TwetenAbstract:Clostridium septicum Alpha-Toxin is secreted as an inactive 46,450-Da proToxin. The proToxin is activated by proteolytic cleavage near the C terminus, which eventually causes the release of a 45-amino-acid fragment. Proteoytic activation and loss of the propeptide allow Alpha-Toxin to oligomerize and form pores on the plasma membrane, which results in colloidal-osmotic lysis. Activation may be accomplished in vitro by cleavage with trypsin at Arg367 (J. Ballard, Y. Sokolov, W. L. Yuan, B. L. Kagan, and R. K. Tweten, Mol. Microbiol. 10:627-634, 1993), which is located within the sequence KKRRGKR367S. A conspicuous feature of this site is a recognition site (RGKR) for the eukaryotic protease furin. Pro-Alpha-Toxin (AT[pro]) that was digested with trypsin or recombinant soluble furin yielded the 41,327-Da active form (AT[act]). A mutated Alpha-Toxin in which the furin consensus site was altered to KKRSGSRS at the cleavage site (AT[SGSR]) was cleaved and activated by trypsin but not by furin. In cytotoxicity assays, wild-type Chinese hamster ovary (CHO) and furin-deficient CHO (FD11) cells were killed by AT(pro) but not by AT(SGSR). Both cell types were killed by AT(SGSR) that was preactivated with trypsin. Propidium iodide uptake assays revealed that FD11 cells were approximately 22% less sensitive to AT(pro) than were CHO cells. AT(pro)-induced cell lysis of FD11 cells, assessed by propidium iodide uptake, was partially prevented by leupeptin (5 mM) and completely prevented by antipain (2.5 mM). The inhibition by antipain suggested the presence of cysteine or serine proteases that could also activate AT(pro). These findings demonstrate that furin is involved in the activation of C. septicum Alpha-Toxin on the cell surface but that alternate eukaryotic proteases can also activate the Toxin. Regardless of the activating protease, the furin consensus site appears to be essential for the activation of Alpha-Toxin on the cell surface.
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the propeptide of clostridium septicum Alpha Toxin functions as an intramolecular chaperone and is a potent inhibitor of Alpha Toxin dependent cytolysis
Molecular Microbiology, 1997Co-Authors: Bret R Sellman, Rodney K TwetenAbstract:: Clostridium septicum Alpha Toxin is activated by a proteolytic cleavage at Arg-398 in its carboxy terminus, which yields a 41.3-kDa cytolytically active Toxin and a 5.1-kDa propeptide. Studies were performed to determine when the propeptide dissociated from the Toxin after proteolytic activation of the proToxin (AT(pro)) and to demonstrate the chaperone activity of the propeptide. The propeptide was found to remain associated with the Toxin after activation with trypsin (AT(act)) when analysed by gel filtration or affinity chromatography of a polyhistidine-tagged derivative that contained the polyhistidine tag on the propeptide. The affinity of the propeptide for the Toxin was decreased significantly when a mutation was introduced in which Val-400 was converted to a cysteine residue. This mutation destabilized the interaction of the propeptide with the Toxin and the propeptide was found to dissociate from the Toxin under the same gel-filtration conditions used for the wild-type Toxin. The separation of the propeptide in the V400C mutant did not affect the cytolytic activity of the Toxin and therefore the propeptide was not necessary for cytolytic activity. These data suggested that the propeptide did not dissociate from the main body of the Toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of Alpha Toxin activity, which inhibited the oligomerization of Alpha Toxin into a functional pore. These data suggest that the propeptide does not participate in the final oligomerized complex and that oligomerization appears to displace the propeptide from AT(act). The importance of the propeptide to the solution stability of Alpha Toxin was also demonstrated. When AT(pro) was activated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed. This aggregate, which could be removed by centrifugation at 14,000 x g, was made up of both SDS-sensitive and -resistant aggregates, suggesting that a variety of inactive aggregates formed when the monomers interacted in solution. Significantly higher levels of haemolytic activity (approximately 16-fold) were observed when Alpha Toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propeptide as an intramolecular chaperone that stabilizes the monomeric AT(pro) and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre-pore complex and forms a pore. The data suggest that oligomerization of the Toxin displaces the propeptide from the monomer form of Alpha Toxin and that the propeptide does not participate in, and is not necessary to, the final cytolytic complex.
Bret R Sellman - One of the best experts on this subject based on the ideXlab platform.
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characterization of Alpha Toxin hla gene variants Alpha Toxin expression levels and levels of antibody to Alpha Toxin in hemodialysis and postsurgical patients with staphylococcus aureus bacteremia
Journal of Clinical Microbiology, 2015Co-Authors: Batu K Sharmakuinkel, Bret R Sellman, David E Tabor, Hoyin Mok, Amy Jenkins, Hasan S Jafri, Thomas H Rude, Felicia Ruffin, Wiley A Schell, Lawrence P ParkAbstract:Alpha-Toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro Alpha-Toxin expression of S. aureus bloodstream isolates, anti-Alpha-Toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-Alpha-Toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of Alpha-Toxin by an anti-Alpha-Toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed Alpha-Toxin (173/200; 86.5%). In vitro Alpha-Toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with Alpha-Toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that Alpha-Toxin is highly conserved in clinical S. aureus isolates. Higher in vitro Alpha-Toxin levels were associated with a positive clinical outcome. Although patients infected with Alpha-Toxin-producing S. aureus exhibited higher anti-Alpha-Toxin antibody levels, these levels were not associated with a better clinical outcome in this study.
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expression and properties of an aerolysin clostridium septicum Alpha Toxin hybrid protein
Molecular Microbiology, 1999Co-Authors: Dzung B Diep, Bret R Sellman, Rodney K Tweten, Kim L Nelson, Tracy S Lawrence, Thomas J BuckleyAbstract:Summary Aerolysin is a bilobal channel-forming Toxin secreted by Aeromonas hydrophila. The Alpha Toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas Toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to Alpha Toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than Alpha Toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas Alpha Toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosylphosphatidylinositol-anchored proteins that is located in the large lobe and is also found in Alpha Toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.
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the propeptide of clostridium septicum Alpha Toxin functions as an intramolecular chaperone and is a potent inhibitor of Alpha Toxin dependent cytolysis
Molecular Microbiology, 1997Co-Authors: Bret R Sellman, Rodney K TwetenAbstract:: Clostridium septicum Alpha Toxin is activated by a proteolytic cleavage at Arg-398 in its carboxy terminus, which yields a 41.3-kDa cytolytically active Toxin and a 5.1-kDa propeptide. Studies were performed to determine when the propeptide dissociated from the Toxin after proteolytic activation of the proToxin (AT(pro)) and to demonstrate the chaperone activity of the propeptide. The propeptide was found to remain associated with the Toxin after activation with trypsin (AT(act)) when analysed by gel filtration or affinity chromatography of a polyhistidine-tagged derivative that contained the polyhistidine tag on the propeptide. The affinity of the propeptide for the Toxin was decreased significantly when a mutation was introduced in which Val-400 was converted to a cysteine residue. This mutation destabilized the interaction of the propeptide with the Toxin and the propeptide was found to dissociate from the Toxin under the same gel-filtration conditions used for the wild-type Toxin. The separation of the propeptide in the V400C mutant did not affect the cytolytic activity of the Toxin and therefore the propeptide was not necessary for cytolytic activity. These data suggested that the propeptide did not dissociate from the main body of the Toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of Alpha Toxin activity, which inhibited the oligomerization of Alpha Toxin into a functional pore. These data suggest that the propeptide does not participate in the final oligomerized complex and that oligomerization appears to displace the propeptide from AT(act). The importance of the propeptide to the solution stability of Alpha Toxin was also demonstrated. When AT(pro) was activated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed. This aggregate, which could be removed by centrifugation at 14,000 x g, was made up of both SDS-sensitive and -resistant aggregates, suggesting that a variety of inactive aggregates formed when the monomers interacted in solution. Significantly higher levels of haemolytic activity (approximately 16-fold) were observed when Alpha Toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propeptide as an intramolecular chaperone that stabilizes the monomeric AT(pro) and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre-pore complex and forms a pore. The data suggest that oligomerization of the Toxin displaces the propeptide from the monomer form of Alpha Toxin and that the propeptide does not participate in, and is not necessary to, the final cytolytic complex.
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The propeptide of Clostridium septicum Alpha Toxin functions as an intramolecular chaperone and is a potent inhibitor of Alpha Toxin‐dependent cytolysis
Molecular microbiology, 1997Co-Authors: Bret R Sellman, Rodney K TwetenAbstract:Clostridium septicum Alpha Toxin is activated by a proteolytic cleavage at Arg-398 in its carboxy terminus, which yields a 41.3-kDa cytolytically active Toxin and a 5.1-kDa propeptide. Studies were performed to determine when the propeptide dissociated from the Toxin after proteolytic activation of the proToxin (AT(pro)) and to demonstrate the chaperone activity of the propeptide. The propeptide was found to remain associated with the Toxin after activation with trypsin (AT(act)) when analysed by gel filtration or affinity chromatography of a polyhistidine-tagged derivative that contained the polyhistidine tag on the propeptide. The affinity of the propeptide for the Toxin was decreased significantly when a mutation was introduced in which Val-400 was converted to a cysteine residue. This mutation destabilized the interaction of the propeptide with the Toxin and the propeptide was found to dissociate from the Toxin under the same gel-filtration conditions used for the wild-type Toxin. The separation of the propeptide in the V400C mutant did not affect the cytolytic activity of the Toxin and therefore the propeptide was not necessary for cytolytic activity. These data suggested that the propeptide did not dissociate from the main body of the Toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of Alpha Toxin activity, which inhibited the oligomerization of Alpha Toxin into a functional pore. These data suggest that the propeptide does not participate in the final oligomerized complex and that oligomerization appears to displace the propeptide from AT(act). The importance of the propeptide to the solution stability of Alpha Toxin was also demonstrated. When AT(pro) was activated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed. This aggregate, which could be removed by centrifugation at 14,000 x g, was made up of both SDS-sensitive and -resistant aggregates, suggesting that a variety of inactive aggregates formed when the monomers interacted in solution. Significantly higher levels of haemolytic activity (approximately 16-fold) were observed when Alpha Toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propeptide as an intramolecular chaperone that stabilizes the monomeric AT(pro) and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre-pore complex and forms a pore. The data suggest that oligomerization of the Toxin displaces the propeptide from the monomer form of Alpha Toxin and that the propeptide does not participate in, and is not necessary to, the final cytolytic complex.
Masataka Oda - One of the best experts on this subject based on the ideXlab platform.
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Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin
Toxins, 2015Co-Authors: Masataka Oda, Jun Sakurai, Yutaka Terao, Masahiro NagahamaAbstract:Clostridium perfringens Alpha-Toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-Toxin possesses phospholipase C and sphingomyelinase activities. The Toxin is composed of an N-terminal domain (1–250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251–370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of Alpha-Toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55–93 aa), especially H….SW84Y85….G, plays an important role in the interaction with ganglioside GM1a. The Toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of Alpha-Toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with Alpha-Toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of Alpha-Toxin in detail.
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clostridium perfringens Alpha Toxin recognizes the gm1a trka complex
Journal of Biological Chemistry, 2012Co-Authors: Masataka Oda, Masahiro Nagahama, Keiko Kobayashi, Michiko Kabura, Teruhisa Takagishi, Ayaka Suzue, Kaori Tominaga, Shiori Urano, Keiko Furukawa, Koichi FurukawaAbstract:Clostridium perfringens Alpha-Toxin is the major virulence factor in the pathogenesis of gas gangrene. Alpha-Toxin is a 43-kDa protein with two structural domains; the N-domain contains the catalytic site and coordinates the divalent metal ions, and the C-domain is a membrane-binding site. The role of the exposed loop region (72–93 residues) in the N-domain, however, has been unclear. Here we show that this loop contains a ganglioside binding motif (H … SXWY … G) that is the same motif seen in botulinum neuroToxin and directly binds to a specific conformation of the ganglioside Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer (GM1a) through a carbohydrate moiety. Confocal microscopy analysis using fluorescently labeled BODIPY-GM1a revealed that the Toxin colocalized with GM1a and induced clustering of GM1a on the cell membranes. Alpha-Toxin was only slightly toxic in β1,4-N-acetylgalactosaminyltransferase knock-out mice, which lack the a-series gangliosides that contain GM1a, but was highly toxic in α2,8-sialyltransferase knock-out mice, which lack both b-series and c-series gangliosides, similar to the control mice. Moreover, experiments with site-directed mutants indicated that Trp-84 and Tyr-85 in the exposed Alpha-Toxin loop play an important role in the interaction with GM1a and subsequent activation of TrkA. These results suggest that binding of Alpha-Toxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. Therefore, we conclude that GM1a is the primary cellular receptor for Alpha-Toxin, which can be a potential target for drug developed against this pathogen.
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Clostridium perfringens Alpha-Toxin Recognizes the GM1a/TrkA Complex
The Journal of biological chemistry, 2012Co-Authors: Masataka Oda, Masahiro Nagahama, Keiko Kobayashi, Michiko Kabura, Teruhisa Takagishi, Ayaka Suzue, Kaori Tominaga, Shiori Urano, Keiko Furukawa, Koichi FurukawaAbstract:Clostridium perfringens Alpha-Toxin is the major virulence factor in the pathogenesis of gas gangrene. Alpha-Toxin is a 43-kDa protein with two structural domains; the N-domain contains the catalytic site and coordinates the divalent metal ions, and the C-domain is a membrane-binding site. The role of the exposed loop region (72–93 residues) in the N-domain, however, has been unclear. Here we show that this loop contains a ganglioside binding motif (H … SXWY … G) that is the same motif seen in botulinum neuroToxin and directly binds to a specific conformation of the ganglioside Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer (GM1a) through a carbohydrate moiety. Confocal microscopy analysis using fluorescently labeled BODIPY-GM1a revealed that the Toxin colocalized with GM1a and induced clustering of GM1a on the cell membranes. Alpha-Toxin was only slightly toxic in β1,4-N-acetylgalactosaminyltransferase knock-out mice, which lack the a-series gangliosides that contain GM1a, but was highly toxic in α2,8-sialyltransferase knock-out mice, which lack both b-series and c-series gangliosides, similar to the control mice. Moreover, experiments with site-directed mutants indicated that Trp-84 and Tyr-85 in the exposed Alpha-Toxin loop play an important role in the interaction with GM1a and subsequent activation of TrkA. These results suggest that binding of Alpha-Toxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. Therefore, we conclude that GM1a is the primary cellular receptor for Alpha-Toxin, which can be a potential target for drug developed against this pathogen.
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Role of Phospholipid Metabolism and G Protein in the Action Induced by Clostridium Perfringens Alpha-Toxin
Journal of Glycomics & Lipidomics, 2012Co-Authors: Masahiro Nagahama, Masataka Oda, Sadayuki Ochi, Keiko Kobayashi, Jun SakuraiAbstract:Alpha-Toxin (370 residues) of Clostridium perfringens is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. Alpha-Toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis and lethality. The structure of the Toxin reveals two domains: the N-terminal domain containing the catalytic active site and the C-terminal domain involving the binding to membranes. Recent research data showed that Alpha-Toxin-induced biological activities are responsible for the activation of phospholipid metabolism via a pertussis Toxin (PT)-sensitive GTP-binding protein, Gi. In this review, we summary the role of phospholipid metabolism and G protein in the biological activities induced by Alpha-Toxin. Discussed are activations of the arachidonic acid cascade (Section 1), the phospholipid metabolism (Section 2), the sphingomeylin metabolism (Section 3) and TrkA signaling (Section 4) induced by Alpha-Toxin.
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clostridium perfringens Alpha Toxin characterization and mode of action
Journal of Biochemistry, 2004Co-Authors: Jun Sakurai, Masahiro Nagahama, Masataka OdaAbstract:Clostridium perfringens type A strains that produce Alpha-Toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production. Intramuscular injection of the Toxin or Bacillus subtilis carrying the Alpha-Toxin gene causes myonecrosis and produces histopathological features of the disease. Immunization of mice with Alpha-Toxin or fragments of the Toxin prevents gas gangrene caused by C. perfringens. The Toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis. These biological activities are closely related to PLC and/or SMase activities. However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of Alpha-Toxin. Based on the isolation and characterization of the gene for Alpha-Toxin and a comparison of the Toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of Alpha-Toxin and the mode of action of the Toxin. To provide a better understanding of the role of Alpha-Toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of Alpha-Toxin.
Nadja Hellmann - One of the best experts on this subject based on the ideXlab platform.
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Lipid and Phase Specific Interaction of Alpha Toxin from S.aureus with Lipid Membranes
Biophysical Journal, 2013Co-Authors: Markus Schwiering, Lars Schmuser, Nadja HellmannAbstract:The pore forming Alpha Toxin from S.aureus is secreted by the bacterium as a monomer and assembles up to heptameric oligomers on cellular und artificial membranes, to yield a transmembrane pore. Interaction with artificial lipid membranes requires the presence of a lipid with a phosphocholine headgroup, namely sphingomyelin or phosphatidylcholine. Both represent the major fraction of lipids in the outer plasma membrane: saturated sphingomyelin preferring the liquid ordered phase, and unsaturated phosphatidylcholine with a preference for the liquid ordered phase. Employing liposomes we could show that Alpha Toxin has a higher overall affinity for sphingomyelin compared to phosphatidylcholine if the lipids are in the same phase. Preferred binding to sphingomyelin usually is interpreted as a preference for rafts or domains in the liquid ordered phase, especially if also binding is increased in presence of cholesterol as is also the case for Alpha Toxin. However, this is not necessarily the case. Alpha Toxin preferentially interacts with lipids in the liquid disordered phase as shown by fluorescence spectroscopy and microscopy. Since sphingomyelin is also present in the liquid disordered phase, albeit to a smaller extent compared to the liquid ordered phase, binding could occur both to sphingomyelin or phosphatidylcholine. A further complexity is added by the observation charged lipids further increase Alpha Toxin binding. Thus one can expect that the partition coefficient of Alpha Toxin between liquid ordered and liquid disordered phase is critically dependent on the detailed composition, and might very well be shifted by presence of proteins as is the case in the cellular membranes, without the need of any specific interaction between Alpha Toxin and the protein.Granted by the DFG (SFB 490)
Sucharit Bhakdi - One of the best experts on this subject based on the ideXlab platform.
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Altered pore-forming properties of proteolytically nicked staphylococcal Alpha-Toxin.
The Journal of biological chemistry, 1993Co-Authors: Michael Palmer, Ulrich Weller, Martina Messner, Sucharit BhakdiAbstract:Staphylococcal Alpha-Toxin is a single-chain polypeptide with a molecular weight of 34,000 that hexamerizes in lipid bilayers to form pores of 1-1.5 nm effective diameter in membranes. We demonstrate that limited proteolysis of purified Alpha-Toxin with proteinase K generates a hemolytically active product that yields one major protein band of 17-18 kDa in SDS-polyacrylamide gel electrophoresis. The 17-18-kDa protein band harbors two major fragments of similar size representing the N- and C-terminal halves, which remain associated with each other in non-denaturing buffers but dissociate in 6 M urea. Dissociation in urea leads to loss of hemolytic activity. In contrast, unnicked Alpha-Toxin is not inactivated by urea. Nicked, hemolytically active Alpha-Toxin forms hexamers on erythrocyte membranes and on lymphocytes and monocytes. However, the nicked Toxin can only lyse erythrocytes and fails to permeabilize nucleated cells. Osmotic protection experiments indicate that the size of pores generated by the nicked Toxin is considerably smaller (0.6-0.9 nm effective diameter) than that generated by native Toxin. The collective results do not support a previous proposal that different functions of Alpha-Toxin are contained in separate domains of the molecule.
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Staphylococcus aureus Alpha-Toxin. Dual mechanism of binding to target cells.
The Journal of biological chemistry, 1991Co-Authors: A. Hildebrand, M. Pohl, Sucharit BhakdiAbstract:Staphylococcal Alpha-Toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of Alpha-Toxin to target cells can occur via two different mechanisms. Binding of Alpha-Toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, Toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at Alpha-Toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated Alpha-Toxin from either cell species proved that hemolysis was inevitably associated with the formation of Toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward Alpha-Toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving Toxin oligomerization and pore formation.