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.
Highlights
Cytolethal distending toxins (CDTs) represent an evolutionarily successful family of virulence factors encoded by more than 30 pathogenic γ- and ε-Proteobacteria [1]
To evaluate the relative importance of the CdtA and CdtC subunits in CdtB-mediated cellular intoxication, we first compared the dose-dependent cytotoxicity of CdtAB and CdtB and CdtC (CdtBC) heterodimers in human T-cell (Jurkat), human epithelial (HeLa), and Chinese hamster ovary (CHOpgsA745; “CHO-A745”) cell lines
Cdt subunits were individually overexpressed in E. coli, purified and refolded together at a 1:1:1 molar ratio repurified by size exclusion as previously reported (CDT holotoxin) [18,36], or refolded individually and mixed at a 1:1 ratio at the time of intoxication (CdtAB and CdtBC heterodimers and CdtABC heterotrimer)
Summary
CDTs represent an evolutionarily successful family of virulence factors encoded by more than 30 pathogenic γ- and ε-Proteobacteria [1]. Encoded in a single operon, CDTs form a heterotrimeric “AB2” toxin consisting of CdtA, CdtB, and CdtC subunits [13,14,15]. CdtA and CdtC have been proposed to function together as the two binding “B” moieties of this heterotrimeric AB2 toxin that deliver the active “A” moiety, CdtB, into cells [13,14]. DNase and/or phosphatase activities of CdtB cause the host cell to undergo cell cycle arrest between the G2 and M phase leading to distension and apoptosis [20,21,23,24,25,26,27]. Inhibiting cell cycle and/or induction of apoptosis is predicted to disrupt the normal immune and barrier functions of rapidly dividing eukaryotic cells, including lymphocytes and epithelial cells, providing an advantage to pathogenic bacteria [28,29,30]
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