The Experts below are selected from a list of 162 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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vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
Infection and Immunity, 2017Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W PatonAbstract:ABSTRACT Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans -Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 Toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.
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EcxAB Is a Founding Member of a New Family of Metalloprotease AB5 Toxins with a Hybrid Cholera-like B Subunit.
Structure, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Adrienne W Paton, James C Paton, Jamie Rossjohn, Jérôme Le Nours, Travis Clarke BeddoeAbstract:AB5 Toxins are composed of an enzymatic A subunit that disrupts cellular function associated with a pentameric B subunit required for host cell invasion. EcxAB is an AB5 Toxin isolated from clinical strains of Escherichia coli classified as part of the cholera family due to B subunit homology. Cholera-group Toxins have catalytic ADP-ribosyltransferases as their A subunits, so it was surprising that EcxA did not. We confirmed that EcxAB self-associates as a functional Toxin and obtained its structure. EcxAB is a prototypical member of a hybrid AB5 Toxin family containing metzincin-type metalloproteases as their active A subunit paired to a cholera-like B subunit. Furthermore, EcxA is distinct from previously characterized proteases and thus founds an AB5-associated metzincin family that we term the toxilysins. EcxAB provides the first observation of conserved B subunit usage across different AB5 Toxin families and provides evidence that the intersubunit interface of these Toxins is far more permissive than previously supposed.
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Structural basis of subtilase cytoToxin subAB assembly
Journal of Biological Chemistry, 2013Co-Authors: J. Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Brock P Herdman, E. Byres, S. Troy, 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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cloning expression purification and preliminary x ray diffraction studies of a novel AB5 Toxin
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Jerome Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Travis Clarke BeddoeAbstract:AB5 Toxins are key virulence factors found in a range of pathogenic bacteria. AB5 Toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 Toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 Toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 Toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.
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Cloning, expression, purification and preliminary X‐ray diffraction studies of a novel AB5 Toxin
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Jerome Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Travis Clarke BeddoeAbstract:AB5 Toxins are key virulence factors found in a range of pathogenic bacteria. AB5 Toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 Toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 Toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 Toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.
James C Paton - One of the best experts on this subject based on the ideXlab platform.
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vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
Infection and Immunity, 2017Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W PatonAbstract:ABSTRACT Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans -Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 Toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.
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EcxAB Is a Founding Member of a New Family of Metalloprotease AB5 Toxins with a Hybrid Cholera-like B Subunit.
Structure, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Adrienne W Paton, James C Paton, Jamie Rossjohn, Jérôme Le Nours, Travis Clarke BeddoeAbstract:AB5 Toxins are composed of an enzymatic A subunit that disrupts cellular function associated with a pentameric B subunit required for host cell invasion. EcxAB is an AB5 Toxin isolated from clinical strains of Escherichia coli classified as part of the cholera family due to B subunit homology. Cholera-group Toxins have catalytic ADP-ribosyltransferases as their A subunits, so it was surprising that EcxA did not. We confirmed that EcxAB self-associates as a functional Toxin and obtained its structure. EcxAB is a prototypical member of a hybrid AB5 Toxin family containing metzincin-type metalloproteases as their active A subunit paired to a cholera-like B subunit. Furthermore, EcxA is distinct from previously characterized proteases and thus founds an AB5-associated metzincin family that we term the toxilysins. EcxAB provides the first observation of conserved B subunit usage across different AB5 Toxin families and provides evidence that the intersubunit interface of these Toxins is far more permissive than previously supposed.
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Structural basis of subtilase cytoToxin subAB assembly
Journal of Biological Chemistry, 2013Co-Authors: J. Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Brock P Herdman, E. Byres, S. Troy, 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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cloning expression purification and preliminary x ray diffraction studies of a novel AB5 Toxin
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Jerome Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Travis Clarke BeddoeAbstract:AB5 Toxins are key virulence factors found in a range of pathogenic bacteria. AB5 Toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 Toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 Toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 Toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.
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Cloning, expression, purification and preliminary X‐ray diffraction studies of a novel AB5 Toxin
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Jerome Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Travis Clarke BeddoeAbstract:AB5 Toxins are key virulence factors found in a range of pathogenic bacteria. AB5 Toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 Toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 Toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 Toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.
Hui Wang - One of the best experts on this subject based on the ideXlab platform.
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vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
Infection and Immunity, 2017Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W PatonAbstract:ABSTRACT Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans -Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 Toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.
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the b subunit of an AB5 Toxin produced by salmonella enterica serovar typhi up regulates chemokines cytokines and adhesion molecules in human macrophage colonic epithelial and brain microvascular endothelial cell lines
Infection and Immunity, 2013Co-Authors: Hui Wang, James C Paton, Travis Clarke Beddoe, Brock P Herdman, Trisha J Rogers, Adrienne W PatonAbstract:The principal function of bacterial AB5 Toxin B subunits is to interact with glycan receptors on the surfaces of target cells and mediate the internalization of holoToxin. However, B subunit-receptor interactions also have the potential to impact cell signaling pathways and, in so doing, contribute to pathogenesis independently of the catalytic (toxic) A subunits. Various Salmonella enterica serovars, including Salmonella enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. We therefore examined the capacity of the purified B subunit (ArtB) from S. Typhi to elicit cytokine, chemokine, and adhesion molecule responses in human macrophage (U937), colonic epithelial (HCT-8) cell, and brain microvascular endothelial cell (HBMEC) lines. Secretion of the chemokines monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) was increased in all three tested cell lines, with macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and granulocyte colony-stimulating factor (G-CSF) also significantly increased in U937 cells. ArtB also upregulated the cytokines tumor necrosis factor alpha (TNF-α) and IL-6 in HBMECs and HCT-8 cells, but not in U937 cells, while intercellular adhesion molecule 1 (ICAM-1) was upregulated in HCT-8 and U937 cells and vascular cell adhesion molecule 1 (VCAM-1) was upregulated in HBMECs. Thus, ArtB may contribute to pathogenesis independently of the A subunit by promoting and maintaining a strong inflammatory response at the site of infection.
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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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tissue factor dependent procoagulant activity of subtilase cytoToxin a potent AB5 Toxin produced by shiga toxigenic escherichia coli
The Journal of Infectious Diseases, 2010Co-Authors: Hui Wang, James C Paton, Cheleste M Thorpe, Claudine S Bonder, Adrienne W PatonAbstract:Subtilase cytoToxin (SubAB), produced by certain virulent Shiga toxigenic Escherichia coli strains, causes hemolytic uremic syndrome-like pathology in mice, including extensive microvascular thrombosis. SubAB acts by specifically cleaving the essential endoplasmic reticulum chaperone binding immunoglobulin protein (BiP). BiP has been reported to inhibit the activation of tissue factor (TF), the major initiator of extrinsic coagulation. We hypothesized that the apparent prothrombotic effect of SubAB in vivo may involve the stimulation of TF-dependent procoagulant activity. TF-dependent procoagulant activity, TF messenger RNA (mRNA) levels, and BiP cleavage were therefore examined in human macrophage cells and primary human umbilical vein endothelial cells exposed to SubAB. In both types of cells, SubAB significantly increased TF-dependent procoagulant activity, induced TF mRNA expression, and mediated BiP cleavage. No effects were seen when cells were treated with a nonproteolytic mutant Toxin, SubA A272 B. Our results suggest that the procoagulant effect of SubAB may be dependent on both the up-regulation of TF expression and the activation of TF by means of BiP cleavage.
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pathologic changes in mice induced by subtilase cytoToxin a potent new escherichia coli AB5 Toxin that targets the endoplasmic reticulum
The Journal of Infectious Diseases, 2007Co-Authors: Hui Wang, James C Paton, Adrienne W PatonAbstract:Subtilase cytoToxin (SubAB) is the prototype of a recently discovered AB(5) cytoToxin family produced by certain strains of Shiga toxigenic Escherichia coli (STEC). The catalytic A subunit is a highly specific subtilase-like serine protease that cleaves the endoplasmic reticulum chaperone BiP. The Toxin is lethal for mice, but the pathology it induces is poorly understood. Here, we show that intraperitoneal injection of SubAB causes microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment in mice--characteristics typical of Shiga Toxin-induced hemolytic uremic syndrome. SubAB caused extensive microvascular thrombosis and other histologic damage in the brain, kidneys, and liver, as well as dramatic splenic atrophy. Peripheral blood leukocyte levels were increased at 24 h; there was also significant neutrophil infiltration in the liver, kidneys, and spleen and Toxin-induced apoptosis at these sites. These findings raise the possibility that SubAB directly contributes to pathology in humans infected with strains of STEC that produce both Shiga Toxin and SubAB.
Travis Clarke Beddoe - One of the best experts on this subject based on the ideXlab platform.
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vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
Infection and Immunity, 2017Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W PatonAbstract:ABSTRACT Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans -Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 Toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.
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EcxAB Is a Founding Member of a New Family of Metalloprotease AB5 Toxins with a Hybrid Cholera-like B Subunit.
Structure, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Adrienne W Paton, James C Paton, Jamie Rossjohn, Jérôme Le Nours, Travis Clarke BeddoeAbstract:AB5 Toxins are composed of an enzymatic A subunit that disrupts cellular function associated with a pentameric B subunit required for host cell invasion. EcxAB is an AB5 Toxin isolated from clinical strains of Escherichia coli classified as part of the cholera family due to B subunit homology. Cholera-group Toxins have catalytic ADP-ribosyltransferases as their A subunits, so it was surprising that EcxA did not. We confirmed that EcxAB self-associates as a functional Toxin and obtained its structure. EcxAB is a prototypical member of a hybrid AB5 Toxin family containing metzincin-type metalloproteases as their active A subunit paired to a cholera-like B subunit. Furthermore, EcxA is distinct from previously characterized proteases and thus founds an AB5-associated metzincin family that we term the toxilysins. EcxAB provides the first observation of conserved B subunit usage across different AB5 Toxin families and provides evidence that the intersubunit interface of these Toxins is far more permissive than previously supposed.
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Structural basis of subtilase cytoToxin subAB assembly
Journal of Biological Chemistry, 2013Co-Authors: J. Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Brock P Herdman, E. Byres, S. Troy, 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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cloning expression purification and preliminary x ray diffraction studies of a novel AB5 Toxin
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Jerome Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Travis Clarke BeddoeAbstract:AB5 Toxins are key virulence factors found in a range of pathogenic bacteria. AB5 Toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 Toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 Toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 Toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.
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Cloning, expression, purification and preliminary X‐ray diffraction studies of a novel AB5 Toxin
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Jerome Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Travis Clarke BeddoeAbstract:AB5 Toxins are key virulence factors found in a range of pathogenic bacteria. AB5 Toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 Toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 Toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 Toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.
Brock P Herdman - One of the best experts on this subject based on the ideXlab platform.
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vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
Infection and Immunity, 2017Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W PatonAbstract:ABSTRACT Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans -Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 Toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.
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Structural basis of subtilase cytoToxin subAB assembly
Journal of Biological Chemistry, 2013Co-Authors: J. Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Brock P Herdman, E. Byres, S. Troy, 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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the b subunit of an AB5 Toxin produced by salmonella enterica serovar typhi up regulates chemokines cytokines and adhesion molecules in human macrophage colonic epithelial and brain microvascular endothelial cell lines
Infection and Immunity, 2013Co-Authors: Hui Wang, James C Paton, Travis Clarke Beddoe, Brock P Herdman, Trisha J Rogers, Adrienne W PatonAbstract:The principal function of bacterial AB5 Toxin B subunits is to interact with glycan receptors on the surfaces of target cells and mediate the internalization of holoToxin. However, B subunit-receptor interactions also have the potential to impact cell signaling pathways and, in so doing, contribute to pathogenesis independently of the catalytic (toxic) A subunits. Various Salmonella enterica serovars, including Salmonella enterica serovar Typhi, encode an AB5 Toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis Toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holoToxin. We therefore examined the capacity of the purified B subunit (ArtB) from S. Typhi to elicit cytokine, chemokine, and adhesion molecule responses in human macrophage (U937), colonic epithelial (HCT-8) cell, and brain microvascular endothelial cell (HBMEC) lines. Secretion of the chemokines monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) was increased in all three tested cell lines, with macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and granulocyte colony-stimulating factor (G-CSF) also significantly increased in U937 cells. ArtB also upregulated the cytokines tumor necrosis factor alpha (TNF-α) and IL-6 in HBMECs and HCT-8 cells, but not in U937 cells, while intercellular adhesion molecule 1 (ICAM-1) was upregulated in HCT-8 and U937 cells and vascular cell adhesion molecule 1 (VCAM-1) was upregulated in HBMECs. Thus, ArtB may contribute to pathogenesis independently of the A subunit by promoting and maintaining a strong inflammatory response at the site of infection.
