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Induction of Cell Cycle Arrest in Lymphocytes by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Requires Three Subunits for Maximum Activity¹

Bruce J. Shenker^2,*, Dave Besack^*, Terry McKay^*, Lisa Pankoski^*, Ali Zekavat^* and Donald R. Demuth

Departments of ^* Pathology and Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene. In this study, we used rCdt peptides to study the contribution of each subunit to toxin activity. As previously reported, CdtB is the only Cdt subunit that is capable of inducing cell cycle arrest by itself. Although CdtA and CdtC do not exhibit activity alone, each subunit is able to significantly enhance the ability of CdtB to induce G₂ arrest in Jurkat cells; these effects were dependent upon protein concentration. Moreover, the combined addition of both CdtA and CdtC increased the ED₅₀ for CdtB >7000-fold. In another series of experiments, we demonstrate that the three Cdt peptides are able to form a functional toxin unit on the cell surface. However, these interactions first require that a complex forms between the CdtA and CdtC subunits, indicating that these peptides are required for interaction between the cell and the holotoxin. This conclusion is further supported by experiments in which both Jurkat cells and normal human lymphocytes were protected from Cdt holotoxin-induced G₂ arrest by pre-exposure to CdtA and CdtC. Finally, we have used optical biosensor technology to show that CdtA and CdtC have a strong affinity for one another (10^–7 M). Furthermore, although CdtB is unable to bind to either CdtA or CdtC alone, it is capable of forming a stable complex with CdtA/CdtC. The implications of our results with respect to the function and structure of the Cdt holotoxin are discussed.

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