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Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin

Nature Chemical Biology volume 5, pages 469–478 (2009)Cite this article

Abstract

MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-Å structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.

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Figure 1: Identification of MARTXVc CPD autoprocessing inhibitors.
Figure 2: Structure of activated MARTXVc CPD bound to an aza-peptide epoxide inhibitor.
Figure 3: MARTX CPDs cleave after a P1 leucine.
Figure 4: MARTXVc is processed in a CPD-dependent manner.
Figure 5: Identification of MARTXVc toxin cleavage sites in vitro.
Figure 6: Effect of cleavage site mutations on MARTXVc processing and function.
Figure 7: Chemical inhibition of MARTXVc processing and toxin function.

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References

  1. Satchell, K.J. MARTX, multifunctional autoprocessing repeats-in-toxin toxins. Infect. Immun. 75, 5079–5084 (2007).

    Article  CAS  Google Scholar 

  2. Li, L., Rock, J.L. & Nelson, D.R. Identification and characterization of a repeat-in-toxin gene cluster in Vibrio anguillarum. Infect. Immun. 76, 2620–2632 (2008).

    Article  CAS  Google Scholar 

  3. Lee, B.C. et al. Vibrio vulnificus rtxE is important for virulence, and its expression is induced by exposure to host cells. Infect. Immun. 76, 1509–1517 (2008).

    Article  CAS  Google Scholar 

  4. Lee, J.H. et al. Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice. J. Microbiol. 45, 146–152 (2007).

    CAS  PubMed  Google Scholar 

  5. Liu, M., Alice, A.F., Naka, H. & Crosa, J.H. The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus. Infect. Immun. 75, 3282–3289 (2007).

    Article  CAS  Google Scholar 

  6. Olivier, V., Haines III, G.K., Tan, Y. & Satchell, K.J. Hemolysin and the multifunctional autoprocessing RTX toxin are virulence factors during intestinal infection of mice with Vibrio cholerae El Tor O1 strains. Infect. Immun. 75, 5035–5042 (2007).

    Article  CAS  Google Scholar 

  7. Olivier, V., Salzman, N.H. & Satchell, K.J. Prolonged colonization of mice by Vibrio cholerae El Tor O1 depends on accessory toxins. Infect. Immun. 75, 5043–5051 (2007).

    Article  CAS  Google Scholar 

  8. Cordero, C.L., Sozhamannan, S. & Satchell, K.J. RTX toxin actin cross-linking activity in clinical and environmental isolates of Vibrio cholerae. J. Clin. Microbiol. 45, 2289–2292 (2007).

    Article  CAS  Google Scholar 

  9. Rahman, M.H. et al. Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains. DNA Cell Biol. 27, 347–355 (2008).

    Article  CAS  Google Scholar 

  10. Sheahan, K.L., Cordero, C.L. & Satchell, K.J. Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin. Proc. Natl. Acad. Sci. USA 101, 9798–9803 (2004).

    Article  CAS  Google Scholar 

  11. Sheahan, K.L. & Satchell, K.J. Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae. Cell. Microbiol. 9, 1324–1335 (2007).

    Article  CAS  Google Scholar 

  12. Sheahan, K.L., Cordero, C.L. & Satchell, K.J. Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain. EMBO J. 26, 2552–2561 (2007).

    Article  CAS  Google Scholar 

  13. Lupardus, P.J., Shen, A., Bogyo, M. & Garcia, K.C. Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain. Science 322, 265–268 (2008).

    Article  CAS  Google Scholar 

  14. Egerer, M., Giesemann, T., Jank, T., Satchell, K.J. & Aktories, K. Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J. Biol. Chem. 282, 25314–25321 (2007).

    Article  CAS  Google Scholar 

  15. Giesemann, T., Egerer, M., Jank, T. & Aktories, K. Processing of Clostridium difficile toxins. J. Med. Microbiol. 57, 690–696 (2008).

    Article  CAS  Google Scholar 

  16. Reineke, J. et al. Autocatalytic cleavage of Clostridium difficile toxin B. Nature 446, 415–419 (2007).

    Article  CAS  Google Scholar 

  17. Gordon, V.M. & Leppla, S.H. Proteolytic activation of bacterial toxins: role of bacterial and host cell proteases. Infect. Immun. 62, 333–340 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Arastu-Kapur, S. et al. Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum. Nat. Chem. Biol. 4, 203–213 (2008).

    Article  CAS  Google Scholar 

  19. Powers, J.C., Asgian, J.L., Ekici, O.D. & James, K.E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102, 4639–4750 (2002).

    Article  CAS  Google Scholar 

  20. Prochazkova, K. & Satchell, K.J. Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin. J. Biol. Chem. 283, 23656–23664 (2008).

    Article  CAS  Google Scholar 

  21. Asgian, J.L. et al. Aza-peptide epoxides: a new class of inhibitors selective for clan CD cysteine proteases. J. Med. Chem. 45, 4958–4960 (2002).

    Article  CAS  Google Scholar 

  22. Ganesan, R. et al. Exploring the S4 and S1 prime subsite specificities in caspase-3 with aza-peptide epoxide inhibitors. Biochemistry 45, 9059–9067 (2006).

    Article  CAS  Google Scholar 

  23. Barrett, A.J. & Rawlings, N.D. Evolutionary lines of cysteine peptidases. Biol. Chem. 382, 727–733 (2001).

    Article  CAS  Google Scholar 

  24. Bedard, K.M. & Semler, B.L. Regulation of picornavirus gene expression. Microbes Infect. 6, 702–713 (2004).

    Article  CAS  Google Scholar 

  25. Reed, K.E. & Rice, C.M. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr. Top. Microbiol. Immunol. 242, 55–84 (2000).

    CAS  PubMed  Google Scholar 

  26. Schilling, O. & Overall, C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites. Nat. Biotechnol. 26, 685–694 (2008).

    Article  CAS  Google Scholar 

  27. Stennicke, H.R., Renatus, M., Meldal, M. & Salvesen, G.S. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem. J. 350, 563–568 (2000).

    Article  CAS  Google Scholar 

  28. Eichinger, A. et al. Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold. EMBO J. 18, 5453–5462 (1999).

    Article  CAS  Google Scholar 

  29. Kato, D. et al. Activity-based probes that target diverse cysteine protease families. Nat. Chem. Biol. 1, 33–38 (2005).

    Article  CAS  Google Scholar 

  30. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  31. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  32. Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  Google Scholar 

  33. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  34. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  Google Scholar 

  35. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131–1137 (2003).

    Article  Google Scholar 

  36. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).

    Google Scholar 

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Acknowledgements

We thank P. Gulig (University of Florida) for generously providing Vibrio vulnificus CMCP6 genomic DNA, E. Shank and R. Kolter (Harvard Medical School) for providing the Photorhabdus luminescens TTO1 strain, and M. Blokech and G. Schoolnik (Stanford School of Medicine) for help with V. cholerae strain construction and for providing V. cholerae genomic DNA. P.J.L. is supported by the Damon Runyon Cancer Research Foundation. K.C.G. is supported by the Keck Foundation and the Howard Hughes Medical Institute. M.B. is supported by the Burroughs Wellcome Foundation, the Searle Scholars Program, the US National Institutes of Health National Technology Center for Networks and Pathways (grant U54-RR020843) and the Human Frontier Science Program (grant RGP0024/2006-C).

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Authors and Affiliations

  1. Department of Pathology and Howard Hughes Medical Institute, Stanford School of Medicine, Stanford, California, USA

    Aimee Shen, Victoria E Albrow, Andrew Guzzetta & Matthew Bogyo

  2. Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford School of Medicine, Stanford, California, USA

    Patrick J Lupardus & K Christopher Garcia

  3. Department of Chemistry, Georgia Institute of Technology, Atlanta, Georgia, USA

    James C Powers

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Contributions

The inhibitor screen, synthesis of AS01, AS02 and AS04, protein expression and purification, cleavage assays, FT-MS data analysis, V. cholerae strain construction, actin crosslinking assays, MARTXVc silver staining and western blot analyses were performed by A.S. Crystallization of the MARTX CPD–InsP6–JCP598 complex was performed by A.S. and P.J.L. P.J.L. collected the data, solved and analyzed the structure, and generated the figures of the inhibitor-bound CPD structure. J.C.P. provided the cysteine protease compound library. V.E.A. synthesized JCP598, AS01 and VEA223, guided A.S. in the synthesis of AS01, AS02 and AS04, and assessed the integrity of all compounds described in this paper. A.G. designed the conditions for running samples for FT-MS, ran the samples for FT-MS and provided advice in FT-MS analysis. Creative input and financial support for the project were provided by M.B. The manuscript was written by A.S. and M.B. with advice from P.J.L., V.E.A., K.C.G. and A.G.

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Correspondence to Aimee Shen or Matthew Bogyo.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods (PDF 2078 kb)

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Shen, A., Lupardus, P., Albrow, V. et al. Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat Chem Biol 5, 469–478 (2009). https://doi.org/10.1038/nchembio.178

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