The Journal of Immunology, 2004, 172: 5813-5814.
Copyright © 2004 by The American Association of Immunologists
The Authors Respond
Bruce J. Shenker* and
Donald Demuth
* Department of Pathology University of Pennsylvania School of Dental Medicine Philadelphia, PA 19104
Department of Periodontology, Endodontics and Dental Hygiene Center for Oral Health and Systemic Disease University of Louisville School of Dentistry Louisville, KY 40292
Drs. Frisan and Thelestam raise several interesting points regarding the mode of action of the cytolethal distending toxin and its putative DNase activity. Clearly, CdtB shares low level homology with a large family of metalloenzyme proteins, including DNases. However, based on this homology data, Dlakic (1) suggested that CdtB might represent a phosphatase rather than a nuclease. The evidence supporting the role of CdtB as a nuclease comes largely from studies reporting DNA degradation in cells intoxicated with CdtB. However, our concerns with these studies arise from experimental conditions that were used to demonstrate DNase activity. Many of these studies exposed yeast and mammalian target cells to levels of CdtB (up to 20 µg/ml) that are significantly higher than the level of toxin that has been shown to be required to induce G2 arrest in lymphocytes (ED50, 40 pg/ml; Ref.2). In addition, the level of DNA degradation observed in experiments where CdtB was overexpressed or injected into target cells was very low. Elwell and Dreyfus (3) reported that supercoiled plasmid was degraded by CdtB, but strangely, linear and relaxed forms of the same plasmid preparation were unaffected. In addition, control samples that were not exposed to CdtB also exhibited DNA degradation. In their subsequent paper (4), no significant DNA degradation was detected at levels of toxin that induce G2 arrest in lymphocytes. Furthermore, many of these studies measure DNA degradation at late time points relative to the onset of G2 arrest. Our results show that DNA fragmentation associated with activation of the apoptotic cascade occurs at late time points subsequent to cell cycle arrest. Therefore, we believe that the identity of CdtB as a DNase, and the causative role of nuclease activity in initiating G2 arrest has yet to be firmly established.
Cortes-Bratti et al. (5) describe an elegant series of experiments that suggest that cytolethal distending toxin is internalized in clathrin-coated pits. However, the toxin preparation used in these studies did not contain CdtA. Reports from several labs have now shown that all three toxin polypeptides are required for activity. Furthermore, preliminary studies suggest that CdtA and CdtC may not be immediately internalized and may perform specific functions at the cell surface that facilitate CdtB activity. The nature of these functions and the mechanism of action of CdtA and CdtC remain to be determined. Therefore, we believe that it is premature to exclude the possibility that cell surface signaling events may play a role in initiating and propagating CdtB activity and events leading to cell cycle arrest.
References
- Dlakic, M.. 2001. Is CdtB a nuclease or phosphatase?. Science 291:547A.
- Shenker, B. J., D. Besack, T. McKay, L. Pankoski, A. Zekavat, D. R. Demuth. 2004. Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt): evidence that the holotoxin is composed of three subunits: CdtA, CdtB, and CdtC. J. Immunol. 172:410.[Abstract/Free Full Text]
- Elwell, C. A., L. A. Dryfus. 2000. DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Molec. Micro. 37:952.
- Elwell, C., K. Chao, K. Patel, L. Dreyfus. 2001. Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest. Infect. Immun. 69:3418.[Abstract/Free Full Text]
- Cortes-Bratti, X., E. Chaves-Olarte, T. Lagergård, M. K. Thelestam. 2000. Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi. Infect. Immun. 68:6903.[Abstract/Free Full Text]
