Limits...
Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Dixon SD, Huynh MM, Tamilselvam B, Spiegelman LM, Son SB, Eshraghi A, Blanke SR, Bradley KA - PLoS ONE (2015)

Bottom Line: In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type.Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response.In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Show MeSH

Related in: MedlinePlus

Intoxication Mediated by CdtA and CdtC Subunits.Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664275&req=5

pone.0143977.g001: Intoxication Mediated by CdtA and CdtC Subunits.Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).

Mentions: Surprisingly, the dose response curve and corresponding cellular LD50 values obtained for Ec-CdtAB and Hd-CdtAB heterodimers were similar to those of their respective holotoxins for all three cell lines tested (Fig 1 and Tables 1 and 2). Further, the LD50 value for Hd-CdtBC heterodimer was similar to that of the Hd-CDT holotoxin on Jurkat cells. In contrast, the CdtBC heterodimers derived from both H. ducreyi and E. coli were significantly less potent on HeLa and CHO-A745 cells than their cognate holotoxins (Fig 1 and Tables 1 and 2). Treatment of cells with CdtB in the absence of either CdtA or CdtC did not result in toxicity (data not shown). These data suggest that CdtA and CdtC can each independently support intoxication by the catalytic subunit CdtB, though CdtA is more efficient on the target cell types tested here while the ability of CdtC to support intoxication depends on the source of the toxin as well as on target cell. Further, these data demonstrate that CdtC is not required for cell killing by either Ec- or Hd-CDT in the three cell lines tested here.


Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Dixon SD, Huynh MM, Tamilselvam B, Spiegelman LM, Son SB, Eshraghi A, Blanke SR, Bradley KA - PLoS ONE (2015)

Intoxication Mediated by CdtA and CdtC Subunits.Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664275&req=5

pone.0143977.g001: Intoxication Mediated by CdtA and CdtC Subunits.Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).
Mentions: Surprisingly, the dose response curve and corresponding cellular LD50 values obtained for Ec-CdtAB and Hd-CdtAB heterodimers were similar to those of their respective holotoxins for all three cell lines tested (Fig 1 and Tables 1 and 2). Further, the LD50 value for Hd-CdtBC heterodimer was similar to that of the Hd-CDT holotoxin on Jurkat cells. In contrast, the CdtBC heterodimers derived from both H. ducreyi and E. coli were significantly less potent on HeLa and CHO-A745 cells than their cognate holotoxins (Fig 1 and Tables 1 and 2). Treatment of cells with CdtB in the absence of either CdtA or CdtC did not result in toxicity (data not shown). These data suggest that CdtA and CdtC can each independently support intoxication by the catalytic subunit CdtB, though CdtA is more efficient on the target cell types tested here while the ability of CdtC to support intoxication depends on the source of the toxin as well as on target cell. Further, these data demonstrate that CdtC is not required for cell killing by either Ec- or Hd-CDT in the three cell lines tested here.

Bottom Line: In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type.Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response.In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Show MeSH
Related in: MedlinePlus