The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Adrienne W Paton - One of the best experts on this subject based on the ideXlab platform.
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systemic effects of Subtilase cytotoxin produced by escherichia coli o113 h21
Toxicon, 2017Co-Authors: Abril E Seyahian, Adrienne W Paton, James C. Paton, Gisela Oltra, Federico Ochoa, Santiago Melendi, Ricardo Hermes, Nestor Lago, Mauricio Castro Parodi, Alicia E DamianoAbstract:Abstract Subtilase cytotoxin (SubAB) is a member of the AB 5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli . The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. Importance Uremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.
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differential effects of escherichia coli Subtilase cytotoxin
2014Co-Authors: Hui Wang, James C. Paton, Trisha J Rogers, Adrienne W PatonAbstract:17 Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins 18 produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly 19 specific A subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) 20 chaperone BiP. Our previous in vivo studies have shown that intraperitoneal injection of purified 21 SubAB causes major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as 22 well as profound splenic atrophy. In the current study, we investigated selected chemokine and 23 pro-inflammatory cytokine responses to treatment with SubAB, a non-toxic derivative 24 SubAA272B or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular 25 endothelial (HBMEC) and colonic epithelial (HCT-8) cell lines, at levels of secreted protein, 26 cell-associated protein and gene expression. Stx2 treatment up-regulated expression of 27 chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced 28 significant decreases in secreted IL-8 and MCP-1 in all three tested cell lines and a significant 29 decrease in secreted IL-6 in HBMECs. The down-regulation of secreted chemokines or 30 cytokines was not observed in SubAA272B-treated cells indicating a requirement for BiP cleavage. 31 The down-regulation of secreted chemokines and cytokines by SubAB was not reflected at the 32 mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect. 33 34
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Structural basis of Subtilase cytotoxin subAB assembly
Journal of Biological Chemistry, 2013Co-Authors: Jérôme Le Nours, Brock P Herdman, Eric Byres, Stefanie Troy, Adrienne W Paton, Jamie Rossjohn, James C. Paton, Matthew D. Johnson, Travis Clarke BeddoeAbstract:Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhanceorinhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.
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BiP (Grp78): A Target for Escherichia coli Subtilase Cytotoxin
Heat Shock Proteins, 2013Co-Authors: Adrienne W Paton, Hui Wang, James C. PatonAbstract:Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB5 toxin family produced by certain strains of pathogenic Escherichia coli. Its A subunit is a serine protease belonging to the Peptidase_S8 (Subtilase) family, while the pentameric B subunit binds to cell surface receptor glycans terminating in the sialic acid α2-3-linked N-glycolylneuraminic acid. Receptor binding triggers internalization of the holotoxin and retrograde trafficking to the endoplasmic reticulum (ER), where the A subunit cleaves its only known substrate, the essential Hsp70 family chaperone BiP (also known as GRP78/HSPA5). BiP is a highly conserved protein with diverse functions including the folding of nascent proteins in the ER and maintenance of cellular homeostasis as the master regulator of the ER stress response. BiP is essential for survival of eukaryotes from simple yeasts to higher organisms such as mammals. Consequently, SubAB-mediated BiP cleavage has devastating consequences for the cell, triggering a severe and unresolved ER stress response, ultimately leading to apoptosis. Apart from a likely role in pathogenesis of disease caused by bacteria that produce it, SubAB is also proving to be a useful tool for probing the role of BiP and ER stress in a variety of cellular functions and may have potential as a cancer therapeutic.
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in vivo leukocyte changes induced by escherichia coli Subtilase cytotoxin
Infection and Immunity, 2011Co-Authors: Hui Wang, Adrienne W Paton, Shaun R Mccoll, James C. PatonAbstract:Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga-toxigenic Escherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubAB causes a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramatic splenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes in the peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (including T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peaked at 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistribution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood and peritoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significant increases in the apoptosis rates of CD4(+) T cells, B lymphocytes, and macrophages. These findings indicate that apart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptive arm of the immune system, with potential consequences for disease pathogenesis.
James C. Paton - One of the best experts on this subject based on the ideXlab platform.
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systemic effects of Subtilase cytotoxin produced by escherichia coli o113 h21
Toxicon, 2017Co-Authors: Abril E Seyahian, Adrienne W Paton, James C. Paton, Gisela Oltra, Federico Ochoa, Santiago Melendi, Ricardo Hermes, Nestor Lago, Mauricio Castro Parodi, Alicia E DamianoAbstract:Abstract Subtilase cytotoxin (SubAB) is a member of the AB 5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli . The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. Importance Uremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.
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differential effects of escherichia coli Subtilase cytotoxin
2014Co-Authors: Hui Wang, James C. Paton, Trisha J Rogers, Adrienne W PatonAbstract:17 Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins 18 produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly 19 specific A subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) 20 chaperone BiP. Our previous in vivo studies have shown that intraperitoneal injection of purified 21 SubAB causes major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as 22 well as profound splenic atrophy. In the current study, we investigated selected chemokine and 23 pro-inflammatory cytokine responses to treatment with SubAB, a non-toxic derivative 24 SubAA272B or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular 25 endothelial (HBMEC) and colonic epithelial (HCT-8) cell lines, at levels of secreted protein, 26 cell-associated protein and gene expression. Stx2 treatment up-regulated expression of 27 chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced 28 significant decreases in secreted IL-8 and MCP-1 in all three tested cell lines and a significant 29 decrease in secreted IL-6 in HBMECs. The down-regulation of secreted chemokines or 30 cytokines was not observed in SubAA272B-treated cells indicating a requirement for BiP cleavage. 31 The down-regulation of secreted chemokines and cytokines by SubAB was not reflected at the 32 mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect. 33 34
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Structural basis of Subtilase cytotoxin subAB assembly
Journal of Biological Chemistry, 2013Co-Authors: Jérôme Le Nours, Brock P Herdman, Eric Byres, Stefanie Troy, Adrienne W Paton, Jamie Rossjohn, James C. Paton, Matthew D. Johnson, Travis Clarke BeddoeAbstract:Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhanceorinhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.
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BiP (Grp78): A Target for Escherichia coli Subtilase Cytotoxin
Heat Shock Proteins, 2013Co-Authors: Adrienne W Paton, Hui Wang, James C. PatonAbstract:Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB5 toxin family produced by certain strains of pathogenic Escherichia coli. Its A subunit is a serine protease belonging to the Peptidase_S8 (Subtilase) family, while the pentameric B subunit binds to cell surface receptor glycans terminating in the sialic acid α2-3-linked N-glycolylneuraminic acid. Receptor binding triggers internalization of the holotoxin and retrograde trafficking to the endoplasmic reticulum (ER), where the A subunit cleaves its only known substrate, the essential Hsp70 family chaperone BiP (also known as GRP78/HSPA5). BiP is a highly conserved protein with diverse functions including the folding of nascent proteins in the ER and maintenance of cellular homeostasis as the master regulator of the ER stress response. BiP is essential for survival of eukaryotes from simple yeasts to higher organisms such as mammals. Consequently, SubAB-mediated BiP cleavage has devastating consequences for the cell, triggering a severe and unresolved ER stress response, ultimately leading to apoptosis. Apart from a likely role in pathogenesis of disease caused by bacteria that produce it, SubAB is also proving to be a useful tool for probing the role of BiP and ER stress in a variety of cellular functions and may have potential as a cancer therapeutic.
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in vivo leukocyte changes induced by escherichia coli Subtilase cytotoxin
Infection and Immunity, 2011Co-Authors: Hui Wang, Adrienne W Paton, Shaun R Mccoll, James C. PatonAbstract:Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga-toxigenic Escherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubAB causes a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramatic splenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes in the peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (including T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peaked at 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistribution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood and peritoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significant increases in the apoptosis rates of CD4(+) T cells, B lymphocytes, and macrophages. These findings indicate that apart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptive arm of the immune system, with potential consequences for disease pathogenesis.
Masatoshi Noda - One of the best experts on this subject based on the ideXlab platform.
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fatal hemorrhage induced by Subtilase cytotoxin from shiga toxigenic escherichia coli
Microbial Pathogenesis, 2011Co-Authors: Takeshi Furukawa, Naoko Morinaga, Kinnosuke Yahiro, Joel Moss, Atsushi B Tsuji, Yasuhiro Terasaki, Masaru Miyazaki, Yuh Fukuda, Tsuneo Saga, Masatoshi NodaAbstract:Abstract Subtilase cytotoxin (SubAB) is an AB5 type toxin produced by a subset of Shiga-toxigenic Escherichia coli. The A subunit is a Subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone BiP. The B subunit binds to a receptor on the cell surface. Although SubAB is lethal for mice, the cause of death is not clear. In this study, we demonstrate in mice that SubAB induced small bowel hemorrhage and a coagulopathy characterized by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. SubAB also induced inflammatory changes in the small intestine as detected by 18F-fluoro-2-deoxy- d -glucose positron emission tomography imaging and histochemical analysis. Using RT-PCR and ELISA, SubAB was shown to increase interleukin-6 in a time-dependent manner. Thus, our results indicate that death in SubAB-treated mice may be associated with severe inflammatory response and hemorrhage of the small intestine, accompanied by coagulopathy and IL6 production.
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Subtilase cytotoxin induces apoptosis in hela cells by mitochondrial permeabilization via activation of bax bak independent of c ebf homologue protein chop ire1α or jnk signaling
Microbial Pathogenesis, 2010Co-Authors: Kinnosuke Yahiro, Naoko Morinaga, Joel Moss, Masatoshi NodaAbstract:Subtilase cytotoxin (SubAB) is an AB5 cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a Subtilase-like serine protease and cleaves an endoplasmic reticulum (ER) chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G1 phase, and inducing caspase-dependent apoptosis via mitochondrial membrane damage in Vero cells. Here we investigated the mechanism of mitochondrial permeabilization in HeLa cells. SubAB-induced cytochrome c release into cytosol did not depend on mitochondrial permeability transition pore (PTP), since cyclosporine A did not suppress cytochrome c release. SubAB did not change the expression of anti-apoptotic Bcl-2 or Bcl-XL and pro-apoptotic Bax or Bak, but triggered Bax and Bak conformational changes and association of Bax with Bak. Silencing using siRNA of both bax and bak genes, but not bax, bak, or bim alone, resulted in reduction of cytochrome c release, caspase-3 activation, DNA ladder formation and cytotoxicity, indicating that Bax and Bak were involved in apoptosis. SubAB activated ER transmembrane transducers, Ire1α, and cJun N-terminal kinase (JNK), and induced C/EBF-homologue protein (CHOP). To investigate whether these signals were involved in cytochrome c release by Bax activation, we silenced ire1α, jnk or chop; however, silencing did not decrease SubAB-induced cytochrome c release, suggesting that these signals were not necessary for SubAB-induced mitochondrial permeabilization by Bax activation.
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novel Subtilase cytotoxin produced by shiga toxigenic escherichia coli induces apoptosis in vero cells via mitochondrial membrane damage
Infection and Immunity, 2009Co-Authors: Gen Matsuura, Naoko Morinaga, Kinnosuke Yahiro, Joel Moss, Reiko Komine, Hideo Yoshida, Masatoshi NodaAbstract:Subtilase cytotoxin (SubAB) is an AB5 cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a Subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G1 phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.
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Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1
Cellular microbiology, 2007Co-Authors: Naoko Morinaga, Matsuura, Kinnosuke Yahiro, Joel Moss, Masatoshi NodaAbstract:Subtilase cytotoxin (SubAB) is a AB(5) type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a Subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha). The phosphorylation of PERK and eIF2alpha was maximal at 30-60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.
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Two Distinct Cytotoxic Activities of Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli
Infection and Immunity, 2006Co-Authors: Naoko Morinaga, Matsuura, Kinnosuke Yahiro, Masaharu Watanabe, Fumio Nomura, Joel Moss, Masatoshi NodaAbstract:Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a Subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 μg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was ∼1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a Subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.
Herbert Schmidt - One of the best experts on this subject based on the ideXlab platform.
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variants of escherichia coli Subtilase cytotoxin subunits show differences in complex formation in vitro
Toxins, 2019Co-Authors: Maike Krause, Holger Barth, Katharina Sessler, Anna Kaziales, Richard Grahl, Sabrina Noettger, Herbert SchmidtAbstract:The Subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant Subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 μmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.
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transcription of the Subtilase cytotoxin gene subab1 in shiga toxin producing escherichia coli is dependent on hfq and hns
Applied and Environmental Microbiology, 2019Co-Authors: Laura Heinisch, Maike Krause, Katharina Zoric, Herbert SchmidtAbstract:Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the Subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given on the role of the global regulatory protein genes hfq and hns on subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1 and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phase in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. This data showed that the expression of subAB1, stx2a and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coli. Importance: Shiga toxin-producing Escherichia coli (STEC) are responsible for outbreaks of foodborne diseases such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed among other factors to the production of toxins. Recently, the Subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although, the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wildtype STEC strain TS18/08.
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cytotoxic and apoptotic effects of recombinant Subtilase cytotoxin variants of shiga toxin producing escherichia coli
Infection and Immunity, 2015Co-Authors: Joschua Funk, N Biber, M Schneider, Elisabeth Hauser, Stefanie Enzenmuller, C Fortsch, Holger Barth, Herbert SchmidtAbstract:ABSTRACT In this study, the cytotoxicity of the recently described Subtilase variant SubAB 2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB 1 and the chromosome-encoded SubAB 2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the Subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD 50 ) differ for the individual variants. Highest cytotoxicity was shown for SubAB 1 . Moreover, hybrids of subunits from different Subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA 1 exhibited cytotoxic effects in the absence of the respective B 1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA 1 alone induced apoptosis, while the B 1 subunit alone did not induce cell death.
Andreas Schaller - One of the best experts on this subject based on the ideXlab platform.
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Subtilase activity in the intrusive cells mediates haustorium maturation in parasitic plants
2020Co-Authors: Satoshi Ogawa, Takanori Wakatake, Thomas Spallek, Juliane K. Ishida, Ryosuke Sano, Tetsuya Kurata, Taku Demura, Satoko Yoshida, Yasunori Ichihashi, Andreas SchallerAbstract:ABSTRACT Parasitic plants that infect crops are devastating to agriculture throughout the world. They develop a unique inducible organ called the haustorium, which connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically towards the host vasculature. Then, some of the intrusive cells re-differentiate to form a xylem bridge that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells are unknown. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-Sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and used promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of an SBT inhibitor in the intrusive cells inhibited their development, inhibited the development of the xylem bridge, and reduced auxin response levels near the site where the xylem bridge normally develops. Therefore, we propose that Subtilase activity plays an important role in haustorium development in this parasitic plant. One sentence summary Tissue-specific analysis showed that the Subtilases specifically expressed in intrusive cells regulate auxin-mediated host-parasite connections in the parasitic plant Phtheirospermum japonicum.
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a two way molecular dialogue between embryo and endosperm is required for seed development
Science, 2020Co-Authors: Nicolas M Doll, Andreas Schaller, Annick Stintzi, S Royek, Satoshi Fujita, Satohiro Okuda, Sophy Chamot, Thomas Widiez, Michael Hothorn, Niko GeldnerAbstract:The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination, it protects the seedling from water loss and is, thus, critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the ABNORMAL LEAF SHAPE1 Subtilase and the two GASSHO receptor-like kinases. We show that a sulfated peptide, TWISTED SEED1 (TWS1), acts as a GASSHO ligand. Cuticle surveillance depends on the action of the Subtilase, which, unlike the TWS1 precursor and the GASSHO receptors, is not produced in the embryo but in the neighboring endosperm. Subtilase-mediated processing of the embryo-derived TWS1 precursor releases the active peptide, triggering GASSHO-dependent cuticle reinforcement in the embryo. Thus, a bidirectional molecular dialogue between embryo and endosperm safeguards cuticle integrity before germination.
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a two way molecular dialogue between embryo and endosperm required for seed development
bioRxiv, 2019Co-Authors: Nicolas M Doll, Andreas Schaller, Annick Stintzi, S Royek, Satoshi Fujita, Satohiro Okuda, Thomas Widiez, Michael Hothorn, Niko Geldner, Gwyneth C IngramAbstract:Abstract The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination it protects the seedling from water loss and is thus critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the protease ALE1 and the receptor-like kinases GSO1 and GSO2. We show that a sulfated peptide, TWISTED SEED1 (TWS1) acts as a GSO1/GSO2 ligand. Cuticle surveillance depends on the action of ALE1 which, unlike TWS1 and GSO1/2, is not produced in the embryo but in the neighboring endosperm. Cleavage of an embryo-derived TWS1 precursor by ALE1 releases the active peptide, triggering GSO1/2-dependent cuticle reinforcement in the embryo. A bidirectional molecular dialogue between embryo and endosperm thus safeguards cuticle integrity prior to germination. One Sentence Summary Subtilase-mediated activation of the TWISTED SEED1 peptide provides spatial cues during embryo cuticle integrity monitoring.
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a novel Subtilase inhibitor in plants shows structural and functional similarities to protease propeptides
Journal of Biological Chemistry, 2017Co-Authors: Mathias Hohl, Annick Stintzi, Andreas SchallerAbstract:The propeptides of subtilisin-like serine proteinases (Subtilases, SBTs) serve dual functions as intramolecular chaperones that are required for enzyme folding and as inhibitors of the mature proteases. SBT propeptides are homologous to the I9 family of protease inhibitors that have only been described in fungi. Here we report the identification and characterization of subtilisin propeptide-like inhibitor 1 (SPI-1) from Arabidopsis thaliana. Sequence similarity and the shared β-α-β-β-α-β core structure identified SPI-1 as a member of the I9 inhibitor family and as the first independent I9 inhibitor in higher eukaryotes. SPI-1 was characterized as a high-affinity, tight-binding inhibitor of Arabidopsis Subtilase SBT4.13 with Kd and Ki values in the picomolar range. SPI-1 acted as a stable inhibitor of SBT4.13 over the physiologically relevant range of pH, and its inhibitory profile included many other SBTs from plants but not bovine chymotrypsin or bacterial subtilisin A. Upon binding to SBT4.13, the C-terminal extension of SPI-1 was proteolytically cleaved. The last four amino acids at the newly formed C terminus of SPI-1 matched both the cleavage specificity of SBT4.13 and the consensus sequence of Arabidopsis SBTs at the junction of the propeptide with the catalytic domain. The data suggest that the C terminus of SPI-1 acts as a competitive inhibitor of target proteases as it remains bound to the active site in a product-like manner. SPI-1 thus resembles SBT propeptides with respect to its mode of protease inhibition. However, in contrast to SBT propeptides, SPI-1 could not substitute as a folding assistant for SBT4.13.
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structural basis for ca2 independence and activation by homodimerization of tomato Subtilase 3
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Christian Ottmann, Rolf Rose, Franziska Huttenlocher, Anna Cedzich, Patrick Hauske, Markus Kaiser, Robert Huber, Andreas SchallerAbstract:Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for Subtilases in plant biology. We report the first structure of a plant Subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal seven-stranded jelly-roll fibronectin (Fn) III-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although Ca2+-binding sites are conserved and critical for stability in other Subtilases, SBT3 was found to be Ca2+-free and its thermo stability is Ca2+-independent.
