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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.

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Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.(A) CHO-A745 cells were intoxicated as in Fig 1 except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H2AX (anti-pH2AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH2AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 104 cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.
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pone.0143977.g002: Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.(A) CHO-A745 cells were intoxicated as in Fig 1 except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H2AX (anti-pH2AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH2AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 104 cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.

Mentions: Although CdtC is not required for CDT-mediated cytotoxicity, this does not preclude a role for CdtC during binding or entry of the CDT holotoxin. Indeed, Damek-Poprawa and colleagues reported that CdtC from Aggregatibacter actinomycetemcomintans colocalizes with CdtB in the endolysosomal network and ER, and thus may influence intracellular trafficking [40]. CDTs from E. coli and H. ducreyi take distinct pathways in CHO-A745 and HeLa cells to traffic from the plasma membrane to the lumen of the ER [18,59]. Specifically, intoxication by Hd-CDT is inhibited by lysosomotropic agents that neutralize late endosome pH and by dominant negative Rab7, indicating that this toxin traffics through a late endosome prior to accessing the Golgi and ER [17,18]. Indeed, Hd-CdtB co-localizes with the late endosomal marker Rab9 [18]. In contrast, intoxication by Ec-CDT is unaltered in the presence of these inhibitors and does not colocalize with Rab9, indicating a direct early endosome to Golgi trafficking pathway [18]. These distinct trafficking pathways can be readily assessed using the small molecule EGA that prevents transport from early to late endosomes [59]. EGA blocks intoxication by Hd-CDT holotoxin, but does not block intoxication by Ec-CDT holotoxin [59](Fig 2A and Tables 1 and 2). To explore potential roles of CdtC in trafficking, we next examined sensitivity of CdtAB and CdtBC dimers to EGA. Consistent with its effect on holotoxin, EGA blocked intoxication by both Hd-CdtAB and Hd-CdtBC (Fig 2A and Tables 1 and 2). Surprisingly, EGA provided a strong block to intoxication by Ec-CdtAB but not to Ec-CDT holotoxin (Fig 2A and Tables 1 and 2). These results reveal that sensitivity or resistance of intoxication to EGA corresponds to the absence or presence of Ec-CdtC respectively, suggesting that Ec-CdtC alters the trafficking of toxin.


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)

Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.(A) CHO-A745 cells were intoxicated as in Fig 1 except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H2AX (anti-pH2AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH2AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 104 cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.
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pone.0143977.g002: Ec-CdtC Dictates Resistance to EGA and Alters Intracellular Trafficking of Ec-CdtB.(A) CHO-A745 cells were intoxicated as in Fig 1 except that all wells were additionally treated with 12.5 μM EGA. (B) CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with 1μM Ec-CDT holotoxin or 1 μM Ec-CdtAB for 4 or 16 h. Phosphorylated H2AX (anti-pH2AX) was measured by laser scanning cytometry as described in Methods. Signal intensity for pH2AX induced by Ec-CDT holotoxin was set at 100% and used to normalize signal from CdtAB for each time point. Graphs represent average values from three independent experiments, each performed at least 3 times. *p value = 0.0121 calculated by unpaired two-tailed t test (Prism 5, GraphPad). (C, D) CHO-A745 cells were seeded at 2 x 104 cells/well on 8-well chambered slides and allowed to adhere overnight. The next day, cells were incubated on ice with 100 μM Ec-CDT holotoxin, Ec-CdtAB or Ec-CdtBC for 30 min, washed and incubated at 37°C for 60 minutes. Cells were then fixed, stained, and imaged as described in Methods [anti-Ec-CdtB (green) and EEA1 or Rab9 antibody (red)]. White scale bars at the left panel of each treatment indicate 10 μm and the right insert panel indicate 2 μm. Quantification of microscopy results was performed using Pearson's coefficient values indicating colocalization of the Ec-CdtB signal with the EEA1 or Rab9 enriched vesicles. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during three independent experiments and error bars represent standard deviations.
Mentions: Although CdtC is not required for CDT-mediated cytotoxicity, this does not preclude a role for CdtC during binding or entry of the CDT holotoxin. Indeed, Damek-Poprawa and colleagues reported that CdtC from Aggregatibacter actinomycetemcomintans colocalizes with CdtB in the endolysosomal network and ER, and thus may influence intracellular trafficking [40]. CDTs from E. coli and H. ducreyi take distinct pathways in CHO-A745 and HeLa cells to traffic from the plasma membrane to the lumen of the ER [18,59]. Specifically, intoxication by Hd-CDT is inhibited by lysosomotropic agents that neutralize late endosome pH and by dominant negative Rab7, indicating that this toxin traffics through a late endosome prior to accessing the Golgi and ER [17,18]. Indeed, Hd-CdtB co-localizes with the late endosomal marker Rab9 [18]. In contrast, intoxication by Ec-CDT is unaltered in the presence of these inhibitors and does not colocalize with Rab9, indicating a direct early endosome to Golgi trafficking pathway [18]. These distinct trafficking pathways can be readily assessed using the small molecule EGA that prevents transport from early to late endosomes [59]. EGA blocks intoxication by Hd-CDT holotoxin, but does not block intoxication by Ec-CDT holotoxin [59](Fig 2A and Tables 1 and 2). To explore potential roles of CdtC in trafficking, we next examined sensitivity of CdtAB and CdtBC dimers to EGA. Consistent with its effect on holotoxin, EGA blocked intoxication by both Hd-CdtAB and Hd-CdtBC (Fig 2A and Tables 1 and 2). Surprisingly, EGA provided a strong block to intoxication by Ec-CdtAB but not to Ec-CDT holotoxin (Fig 2A and Tables 1 and 2). These results reveal that sensitivity or resistance of intoxication to EGA corresponds to the absence or presence of Ec-CdtC respectively, suggesting that Ec-CdtC alters the trafficking of toxin.

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