The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Bruce J Shenker - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle regulatory protein p21cip1 waf1 is required for Cytolethal Distending Toxin cdt induced apoptosis
Pathogenetics, 2020Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa M Walker, Robert H Weiss, Kathleen BoeszebattagliaAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked Toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo Toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon Toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.
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the Cytolethal Distending Toxin contributes to microbial virulence and disease pathogenesis by acting as a tri perditious Toxin
Frontiers in Cellular and Infection Microbiology, 2016Co-Authors: Monika Damek Scuron, Kathleen Boeszebattaglia, Mensur Dlakic, Bruce J ShenkerAbstract:This review summarizes the current status and recent advances in our understanding of the role that the Cytolethal Distending Toxin (Cdt) plays as a virulence factor in promoting disease by Toxin-producing pathogens. A major focus of this review is on the relationship between structure and function of the individual subunits that comprise the AB2 Cdt holoToxin. In particular, we concentrate on the molecular mechanisms that characterize this Toxin and which account for the ability of Cdt to intoxicate multiple cell types by utilizing a ubiquitous binding partner on the cell membrane. Furthermore, we propose a paradigm shift for the molecular mode of action by which the active Cdt subunit, CdtB, is able to block a key signaling cascade and thereby lead to outcomes based upon programming and the role of the phosphatidylinositol 3-kinase (PI-3K) in a variety of cells. Based upon the collective Cdt literature, we now propose that Cdt is a unique and potent virulence factor capable of acting as a tri-perditious Toxin that impairs host defenses by: 1) disrupting epithelial barriers; 2) suppressing acquired immunity; 3) promoting pro-inflammatory responses. Thus Cdt plays a key role in facilitating the early stages of infection and the later stages of disease progression by contributing to persistence and impairing host elimination.
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the toxicity of the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin correlates with its phosphatidylinositol 3 4 5 triphosphate phosphatase activity
Cellular Microbiology, 2016Co-Authors: Bruce J Shenker, Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur DlakicAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the Toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, Toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that Toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunoToxin and induce cell cycle arrest in target cells such as lymphocytes.
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the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin active subunit cdtb contains a cholesterol recognition sequence required for Toxin binding and subunit internalization
Infection and Immunity, 2015Co-Authors: Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur Dlakic, Patrik Nygren, Bruce J ShenkerAbstract:Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as Toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.
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aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin activates the nlrp3 inflammasome in human macrophages leading to the release of proinflammatory cytokines
Infection and Immunity, 2015Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, David M Ojcius, Monika Damek Scuron, Kathleen BoeszebattagliaAbstract:The Cytolethal Distending Toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active Toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the Toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active Toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.
Kathleen Boeszebattaglia - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle regulatory protein p21cip1 waf1 is required for Cytolethal Distending Toxin cdt induced apoptosis
Pathogenetics, 2020Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa M Walker, Robert H Weiss, Kathleen BoeszebattagliaAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked Toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo Toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon Toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.
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the Cytolethal Distending Toxin contributes to microbial virulence and disease pathogenesis by acting as a tri perditious Toxin
Frontiers in Cellular and Infection Microbiology, 2016Co-Authors: Monika Damek Scuron, Kathleen Boeszebattaglia, Mensur Dlakic, Bruce J ShenkerAbstract:This review summarizes the current status and recent advances in our understanding of the role that the Cytolethal Distending Toxin (Cdt) plays as a virulence factor in promoting disease by Toxin-producing pathogens. A major focus of this review is on the relationship between structure and function of the individual subunits that comprise the AB2 Cdt holoToxin. In particular, we concentrate on the molecular mechanisms that characterize this Toxin and which account for the ability of Cdt to intoxicate multiple cell types by utilizing a ubiquitous binding partner on the cell membrane. Furthermore, we propose a paradigm shift for the molecular mode of action by which the active Cdt subunit, CdtB, is able to block a key signaling cascade and thereby lead to outcomes based upon programming and the role of the phosphatidylinositol 3-kinase (PI-3K) in a variety of cells. Based upon the collective Cdt literature, we now propose that Cdt is a unique and potent virulence factor capable of acting as a tri-perditious Toxin that impairs host defenses by: 1) disrupting epithelial barriers; 2) suppressing acquired immunity; 3) promoting pro-inflammatory responses. Thus Cdt plays a key role in facilitating the early stages of infection and the later stages of disease progression by contributing to persistence and impairing host elimination.
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the toxicity of the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin correlates with its phosphatidylinositol 3 4 5 triphosphate phosphatase activity
Cellular Microbiology, 2016Co-Authors: Bruce J Shenker, Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur DlakicAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the Toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, Toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that Toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunoToxin and induce cell cycle arrest in target cells such as lymphocytes.
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the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin active subunit cdtb contains a cholesterol recognition sequence required for Toxin binding and subunit internalization
Infection and Immunity, 2015Co-Authors: Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur Dlakic, Patrik Nygren, Bruce J ShenkerAbstract:Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as Toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.
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aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin activates the nlrp3 inflammasome in human macrophages leading to the release of proinflammatory cytokines
Infection and Immunity, 2015Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, David M Ojcius, Monika Damek Scuron, Kathleen BoeszebattagliaAbstract:The Cytolethal Distending Toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active Toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the Toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active Toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.
Ali Zekavat - One of the best experts on this subject based on the ideXlab platform.
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the cell cycle regulatory protein p21cip1 waf1 is required for Cytolethal Distending Toxin cdt induced apoptosis
Pathogenetics, 2020Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa M Walker, Robert H Weiss, Kathleen BoeszebattagliaAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked Toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo Toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon Toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.
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the toxicity of the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin correlates with its phosphatidylinositol 3 4 5 triphosphate phosphatase activity
Cellular Microbiology, 2016Co-Authors: Bruce J Shenker, Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur DlakicAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the Toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, Toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that Toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunoToxin and induce cell cycle arrest in target cells such as lymphocytes.
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the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin active subunit cdtb contains a cholesterol recognition sequence required for Toxin binding and subunit internalization
Infection and Immunity, 2015Co-Authors: Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur Dlakic, Patrik Nygren, Bruce J ShenkerAbstract:Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as Toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.
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aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin activates the nlrp3 inflammasome in human macrophages leading to the release of proinflammatory cytokines
Infection and Immunity, 2015Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, David M Ojcius, Monika Damek Scuron, Kathleen BoeszebattagliaAbstract:The Cytolethal Distending Toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active Toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the Toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active Toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.
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blockade of the pi 3k signalling pathway by the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin induces macrophages to synthesize and secrete pro inflammatory cytokines
Cellular Microbiology, 2014Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, Mensur Dlakic, Kathleen BoeszebattagliaAbstract:Summary The Aggregatibactor actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP-1- and monocyte-derived macrophages were found not to be susceptible to Cdt-induced apoptosis. Nonetheless, the Toxin was capable of binding to macrophages and perturbing PI-3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro-inflammatory cytokine production including increased expression and release of IL-1β, TNFα and IL-6. Furthermore, treatment of cells with either TLR-2, -3 or -4 agonists in the presence of Cdt resulted in an augmented pro-inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt-induced pro-inflammatory cytokine response suggesting a pivotal role for PI-3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms.
Mensur Dlakic - One of the best experts on this subject based on the ideXlab platform.
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the Cytolethal Distending Toxin contributes to microbial virulence and disease pathogenesis by acting as a tri perditious Toxin
Frontiers in Cellular and Infection Microbiology, 2016Co-Authors: Monika Damek Scuron, Kathleen Boeszebattaglia, Mensur Dlakic, Bruce J ShenkerAbstract:This review summarizes the current status and recent advances in our understanding of the role that the Cytolethal Distending Toxin (Cdt) plays as a virulence factor in promoting disease by Toxin-producing pathogens. A major focus of this review is on the relationship between structure and function of the individual subunits that comprise the AB2 Cdt holoToxin. In particular, we concentrate on the molecular mechanisms that characterize this Toxin and which account for the ability of Cdt to intoxicate multiple cell types by utilizing a ubiquitous binding partner on the cell membrane. Furthermore, we propose a paradigm shift for the molecular mode of action by which the active Cdt subunit, CdtB, is able to block a key signaling cascade and thereby lead to outcomes based upon programming and the role of the phosphatidylinositol 3-kinase (PI-3K) in a variety of cells. Based upon the collective Cdt literature, we now propose that Cdt is a unique and potent virulence factor capable of acting as a tri-perditious Toxin that impairs host defenses by: 1) disrupting epithelial barriers; 2) suppressing acquired immunity; 3) promoting pro-inflammatory responses. Thus Cdt plays a key role in facilitating the early stages of infection and the later stages of disease progression by contributing to persistence and impairing host elimination.
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the toxicity of the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin correlates with its phosphatidylinositol 3 4 5 triphosphate phosphatase activity
Cellular Microbiology, 2016Co-Authors: Bruce J Shenker, Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur DlakicAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the Toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, Toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that Toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunoToxin and induce cell cycle arrest in target cells such as lymphocytes.
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the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin active subunit cdtb contains a cholesterol recognition sequence required for Toxin binding and subunit internalization
Infection and Immunity, 2015Co-Authors: Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur Dlakic, Patrik Nygren, Bruce J ShenkerAbstract:Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as Toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.
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blockade of the pi 3k signalling pathway by the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin induces macrophages to synthesize and secrete pro inflammatory cytokines
Cellular Microbiology, 2014Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, Mensur Dlakic, Kathleen BoeszebattagliaAbstract:Summary The Aggregatibactor actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP-1- and monocyte-derived macrophages were found not to be susceptible to Cdt-induced apoptosis. Nonetheless, the Toxin was capable of binding to macrophages and perturbing PI-3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro-inflammatory cytokine production including increased expression and release of IL-1β, TNFα and IL-6. Furthermore, treatment of cells with either TLR-2, -3 or -4 agonists in the presence of Cdt resulted in an augmented pro-inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt-induced pro-inflammatory cytokine response suggesting a pivotal role for PI-3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms.
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a novel mode of action for a microbial derived immunoToxin the Cytolethal Distending Toxin subunit b exhibits phosphatidylinositol 3 4 5 triphosphate phosphatase activity
Journal of Immunology, 2007Co-Authors: Bruce J Shenker, Dave Besack, Lisa P Walker, Mensur Dlakic, Eileen K Jaffe, Ed Labelle, Kathleen BoeszebattagliaAbstract:The Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is a potent immunoToxin that induces G2 arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P3 to PI-3,4-P2 and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G2 arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P3 in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P3 levels were more sensitive to the Toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P3. Finally, reduction of Jurkat cell PI-3,4,5-P3 synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from Toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P3 phosphatase activity, but also that toxicity in lymphocytes is related to this activity.
Lisa P Walker - One of the best experts on this subject based on the ideXlab platform.
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the toxicity of the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin correlates with its phosphatidylinositol 3 4 5 triphosphate phosphatase activity
Cellular Microbiology, 2016Co-Authors: Bruce J Shenker, Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur DlakicAbstract:The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity, leading us to propose that Cdt toxicity is the result of PIP3 depletion and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signalling. To further explore this relationship, we have focused our analysis on identifying residues that comprise the catalytic pocket and are critical to substrate binding rather than catalysis. In this context, we have generated several CdtB mutants and demonstrate that, in each instance, the ability of the Toxin to induce cell cycle arrest correlates with retention of phosphatase activity. We have also assessed the effect of Cdt on downstream components of the PI-3K signalling pathway. In addition to depletion of intracellular concentrations of PIP3, Toxin-treated lymphocytes exhibit decreases in pAkt and pGSK3β. Further analysis indicates that Toxin-treated cells exhibit a concomitant loss in Akt activity and increase in GSK3β kinase activity consistent with observed changes in their phosphorylation status. We demonstrate that cell susceptibility to Cdt is dependent upon dephosphorylation and concomitant activation of GSK3β. Finally, we demonstrate that, in addition to lymphocytes, HeLa cells exposed to a CdtB mutant that retains phosphatase activity and not DNase activity undergo G2 arrest in the absence of H2AX phosphorylation. Our results provide further insight into the mode of action by which Cdt may function as an immunoToxin and induce cell cycle arrest in target cells such as lymphocytes.
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the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin active subunit cdtb contains a cholesterol recognition sequence required for Toxin binding and subunit internalization
Infection and Immunity, 2015Co-Authors: Kathleen Boeszebattaglia, Ali Zekavat, Lisa P Walker, Monika Damek Scuron, Mensur Dlakic, Patrik Nygren, Bruce J ShenkerAbstract:Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as Toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.
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aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin activates the nlrp3 inflammasome in human macrophages leading to the release of proinflammatory cytokines
Infection and Immunity, 2015Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, David M Ojcius, Monika Damek Scuron, Kathleen BoeszebattagliaAbstract:The Cytolethal Distending Toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active Toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the Toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active Toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.
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blockade of the pi 3k signalling pathway by the aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin induces macrophages to synthesize and secrete pro inflammatory cytokines
Cellular Microbiology, 2014Co-Authors: Bruce J Shenker, Ali Zekavat, Lisa P Walker, Mensur Dlakic, Kathleen BoeszebattagliaAbstract:Summary The Aggregatibactor actinomycetemcomitans Cytolethal Distending Toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP-1- and monocyte-derived macrophages were found not to be susceptible to Cdt-induced apoptosis. Nonetheless, the Toxin was capable of binding to macrophages and perturbing PI-3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro-inflammatory cytokine production including increased expression and release of IL-1β, TNFα and IL-6. Furthermore, treatment of cells with either TLR-2, -3 or -4 agonists in the presence of Cdt resulted in an augmented pro-inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt-induced pro-inflammatory cytokine response suggesting a pivotal role for PI-3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms.
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Cytolethal Distending Toxin induced cell cycle arrest of lymphocytes is dependent upon recognition and binding to cholesterol
Journal of Biological Chemistry, 2009Co-Authors: Kathleen Boeszebattaglia, Dave Besack, Ali Zekavat, Angela C Brown, Lisa P Walker, Steve Wrenn, Claude Krummenacher, Bruce J ShenkerAbstract:Induction of cell cycle arrest in lymphocytes after exposure to the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. In this study we further demonstrate that the association of Cdt with lymphocyte plasma membranes is dependent upon binding to cholesterol. Depletion of cholesterol resulted in reduced Toxin binding, whereas repletion of cholesterol-depleted cells restored binding. We employed fluorescence resonance energy transfer and surface plasmon resonance to demonstrate that Toxin association with model membranes is dependent upon the concentration of cholesterol; moreover, these interactions were cholesterol-specific as the Toxin failed to interact with model membranes containing stigmasterol, ergosterol, or lanosterol. Further analysis of the Toxin indicated that the CdtC subunit contains a cholesterol recognition/interaction amino acid consensus (CRAC) region. Mutation of the CRAC site resulted in decreased binding of the holoToxin to cholesterol-containing model membranes as well as to the surface of Jurkat cells. The mutant Toxin also exhibited reduced capacity for intracellular transfer of the active Toxin subunit, CdtB, as well as reduced toxicity. Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for Cdt and that this association can be blocked by either depleting membranes of cholesterol or mutation of the CRAC site.
