The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Michael S. Gilmore - One of the best experts on this subject based on the ideXlab platform.
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Structure, Function, and Biology of the Enterococcus faecalis Cytolysin
Toxins, 2013Co-Authors: Daria Van Tyne, Melissa J. Martin, Michael S. GilmoreAbstract: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.
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The Comprehensive Sourcebook of Bacterial Protein Toxins (Third Edition) - CHAPTER 41 – Enterococcus faecalis Cytolysin toxin
The Comprehensive Sourcebook of Bacterial Protein Toxins, 2006Co-Authors: Karen Carniol, Michael S. GilmoreAbstract:The Cytolysin toxin, produced by the Gram-positive bacterium Enterococcus faecalis, is unique among bacterial toxins in its ability to lyse both bacterial cells and eukaryotic cells. This chapter describes what is known about the range of target cells, the molecular determinants of Cytolysin activity, the mobility of the Cytolysin-encoding locus, regulation of the expression of Cytolysin activity, and the role of Cytolysin in enterococcal virulence. The Cytolysin toxin of E.faecalis is a two-subunit hemolysin and bacteriocin capable of lysing a broad range of target cells. The maturation process of the two structural toxin subunits is similar to the maturation process of other lantibiotic bacteriocins, but the regulatory activity of each of these subunits in calibrating toxin expression to reflect cell density and the presence of target cells is novel. Further studies of the components of the Cytolysin locus and the target celltoxin interactions are certain to yield more interesting mechanisms and may inform the development of a therapeutic agent to neutralize this virulence factor.
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Enterococcal Cytolysin: A Novel Two Component Peptide System that Serves as a Bacterial Defense Against Eukaryotic and Prokaryotic Cells
Current protein & peptide science, 2005Co-Authors: Christopher R. Cox, Phillip S. Coburn, Michael S. GilmoreAbstract:The Cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The Cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The Cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the Cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the Cytolysin gene cluster. This complex regulation of Cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.
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Enterococcus faecalis Senses Target Cells and in Response Expresses Cytolysin
Science (New York N.Y.), 2004Co-Authors: Phillip S. Coburn, Wolfgang Haas, Chris M. Pillar, Bradley D. Jett, Michael S. GilmoreAbstract:Many virulent strains of Enterococcus faecalis produce a two-subunit toxin, termed Cytolysin. Cytolysin expression is regulated by one of the subunits (CylLS″) through a quorum-sensing autoinduction mechanism. We found that when target cells are absent, the other subunit (CylLL″) forms a complex with CylLS″, blocking it from autoinducing the operon. When target cells are present, however, CylLL″ binds preferentially to the target, allowing free CylLS″ to accumulate above the induction threshold. Thus, enterococci use CylLL″ to actively probe the environment for target cells, and when target cells are detected, allows the organism to express high levels of Cytolysin in response.
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Structure and DNA-binding properties of the Cytolysin regulator CylR2 from Enterococcus faecalis
The EMBO journal, 2004Co-Authors: Sigrun Rumpel, Michael S. Gilmore, Chris M. Pillar, Adelia Razeto, Vinesh Vijayan, Austin Taylor, Karin Giller, Stefan Becker, Markus ZweckstetterAbstract:Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called Cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the Cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix–turn–helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel b-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the Cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the Cytolysin operon transcript.
Hyung-rho Kim - One of the best experts on this subject based on the ideXlab platform.
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A Calcium-Calmodulin Antagonist Blocks Experimental Vibrio vulnificus Cytolysin-Induced Lethality in an Experimental Mouse Model
Infection and immunity, 2004Co-Authors: Young-rae Lee, Hyung-rho Kim, Hye-won Rho, Jin-woo Park, Kwang-hyun Park, Bon-sun Koo, Zhao-zhen Lin, Young-jong Kho, Eun-kyung SongAbstract:We demonstrated that trifluoperazine, a calcium-calmodulin antagonist, blocked the hyperpermeability induced by Vibrio vulnificus Cytolysin in in vitro-modeled endothelium and prevented the deaths of mice. Furthermore, compared to tetracycline alone, tetracycline combined with trifluoperazine enhanced the survival rate of V. vulnificus-infected mice, indicating the role of the Cytolysin as an important factor in pathogenesis.
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Cytotoxic mechanism of vibrio vulnificus Cytolysin in CPAE cells
Life sciences, 2002Co-Authors: Hye-won Rho, Jin-woo Park, Jong-suk Kim, Byung-hyun Park, Min-ji Choi, Ji-na Lee, Hee-sook Sohn, Hyung-rho KimAbstract:Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The Cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus Cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of Cytolysin in vivo. V. vulnificus Cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus Cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus Cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.
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Induction of nitric oxide synthase expression by Vibrio vulnificus Cytolysin.
Biochemical and biophysical research communications, 2002Co-Authors: Mi-kyung Kang, Hyung-rho Kim, Hye-won Rho, Jong-suk Kim, Byung-hyun Park, Eun-chung Jhee, Bon-sun Koo, Jeong-yeh Yang, Jin-woo ParkAbstract:Abstract The pore-forming Cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-γ. Increased NO production was not affected by polymyxin B, and heat inactivation of Cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of Cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy- d -[3H]glucose. At the higher concentrations of Cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFκB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of Cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.
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Species-specific hemolysis by Vibrio vulnificus Cytolysin
Experimental & Molecular Medicine, 1996Co-Authors: Mee‐ree Chae, Jin-woo Park, Hyung-nam Kim, Kwang-hyun Park, Mee-ae Kim, Dong-yun Kim, Hyung-rho KimAbstract:Cytolysin has been incriminated as one of the important virulence determinants in V. vulnificus infection that causes septicemia and serious wound infection. Hemolysis by V. vulnificus Cytolysin is colloid-osmotic in nature, and Cytolysins after binding to membrane oligomerize to form small pores on erythrocyte membrane. The hemolytic sensitivity by V. vulnificus Cytolysin displayed a great variation between the species. Among the species studied, the half lytic doses of Cytolysin for sheep, mouse and human erythrocyte suspensions were 0.40, 0.96 and 2.46 hemolytic units, respectively. There was no significant difference between the species in both the binding and pore formation. Under the hypotonic condition, the stability of erythrocytes from sheep, mouse and human was inversely correlated with the hemolytic susceptibility to the Cytolysin. Our results indicate that species difference in hemolytic susceptibility to V. vulnificus Cytolysin is not dependent on the binding or pore formation, but rather dependent on the unique osmotic stability of erythrocyte membranes, which shows a strong correlation with the size of cells.
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Pulmonary damage by Vibrio vulnificus Cytolysin.
Infection and immunity, 1996Co-Authors: Jin-woo Park, S N, E S Song, C H Song, M R Chae, B H Park, R W Rho, S D Park, Hyung-rho KimAbstract:Vibrio vulnificus is an estuarine bacterium that causes septicemia and serious wound infection. Cytolysin produced by V. vulnificus has been incriminated as one of the important virulence determinants of bacterial infection. Cytolysin (8 hemolytic units) given intravenously to mice via their tail veins caused severe hemoconcentration and lethality. Cytolysin treatment greatly increased pulmonary wet weight and vascular permeability as measured by (125)I-labeled albumin leakage without affecting those factors of other organs significantly. Blood neutrophils were markedly decreased in number after Cytolysin injection, with a concomitant increase in the level of pulmonary myeloperoxidase activity, indicating that Cytolysin-induced neutropenia might be due to pulmonary sequestration of neutrophils. By microscopic examination, severe perivascular edema and neutrophil infiltration were evident in lung tissues. These results suggest that increased vascular permeability and neutrophil sequestration in the lungs are important factors in lethal activity by Cytolysin.
Jong-suk Kim - One of the best experts on this subject based on the ideXlab platform.
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Vibrio vulnificus Cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus Cytolysin.
Experimental & molecular medicine, 2002Co-Authors: Byeong-soo Kim, Jong-suk KimAbstract:Vibrio vulnificus Cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the Cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The Cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentration- and time-dependent manner. The increase of adhesiveness occurred within several minutes after the Cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that Cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the Cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.
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Cytotoxic mechanism of vibrio vulnificus Cytolysin in CPAE cells
Life sciences, 2002Co-Authors: Hye-won Rho, Jin-woo Park, Jong-suk Kim, Byung-hyun Park, Min-ji Choi, Ji-na Lee, Hee-sook Sohn, Hyung-rho KimAbstract:Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The Cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus Cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of Cytolysin in vivo. V. vulnificus Cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus Cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus Cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.
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Induction of nitric oxide synthase expression by Vibrio vulnificus Cytolysin.
Biochemical and biophysical research communications, 2002Co-Authors: Mi-kyung Kang, Hyung-rho Kim, Hye-won Rho, Jong-suk Kim, Byung-hyun Park, Eun-chung Jhee, Bon-sun Koo, Jeong-yeh Yang, Jin-woo ParkAbstract:Abstract The pore-forming Cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-γ. Increased NO production was not affected by polymyxin B, and heat inactivation of Cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of Cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy- d -[3H]glucose. At the higher concentrations of Cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFκB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of Cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.
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Cytotoxicity of Vibrio vulnificus Cytolysin on pulmonary endothelial cells
Experimental & Molecular Medicine, 1997Co-Authors: Jong-suk KimAbstract:The Cytolysin produced by V. vulnificus has been known to cause lethality by increasing pulmonary vascular permeability in mice. In the present study, its cytotoxic mechanism on CPAE cell, a cell line of pulmonary endothelial cell, has been investigated. The Cytolysin rapidly bound on CPAE cells and killed approximately 80% of the cells at 1.0 HU as determined by trypan blue exclusion test. The death of CPAE cells was associated with the formation of transmembrane pore evidenced by rapid flow of monovalent ions in patch clamp of CPAE cell membrane. The Cytolysin decreased cellular ATP levels to less than 30% of control at 0.25 HU. The incubation of Cytolysin in the presence of cholesterol completely inhibited its cytotoxic effect on CPAE cells, suggesting that cholesterol on CPAE cell membrane is the probable binding site for Cytolysin. These results suggest that the death of CPAE cell induced by Cytolysin is due to the depletion of cellular ATP levels by the formation of small transmembrane pores, which are permeable to monovalent ions, but not to LDH.
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Hemolytic mechanism of Cytolysin produced from V. vulnificus
Life sciences, 1993Co-Authors: Hyung-rho Kim, Hye-won Rho, Mi-hee Jeong, Jin-woo Park, Jong-suk Kim, Byung-hyun Park, Uh-hyun Kim, Seok-don ParkAbstract:The characteristics of hemolytic action of Cytolysin produced from V. vulnificus were investigated in mouse erythrocytes. The Cytolysin bound erythrocyte membranes in temperature-independent manner and then lysed cells temperature-dependently. Hemoglobin release by the Cytolysin was completely inhibited by the presence of raffinose or melezitose, but K+ release was not affected. The Cytolysin-induced hemolysis was always accompanied with the conversion of membrane-bound Cytolysin into an oligomer of 210 kDa, corresponding to a tetramer of native Cytolysins. Nonesterified cholesterol inactivated the Cytolysin by converting active monomeric Cytolysin into inactive oligomer. The results suggest that the Cytolysin lyses erythrocytes due to the formation of small pores on erythrocyte membrane by cholesterol-mediated oligomerization of the Cytolysin.
Jin-woo Park - One of the best experts on this subject based on the ideXlab platform.
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A Calcium-Calmodulin Antagonist Blocks Experimental Vibrio vulnificus Cytolysin-Induced Lethality in an Experimental Mouse Model
Infection and immunity, 2004Co-Authors: Young-rae Lee, Hyung-rho Kim, Hye-won Rho, Jin-woo Park, Kwang-hyun Park, Bon-sun Koo, Zhao-zhen Lin, Young-jong Kho, Eun-kyung SongAbstract:We demonstrated that trifluoperazine, a calcium-calmodulin antagonist, blocked the hyperpermeability induced by Vibrio vulnificus Cytolysin in in vitro-modeled endothelium and prevented the deaths of mice. Furthermore, compared to tetracycline alone, tetracycline combined with trifluoperazine enhanced the survival rate of V. vulnificus-infected mice, indicating the role of the Cytolysin as an important factor in pathogenesis.
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Cytotoxic mechanism of vibrio vulnificus Cytolysin in CPAE cells
Life sciences, 2002Co-Authors: Hye-won Rho, Jin-woo Park, Jong-suk Kim, Byung-hyun Park, Min-ji Choi, Ji-na Lee, Hee-sook Sohn, Hyung-rho KimAbstract:Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The Cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus Cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of Cytolysin in vivo. V. vulnificus Cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus Cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus Cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.
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Induction of nitric oxide synthase expression by Vibrio vulnificus Cytolysin.
Biochemical and biophysical research communications, 2002Co-Authors: Mi-kyung Kang, Hyung-rho Kim, Hye-won Rho, Jong-suk Kim, Byung-hyun Park, Eun-chung Jhee, Bon-sun Koo, Jeong-yeh Yang, Jin-woo ParkAbstract:Abstract The pore-forming Cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-γ. Increased NO production was not affected by polymyxin B, and heat inactivation of Cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of Cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy- d -[3H]glucose. At the higher concentrations of Cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFκB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of Cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.
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Species-specific hemolysis by Vibrio vulnificus Cytolysin
Experimental & Molecular Medicine, 1996Co-Authors: Mee‐ree Chae, Jin-woo Park, Hyung-nam Kim, Kwang-hyun Park, Mee-ae Kim, Dong-yun Kim, Hyung-rho KimAbstract:Cytolysin has been incriminated as one of the important virulence determinants in V. vulnificus infection that causes septicemia and serious wound infection. Hemolysis by V. vulnificus Cytolysin is colloid-osmotic in nature, and Cytolysins after binding to membrane oligomerize to form small pores on erythrocyte membrane. The hemolytic sensitivity by V. vulnificus Cytolysin displayed a great variation between the species. Among the species studied, the half lytic doses of Cytolysin for sheep, mouse and human erythrocyte suspensions were 0.40, 0.96 and 2.46 hemolytic units, respectively. There was no significant difference between the species in both the binding and pore formation. Under the hypotonic condition, the stability of erythrocytes from sheep, mouse and human was inversely correlated with the hemolytic susceptibility to the Cytolysin. Our results indicate that species difference in hemolytic susceptibility to V. vulnificus Cytolysin is not dependent on the binding or pore formation, but rather dependent on the unique osmotic stability of erythrocyte membranes, which shows a strong correlation with the size of cells.
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Pulmonary damage by Vibrio vulnificus Cytolysin.
Infection and immunity, 1996Co-Authors: Jin-woo Park, S N, E S Song, C H Song, M R Chae, B H Park, R W Rho, S D Park, Hyung-rho KimAbstract:Vibrio vulnificus is an estuarine bacterium that causes septicemia and serious wound infection. Cytolysin produced by V. vulnificus has been incriminated as one of the important virulence determinants of bacterial infection. Cytolysin (8 hemolytic units) given intravenously to mice via their tail veins caused severe hemoconcentration and lethality. Cytolysin treatment greatly increased pulmonary wet weight and vascular permeability as measured by (125)I-labeled albumin leakage without affecting those factors of other organs significantly. Blood neutrophils were markedly decreased in number after Cytolysin injection, with a concomitant increase in the level of pulmonary myeloperoxidase activity, indicating that Cytolysin-induced neutropenia might be due to pulmonary sequestration of neutrophils. By microscopic examination, severe perivascular edema and neutrophil infiltration were evident in lung tissues. These results suggest that increased vascular permeability and neutrophil sequestration in the lungs are important factors in lethal activity by Cytolysin.
Pierre A. Henkart - One of the best experts on this subject based on the ideXlab platform.
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Synergistic roles of granzymes A and B in mediating target cell death by rat basophilic leukemia mast cell tumors also expressing Cytolysin/perforin.
The Journal of experimental medicine, 1995Co-Authors: Hiroo Nakajima, Heidi L. Park, Pierre A. HenkartAbstract:We have studied the cytotoxic activity of rat basophilic leukemia (RBL) cells transfected with cDNAs for the cytotoxic T lymphocyte (CTL) granule components, Cytolysin (perforin), granzyme A, and granzyme B. With red cell targets, Cytolysin expression conferred potent hemolytic activity, which was not influenced by coexpression of granzymes. With tumor targets, RBL cells expressing Cytolysin alone were weakly cytotoxic, but both cytolytic and nucleolytic activity were enhanced by coexpression of granzyme B. RBL cells expressing all three CTL granule components showed still higher cytotoxic activities, with apoptotic target death. Analysis of the cytotoxic activity of individual transfectant clones showed that cytolytic and nucleolytic activity correlated with granzyme expression but was independent of Cytolysin expression within the range examined. A synergism between granzymes A and B was apparent when the triple transfectant was compared with RBL cells expressing Cytolysin and one granzyme. These data implicate granzymes as the major mediators of tumor target damage by cytotoxic lymphocytes.
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Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both Cytolysin and granzyme A.
Cell, 1992Co-Authors: John W. Shiver, Pierre A. HenkartAbstract:Abstract The noncytotoxic rat mast cell tumor line RBL was transfected with genes for the cytotoxic lymphocyte granule proteins Cytolysin (perforin) and granzyme A, giving transfectants with mRNA and protein expression levels comparable with cloned cytotoxic T lymphocytes. Both RBL-Cytolysin and RBL-Cytolysin-granzyme A transfectants showed extremely potent killing of red cell targets and lysed 20%–60% of EL4 lymphoma targets at an effector-to-target ratio of 30. RBL transfectants expressing only granzyme A were not cytotoxic. Significant EL4 DNA breakdown accompanying lysis was observed only with RBL that was transfected with both Cytolysin and granzyme A. These results support the granule-exocytosis model for lymphocyte cytotoxicity and show that effector granzyme A plays a role in target cell DNA breakdown.
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a noncytotoxic mast cell tumor line exhibits potent ige dependent cytotoxicity after transfection with the Cytolysin perforin gene
Cell, 1991Co-Authors: John W. Shiver, Pierre A. HenkartAbstract:Abstract To test the granule exocytosis model for lymphocyte cytotoxicity, we have expressed the gene for the cytotoxic lymphocyte granule protein Cytolysin (perforin) in the noncytotoxic rat basophilic leukemia (RBL) cell line, which undergoes granule exocytosis when its high affinity IgE receptor is cross-linked. Homogenates of RBL-Cytolysin (RBL-cy) transfectants showed a callum-dependent hemolytic activity in dense granule fractions, demonstrating that the expressed Cytolysin protein was correctly targeted to secretory granules. RBL-cy transfectants showed a potent and calclum-dependent cytolytic activity against IgE-coated RBCs, while the parental RBL line was not cytotoxic under these conditions. RBL-cy cells did not lyse non-IgE-coated RBCs copelleted with targets: this sparing of "innocent bystanders" parallels cytotoxic T lymphocyte lysis and suggests a polarized secretion of Cytolysin. In contrast to RBC targets, IgE-coated tumor cells were much less sensitive to lysis by RBL-cy transfectants.
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Characterization of non-lytic Cytolysin-membrane intermediates
Molecular immunology, 1991Co-Authors: Alexander E. Kuta, Craig W. Reynolds, C.linsey Bashford, Charles A. Pasternak, Pierre A. HenkartAbstract:In order to understand the nature of Cytolysin-membrane interactions, the characteristics of stable, non-lytic Cytolysin-target cell intermediates formed at low ionic strength, neutral pH, and at physiological ionic strength, pH 6.0, were examined. Protease treatment of Cytolysin-RBC intermediates formed at low ionic strength inhibited subsequent hemolysis when the intermediates were exposed to physiological ionic strength and pH. Similarly, when such intermediates were treated with anti-granule and anti-Cytolysin antibodies a significant dose-dependent inhibition of hemolysis was observed. These results suggested that in this non-lytic state the Cytolysin molecule was exposed on the RBC surface. If low ionic strength or pH 6.0 generated intermediates were washed in 0.5 M NaCl, hemolytic activity was greatly reduced and Cytolysin activity could be recovered from the medium. In addition to RBC, both murine (Yac-1 and Lettre ascites) and human (K562) tumor targets formed Cytolysin-target cell intermediates at low ionic strength and at low pH. Multilamellar vesicles composed of either phosphatidylcholine, sphingomyelin or phosphatidylserine inhibited the binding of Cytolysin to RBC at both low ionic strength and pH 6.0 indicating a lack of polar head group specificity for Cytolysin binding.
