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Assembly and function of a bacterial genotoxin

Nature volume 429, pages 429–433 (2004)Cite this article

Abstract

The tripartite cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells1,2. The subunits CdtA and CdtC associate with the nuclease CdtB to form a holotoxin that translocates CdtB into the host cell, where it acts as a genotoxin by creating DNA lesions3,4,5,6,7. Here we show that the crystal structure of the holotoxin from Haemophilus ducreyi reveals that CDT consists of an enzyme of the DNase-I family, bound to two ricin-like lectin domains. CdtA, CdtB and CdtC form a ternary complex with three interdependent molecular interfaces, characterized by globular, as well as extensive non-globular, interactions. The lectin subunits form a deeply grooved, highly aromatic surface that we show to be critical for toxicity. The holotoxin possesses a steric block of the CdtB active site by means of a non-globular extension of the CdtC subunit, and we identify putative DNA binding residues in CdtB that are essential for toxin activity.

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Figure 1: Assembly and features of the heterotrimeric cytolethal distending toxin.
Figure 2: Two non-globular extensions in the holotoxin.
Figure 3: Structure-based mutagenesis of the lectin subunits.
Figure 4: Comparison of CdtB with DNase I and structure-based mutants.

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References

  1. Johnson, W. M. & Lior, H. A new heat-labile cytolethal distending toxin (CLDT) produced by Campylobacter spp. Microb. Pathog. 4, 115–126 (1988)

    Article  CAS  Google Scholar 

  2. Pickett, C. L. & Whitehouse, C. A. The cytolethal distending toxin family. Trends Microbiol. 7, 292–297 (1999)

    Article  CAS  Google Scholar 

  3. De Rycke, J. & Oswald, E. Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation? FEMS Microbiol. Lett. 203, 141–148 (2001)

    Article  CAS  Google Scholar 

  4. Lara-Tejero, M. & Galan, J. E. Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions. Trends Microbiol. 10, 147–152 (2002)

    Article  CAS  Google Scholar 

  5. Lara-Tejero, M. & Galan, J. E. CdtA, CdtB, and CdtC form a tripartite complex that is required for cytolethal distending toxin activity. Infect. Immun. 69, 4358–4365 (2001)

    Article  CAS  Google Scholar 

  6. Cortes-Bratti, X., Chaves-Olarte, E., Lagergard, T. & Thelestam, M. Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi. Infect. Immun. 68, 6903–6911 (2000)

    Article  CAS  Google Scholar 

  7. Frisan, T., Cortes-Bratti, X., Chaves-Olarte, E., Stenerlow, B. & Thelestam, M. The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA. Cell. Microbiol. 5, 695–707 (2003)

    Article  CAS  Google Scholar 

  8. Frisan, T., Cortes-Bratti, X. & Thelestam, M. Cytolethal distending toxins and activation of DNA damage-dependent checkpoint responses. Int. J. Med. Microbiol. 291, 495–499 (2002)

    Article  CAS  Google Scholar 

  9. Elwell, C. A. & Dreyfus, L. A. DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Mol. Microbiol. 37, 952–963 (2000)

    Article  CAS  Google Scholar 

  10. Lara-Tejero, M. & Galan, J. E. A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein. Science 290, 354–357 (2000)

    Article  ADS  CAS  Google Scholar 

  11. Elwell, C., Chao, K., Patel, K. & Dreyfus, L. Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest. Infect. Immun. 69, 3418–3422 (2001)

    Article  CAS  Google Scholar 

  12. Mao, X. & DiRienzo, J. M. Functional studies of the recombinant subunits of a cytolethal distending holotoxin. Cell. Microbiol. 4, 245–255 (2002)

    Article  CAS  Google Scholar 

  13. Cortes-Bratti, X., Frisan, T. & Thelestam, M. The cytolethal distending toxins induce DNA damage and cell cycle arrest. Toxicon 39, 1729–1736 (2001)

    Article  CAS  Google Scholar 

  14. Hassane, D. C., Lee, R. B., Mendenhall, M. D. & Pickett, C. L. Cytolethal distending toxin demonstrates genotoxic activity in a yeast model. Infect. Immun. 69, 5752–5759 (2001)

    Article  CAS  Google Scholar 

  15. Cortes-Bratti, X., Karlsson, C., Lagergard, T., Thelestam, M. & Frisan, T. The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways. J. Biol. Chem. 276, 5296–5302 (2001)

    Article  CAS  Google Scholar 

  16. Alby, F. et al. Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase. FEBS Lett. 491, 261–265 (2001)

    Article  CAS  Google Scholar 

  17. Deng, K., Latimer, J. L., Lewis, D. A. & Hansen, E. J. Investigation of the interaction among the components of the cytolethal distending toxin of Haemophilus ducreyi. Biochem. Biophys. Res. Commun. 285, 609–615 (2001)

    Article  CAS  Google Scholar 

  18. Lewis, D. A. et al. Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems. Infect. Immun. 69, 5626–5634 (2001)

    Article  CAS  Google Scholar 

  19. Comayras, C. et al. Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation. Infect. Immun. 65, 5088–5095 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Escalas, N. et al. Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase. Exp. Cell Res. 257, 206–212 (2000)

    Article  CAS  Google Scholar 

  21. Nishikubo, S. et al. An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus. J. Biol. Chem. 278, 50671–50681 (2003)

    Article  CAS  Google Scholar 

  22. Montfort, W. et al. The three-dimensional structure of ricin at 2.8 A. J. Biol. Chem. 262, 5398–5403 (1987)

    CAS  PubMed  Google Scholar 

  23. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993)

    Article  CAS  Google Scholar 

  24. Lee, R. B., Hassane, D. C., Cottle, D. L. & Pickett, C. L. Interactions of Campylobacter jejuni cytolethal distending toxin subunits CdtA and CdtC with HeLa cells. Infect. Immun. 71, 4883–4890 (2003)

    Article  CAS  Google Scholar 

  25. Deng, K. & Hansen, E. J. A CdtA-CdtC complex can block killing of HeLa cells by Haemophilus ducreyi cytolethal distending toxin. Infect. Immun. 71, 6633–6640 (2003)

    Article  CAS  Google Scholar 

  26. Shenker, B. J. et al. Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt): evidence that the holotoxin is composed of three subunits: CdtA, CdtB, and CdtC. J. Immunol. 172, 410–417 (2004)

    Article  CAS  Google Scholar 

  27. Suck, D., Lahm, A. & Oefner, C. Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I. Nature 332, 464–468 (1988)

    Article  ADS  CAS  Google Scholar 

  28. Weston, S. A., Lahm, A. & Suck, D. X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution. J. Mol. Biol. 226, 1237–1256 (1992)

    Article  CAS  Google Scholar 

  29. Jones, S. J., Worrall, A. F. & Connolly, B. A. Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I. J. Mol. Biol. 264, 1154–1163 (1996)

    Article  CAS  Google Scholar 

  30. Pan, C. Q., Ulmer, J. S., Herzka, A. & Lazarus, R. A. Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence. Protein Sci. 7, 628–636 (1998)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank H. Mueller and T. Radhakannan for access to and assistance with crystallographic equipment, and S. Mazel for access to a flow cytometer. This work was funded by research funds to C.E.S. from the Rockefeller University.Authors' contributions D.N.—cloning of wild-type and mutant CDT holotoxin and CdtB, protein purification, activity assays, and crystallography, and Y.H.—mutant CdtA cloning and purification of mutant CdtA containing holotoxin.

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  1. Laboratory of Structural Microbiology, The Rockefeller University, New York, 10021, USA

    Dragana Nešić, Yun Hsu & C. Erec Stebbins

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  1. Dragana Nešić
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Correspondence to C. Erec Stebbins.

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Supplementary Methods

The crystallographic methods (DOC 33 kb)

Supplementary Table

Summary of crystallographic analysis (PDF 57 kb)

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Nešić, D., Hsu, Y. & Stebbins, C. Assembly and function of a bacterial genotoxin. Nature 429, 429–433 (2004). https://doi.org/10.1038/nature02532

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