Induction of cell cycle arrest in lymphocytes by Actinobacillus actinomycetemcomitans cytolethal distending toxin requires three subunits for maximum activity.
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.
Shenker
Bruce J
BJ
Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA. shenker@pobox.dental.upenn.edu
Besack
Dave
D
McKay
Terry
T
Pankoski
Lisa
L
Zekavat
Ali
A
Demuth
Donald R
DR
D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry / Q000276:immunology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D002453:Cell Cycle / Q000276:immunology*; D016923:Cell Death / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D004306:Dose-Response Relationship, Immunologic; D006131:Growth Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D019169:Jurkat Cells; D016131:Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology*; D010455:Peptides / Q000378:metabolism / Q000633:toxicity; D011485:Protein Binding / Q000276:immunology; D011499:Protein Processing, Post-Translational / Q000276:immunology; D021122:Protein Subunits / Q000378:metabolism / Q000633:toxicity*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D006131:Growth Inhibitors; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; C447141:cytolethal distending toxin, Actinobacillus actinomycetemcomitans