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AB5 Toxin

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Adrienne W Paton – One of the best experts on this subject based on the ideXlab platform.

  • vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
    Infection and Immunity, 2017
    Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W Paton
    Abstract:

    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.

  • EcxAB Is a Founding Member of a New Family of Metalloprotease AB5 Toxins with a Hybrid Cholera-like B Subunit.
    Structure, 2013
    Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Adrienne W Paton, James C Paton, Jamie Rossjohn, Jérôme Le Nours, Travis Clarke Beddoe
    Abstract:

    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.

  • Structural basis of subtilase cytoToxin subAB assembly
    Journal of Biological Chemistry, 2013
    Co-Authors: J. Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Brock P Herdman, E. Byres, S. Troy, Matthew D. Johnson, Travis Clarke Beddoe
    Abstract:

    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.

James C Paton – One of the best experts on this subject based on the ideXlab platform.

  • vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
    Infection and Immunity, 2017
    Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W Paton
    Abstract:

    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.

  • EcxAB Is a Founding Member of a New Family of Metalloprotease AB5 Toxins with a Hybrid Cholera-like B Subunit.
    Structure, 2013
    Co-Authors: Natasha Mayyoke Ng, Dene R Littler, Adrienne W Paton, James C Paton, Jamie Rossjohn, Jérôme Le Nours, Travis Clarke Beddoe
    Abstract:

    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.

  • Structural basis of subtilase cytoToxin subAB assembly
    Journal of Biological Chemistry, 2013
    Co-Authors: J. Le Nours, Adrienne W Paton, James C Paton, Jamie Rossjohn, Brock P Herdman, E. Byres, S. Troy, Matthew D. Johnson, Travis Clarke Beddoe
    Abstract:

    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.

Hui Wang – One of the best experts on this subject based on the ideXlab platform.

  • vacuolation activity and intracellular trafficking of artb the binding subunit of an AB5 Toxin produced by salmonella enterica serovar typhi
    Infection and Immunity, 2017
    Co-Authors: Brock P Herdman, Travis Clarke Beddoe, James C Paton, Hui Wang, Adrienne W Paton
    Abstract:

    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.

  • 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, 2013
    Co-Authors: Hui Wang, Travis Clarke Beddoe, James C Paton, Brock P Herdman, Trisha J Rogers, Adrienne W Paton
    Abstract:

    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.

  • BiP (Grp78): A Target for Escherichia coli Subtilase CytoToxin
    Heat Shock Proteins, 2013
    Co-Authors: Adrienne W Paton, Hui Wang, James C Paton
    Abstract:

    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.