Abstract

Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.

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Data availability

The data that support the findings in this study are available from the corresponding author on request. Coordinates and structure factors for the ChlaDUB1~Ub, ChlaDUB1~CoA and C.a. ChlaDUB structures have been deposited with the protein data bank, accession codes 6GZS, 6GZT and 6GZU respectively.

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References

  1. 1.

    Lin, Y. H. & Machner, M. P. Exploitation of the host cell ubiquitin machinery by microbial effector proteins. J. Cell Sci. 130, 1985–1996 (2017).

  2. 2.

    Bastidas, R. J. & Valdivia, R. H. Emancipating Chlamydia: advances in the genetic manipulation of a recalcitrant pathogen. Microbiol. Mol. Biol. Rev. 80, 411–427 (2016).

  3. 3.

    Rytkönen, A. et al. SseL, a Salmonella deubiquitinase required for macrophage killing and virulence. Proc. Natl Acad. Sci. USA 104, 3502–3507 (2007).

  4. 4.

    Misaghi, S. et al. Chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection. Mol. Microbiol. 61, 142–150 (2006).

  5. 5.

    Catic, A., Misaghi, S., Korbel, G. A. & Ploegh, H. L. ElaD, a deubiquitinating protease expressed by E. coli. PLoS ONE 2, e381 (2007).

  6. 6.

    Chosed, R. et al. Structural analysis of Xanthomonas XopD provides insights into substrate specificity of ubiquitin-like protein proteases. J. Biol. Chem. 282, 6773–6782 (2007).

  7. 7.

    Mukherjee, S. et al. Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation. Science 312, 1211–1214 (2006).

  8. 8.

    Mittal, R., Peak-Chew, S. Y. & McMahon, H. T. Acetylation of MEK2 and IκB kinase (IKK) activation loop residues by YopJ inhibits signaling. Proc. Natl Acad. Sci. USA 103, 18574–18579 (2006).

  9. 9.

    Jones, R. M. et al. Salmonella AvrA coordinates suppression of host immune and apoptotic defenses via JNK pathway blockade. Cell Host Microbe 3, 233–244 (2008).

  10. 10.

    Sheedlo, M. J. et al. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc. Natl Acad. Sci. USA 112, 15090–15095 (2015).

  11. 11.

    Pruneda, J. N. et al. Molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases. Mol. Cell 63, 261–276 (2016).

  12. 12.

    Corn, J. E. & Vucic, D. Ubiquitin in inflammation: the right linkage makes all the difference. Nat. Struct. Mol. Biol. 21, 297–300 (2014).

  13. 13.

    Le Negrate, G. et al. ChlaDub1 of Chlamydia trachomatis suppresses NF-kappaB activation and inhibitis IkappaBalpha ubiquitination and degradation. Cell. Microbiol. 10, 1879–1892 (2008).

  14. 14.

    Mesquita, F. S. et al. The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. PLoS Pathog. 8, e1002743 (2012).

  15. 15.

    Fischer, A. et al. Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense. eLife 6, e21465 (2017).

  16. 16.

    Zhang, Z. M. et al. Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family. Nat. Struct. Mol. Biol. 23, 847–852 (2016).

  17. 17.

    Mittal, R., Peak-Chew, S. Y., Sade, R. S., Vallis, Y. & McMahon, H. T. The acetyltransferase activity of the bacterial toxin YopJ of Yersinia is activated by eukaryotic host cell inositol hexakisphosphate. J. Biol. Chem. 285, 19927–19934 (2010).

  18. 18.

    Reverter, D. & Lima, C. D. A basis for SUMO protease specificity provided by analysis of human senp2 and a senp2-SUMO complex. Structure 12, 1519–1531 (2004).

  19. 19.

    Reverter, D. et al. Structure of a complex between NEDD8 and the Ulp/Senp protease family member Den1. J. Mol. Biol. 345, 141–151 (2005).

  20. 20.

    Shen, L. et al. Structural basis of NEDD8 ubiquitin discrimination by the deNEDDylating enzyme NEDP1. EMBO J. 24, 1341–1351 (2005).

  21. 21.

    Fullam, E. et al. Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase. J. Mol. Biol. 375, 178–191 (2008).

  22. 22.

    Sixt, B. S. & Valdivia, R. H. Molecular genetic analysis of Chlamydia species. Annu. Rev. Microbiol. 70, 179–198 (2016).

  23. 23.

    Kokes, M. et al. Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia. Cell Host Microbe 17, 716–725 (2015).

  24. 24.

    Heuer, D. et al. Chlamydia causes fragmentation of the Golgi compartment to ensure reproduction. Nature 457, 731–735 (2009).

  25. 25.

    Dumoux, M. & Hayward, R. D. Membrane contact sites between pathogen-containing compartments and host organelles. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1861, 895–899 (2016).

  26. 26.

    Wang, X., Hybiske, K. & Stephens, R. S. Direct visualization of the expression and localization of chlamydial effector proteins within infected host cells. Pathog. Dis. 76, fty011 (2018).

  27. 27.

    Henderson, B. An overview of protein moonlighting in bacterial infection. Biochem. Soc. Trans. 42, 1720–1727 (2014).

  28. 28.

    Wesolowski, J. et al. Chlamydia hijacks ARF GTPases to coordinate microtubule posttranslational modifications and Golgi complex repositioning. mBio 8, e02280–16 (2017).

  29. 29.

    Rejman Lipinski, A. et al. Rab6 and Rab11 regulate Chlamydia trachomatis development and golgin-84-dependent Golgi fragmentation. PLoS Pathog. 5, e1000615 (2009).

  30. 30.

    Berrow, N. S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007).

  31. 31.

    Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  32. 32.

    Ekkebus, R. et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 135, 2867–2870 (2013).

  33. 33.

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  34. 34.

    Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution?. Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

  35. 35.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  36. 36.

    Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582–601 (2009).

  37. 37.

    Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

  38. 38.

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

  39. 39.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  40. 40.

    Nguyen, B. D. & Valdivia, R. H. Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches. Proc. Natl Acad. Sci. USA 109, 1263–1268 (2012).

  41. 41.

    Chen, Y. S. et al. The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosinephosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling. PLoS Pathog. 10, e1003954 (2014).

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Acknowledgements

We thank members of our laboratories for reagents and advice, particularly Lee Dolat (Duke University) for his contribution to some preliminary Chlamydia infection work. Access to DLS was supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (contract no. 283570). Work in the D.K. lab was funded by the Medical Research Council (grant no. U105192732), the European Research Council (grant no. 724804), and the Lister Institute for Preventive Medicine. J.N.P. was supported on an EMBO Long-Term Fellowship. Work in the R.H.V. lab was funded by the National Institute of Health (grant no. R01AI100759 to R.H.V.) and the National Institute of Allergy and Infectious Diseases (grant no. STI CRC U19 AI084044 to R.J.B. and R.H.V.). E.B. was supported by North West Cancer Research. B.S. was supported by the Medical Research Council.

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Author notes

  1. These authors contributed equally: Robert J. Bastidas, Erithelgi Bertsoulaki.

Affiliations

  1. Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, UK

    • Jonathan N. Pruneda
    • , Kirby N. Swatek
    •  & David Komander
  2. Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA

    • Robert J. Bastidas
    •  & Raphael H. Valdivia
  3. Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK

    • Erithelgi Bertsoulaki
    • , Michael J. Clague
    •  & Sylvie Urbé
  4. Division of Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK

    • Balaji Santhanam

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Contributions

Conceptualization was by J.N.P. and D.K. The investigation was carried out by J.N.P., R.J.B., E.B., K.N.S. and B.S. The methodology was done by R.J.B., R.H.V., M.J.C. and S.U. The writing was by J.N.P. and D.K. Funding acquisition was by D.K., R.H.V., R.J.B., S.U. and M.J.C.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to David Komander.

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DOI

https://doi.org/10.1038/s41564-018-0271-y