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Chapter 18 : Cytolethal Distending Toxin

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Abstract:

This chapter examines the molecular biology and biochemistry of cytolethal distending toxin (CDT) and its molecular mode of action. Transcript analysis of the gene cluster (Hd-cdtABC) indicated that the genes are organized in an operon. The operon encodes three CDT polypeptides, CdtA, CdtB, and CdtC. All three proteins bear apparent signal peptide coding sequences consistent with secretion across the inner membrane by the general export pathway. Biochemical evidence for the requirement of CdtA, CdtB, and CdtC in biological activity has recently been unequivocally demonstrated. Recent findings on the role of CdtB in the mechanism of CDT action, the similarity of CDT action to that of IR, the apparent pathway of CDT internalization, and the reconstitution of biological activity from pure CDT subunits constitute major advances in the understanding of CDT. The apparent cell type specificity for the induction of apoptosis versus cell cycle arrest and the activation of p53 following CDT treatment represent additional parallels between toxin action and radiation treatment. The resolution of this observation will yield not only important information regarding CDT but perhaps also the cellular response to DNA damage in general.

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18

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Cytolethal Distending Toxin
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DNA Synthesis
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Fatty Acid Synthase
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Figures

Image of Figure 1
Figure 1

Effects of CDT treatment on growing cells. (A) Phase-contrast microscopy and (B) cell cycle distribution of Chinese hamster ovary cells treated with Ec-CDT-I. Microscopic examination reveals the dramatic increase in cell size following 48 h of CDT treatment. Control cells were treated with a cell extract from a non-CDT-producing strain. Cell cycle distribution analysis by flow cytometry reveals that CDT cells are completely blocked at the G/M boundary whereas control cells exhibit a normal distribution for cycling cells.

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18
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Image of Figure 2
Figure 2

T7 expression and exclusive radio-labeling of CDT polypeptides. The Ec-CDTII genes were separated by cloning and expressed under a T7 promoter in the presence of rifampicin and S-methionine/cysteine. Total cell extract and supernatant fractions were then prepared and analyzed by SDS-PAGE and autoradiography. The expressed forms of Ec-CDT-II migrated with molecular sizes of approximately 32, 30, and 20 kDa. These results coincide with the calculated molecular sizes for CDT. As expected, CdtA, CdtB, and CdtC also appeared in the supernatant fraction of the cell culture.

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18
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Figure 3

Homology of CdtB to mammalian type I DNase. (A) PSI-BLAST alignment of Ec-CDT-II was performed through the National Center for Biotechnology Information BLAST site (http://www.ncbi.nlm.nih.gov/BLAST/). Iterations were repeated until the sequences converged. Shaded residues correspond to catalytic or metal ion-binding residues. The underlined sequence represents the phosphohydrolases-related motif found in all CdtBs, mammalian type I endonucleases, and sphingomyelinases (B). The CdtBs aligned are designated as shown in Table 1 . Hum. DNaseI, human DNase I; B.c. SMase, sphingomyelinase; S.a. SMase, sphingomyelinase.

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18
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Image of Figure 4
Figure 4

Mitotic checkpoint pathway following DNA damage. DNAdamage following exposure to IR, UV light, or genotoxic chemical is sensed by either ATM or the ATM-related kinase ATR. Once activated, ATM and ATR phosphorylate several substrates that affect growth arrest. Chk2 (Cds1) is phosphorylated by ATM, which in turn phosphorylates the Cdc25 phosphatase on Ser-216. Phosphorylation of Cdc25 on Ser-216 creates a binding site for a 14-3-3 protein that binds to the phosphatase and escorts it to the cytoplasm. Cdc25 is required by the cell to mediate the transition between Gand M phase by activating Cdc2. Cdc2 is dephosphorylated (activated) by Cdc25 in a cycle-regulated manner to allow formation of the active Cdc2/cyclin B complex, thus directing the onset of mitosis. The persistent phosphorylation of Cdc2 following CDT treatment is a result of the Chk2-dependent inactivation of Cdc25 ( ). ATM activation also is responsible for stabilization of the tumor suppressor p53. Both direct phosphorylation of p53 and phosphorylation of MDM2 (a protein that normally escorts p53 to the cytoplasm for degradation) result in a dramatic elevation and activation of p53. Activation of p53 in turn induces expression of p21, a cyclin-dependent kinase inhibitor that blocks activation of Cdc2, thus enforcing the Garrest. In addition, p53 activation results in apoptosis in some cell types (see text and suggested reading for additional background).

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18
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Figure 5

Model for CDT action. According to the current understanding of CDT action, CDT appears to bind to a receptor on the surface of sensitive cells, enters the cell by receptor-mediated endocytosis, and traffics through the cell through early and late endosome, and following involvement of the Golgi, CdtB enters the nucleus. Nuclear CdtB is then responsible for imparting a damaging effect on chromosomal DNA, resulting in growth arrest either by the mitotic checkpoint cascade or DNA damage-mediated apoptosis.

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18
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References

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1. Bates, S.,, and K. H. Vousden. 1999. Mechanisms of p53-mediated apoptosis. Cell. Mol. Life Sci. 55:2837.
2. Cortes-Bratti, X.,, E. Chaves-Olarte,, T. Lagergard,, and M. Thelestam. 2000. Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi. Infect. Immun. 68:69036911.
3. Cortes-Bratti, X.,, T. Frisan,, and M. Thelestam. 2001. The cytolethal distending toxins induce DNA damage and cell cycle arrest. Toxicon 39:17291736.
4. De Rycke, J.,, and E. Oswald. 2001. Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation? FEMS Microbiol. Lett. 203:141148.
5. Elwell, C. A.,, K. Chao,, K. Patel,, and L. A. Dreyfus. 2001. Escherichia coli CdtB mediates the cytolethal distending toxin cell cycle arrest. Infect. Immun. 69:34183422.
6. Elwell, C. A.,, and L. A. Dreyfus. 2000. DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Mol. Microbiol. 37: 952963.
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8. Lara-Tejero, M.,, and J. E. Galan. 2000. A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein. Science 290:354357.
9. Lara-Tejero, M.,, and J. E. Galán. 2001. CdtA, CdtB, and CdtC form a tripartite complex that is required for cytolethal distending toxin activity. Infect. Immun. 69:43584365.
10. Pickett, C. L.,, and C. A. Whitehouse. 1999. The cytolethal distending toxin family. Trends Microbiol. 7:292297.
11. Rotman, G.,, and Y. Shiloh. 1999. ATM: a mediator of multiple responses to genotoxic stress. Oncogene 18:61356144.
12. Shenker, B. J.,, R. H. Hoffmaster,, A. Zekavat,, N. Yamaguchi,, E. T. Lally,, and D. R. Demuth. 2001. Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle. J. Immunol. 167:435441.
13. Smits, V. A.,, and R. H. Medema. 2001. Checking out the G(2)/M transition. Biochim. Biophys. Acta 1519:112.
14. Wilson, J. F. 2002. Elucidating the DNA damage pathway. Scientist 16:3031.
1. Alberts, B.,, A. Johnson,, J. Lewis,, M. Raff,, K. Roberts,, and P. Walter,. 2002. The cell cycle and programmed cell death, p. 9831026. In B. Alberts,, A. Johnson,, J. Lewis,, M. Raff,, K. Roberts,, and P. Walter (ed.), Molecular Biology of the Cell, 4th ed. Garland Science, New York, N.Y.

Tables

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Table 1

CDT-producing bacterial species and CDT genes

Citation: Dreyfus L. 2003. Cytolethal Distending Toxin, p 257-270. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch18

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