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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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Holotoxin Assembly Method Affects Sensitivity to EGA.CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.
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pone.0143977.g004: Holotoxin Assembly Method Affects Sensitivity to EGA.CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.

Mentions: The assembly of CDT heterotrimeric holotoxins is poorly understood. For Ec-CDT, we measured nearly identical dose response curves when CHO-A745 cells were incubated with Ec-CDT comprising purified recombinants forms of CdtA, CdtB, CdtC that were either folded together and further purified by size-exclusion (designated as Ec-holotoxin), or, refolded separately, and then mixed together just prior to addition to the cell monolayers (designated as Ec-CdtABC) (Fig 4, Table 2). As described above, the presence of EGA did not alter the dose response curves of the Ec-holotoxin (Fig 4, Table 2), consistent with the model that cellular cytotoxicity mediated by the toxin assembled from refolding all three subunits together is not dependent on cellular trafficking from early to late endolysosomal compartments. In contrast, for Ec-ABC, the presence of EGA resulted in an approximate 8-fold increase in the LD50 (Fig 4, Table 2). The trend towards EGA-sensitivity by toxin assembled from the mixing of the three pre-folded subunits indicates that the method of Ec-CDT toxin assembly influences the interaction of the toxin with host cells. Based on the partial block to intoxication by CdtABC, it is likely that combining toxin subunits at the time of use results in a mixed population of fully assembled holotoxin and CdtAB dimers that traffic through the host endosomal network via different routes.


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)

Holotoxin Assembly Method Affects Sensitivity to EGA.CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143977.g004: Holotoxin Assembly Method Affects Sensitivity to EGA.CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.
Mentions: The assembly of CDT heterotrimeric holotoxins is poorly understood. For Ec-CDT, we measured nearly identical dose response curves when CHO-A745 cells were incubated with Ec-CDT comprising purified recombinants forms of CdtA, CdtB, CdtC that were either folded together and further purified by size-exclusion (designated as Ec-holotoxin), or, refolded separately, and then mixed together just prior to addition to the cell monolayers (designated as Ec-CdtABC) (Fig 4, Table 2). As described above, the presence of EGA did not alter the dose response curves of the Ec-holotoxin (Fig 4, Table 2), consistent with the model that cellular cytotoxicity mediated by the toxin assembled from refolding all three subunits together is not dependent on cellular trafficking from early to late endolysosomal compartments. In contrast, for Ec-ABC, the presence of EGA resulted in an approximate 8-fold increase in the LD50 (Fig 4, Table 2). The trend towards EGA-sensitivity by toxin assembled from the mixing of the three pre-folded subunits indicates that the method of Ec-CDT toxin assembly influences the interaction of the toxin with host cells. Based on the partial block to intoxication by CdtABC, it is likely that combining toxin subunits at the time of use results in a mixed population of fully assembled holotoxin and CdtAB dimers that traffic through the host endosomal network via different routes.

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