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Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis

Nature Microbiology volume 2, Article number: 17047 (2017) Cite this article

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Abstract

Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids1. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems2. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs3. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Å resolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB5, EccC5, EccD5 and EccE5) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.

During the past decade, it has become apparent that the type VII secretion system (T7SS) is essential for mycobacterial virulence and physiology4. The first T7SS to be identified was the 6 kDa early secretory antigenic target (ESAT-6) protein family secretion (ESX)-1 system of the human pathogen Mycobacterium tuberculosis5–7. Four other paralogous and highly conserved ESX systems have since been described in mycobacteria, and are termed ESX-2 to ESX-5. Each ESX system has a defined role within the bacterial cell that is mediated by a diverse repertoire of secreted effector proteins, with each system exclusively secreting its cognate effectors4. Intriguingly, unlike most bacterial secretion systems, such as the type III secretion system (T3SS), the ESX systems mediate the export of folded substrates across the diderm mycobacterial cell envelope, indicating a novel mode of protein transport.

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Figure 1: Functional and structural characterization of the M. xenopi ESX-5 complex.
Figure 2: Three-dimensional reconstruction of the ESX-5 complex.
Figure 3: Topology and localization of the individual components of the ESX-5 complex.
Figure 4: Unique features of the mycobacterial T7SS in comparison to other bacterial secretion systems.

References

  1. Hoffmann, C., Leis, A., Niederweis, M., Plitzko, J. M. & Engelhardt, H. Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc. Natl Acad. Sci. USA 105, 3963–3967 (2008).

    Article  CAS  Google Scholar 

  2. Houben, E. N. G., Korotkov, K. V. & Bitter, W. Take five—type VII secretion systems of mycobacteria. Biochim. Biophys. Acta 1843, 1707–1716 (2014).

    Article  CAS  Google Scholar 

  3. Bitter, W. & Kuijl, C. Targeting bacterial virulence. The coming out of type VII secretion inhibitors. Cell Host Microbe 16, 430–432 (2014).

    Article  CAS  Google Scholar 

  4. Gröschel, M. I., Sayes, F., Simeone, R., Majlessi, L. & Brosch, R. ESX secretion systems mycobacterial evolution to counter host immunity. Nat. Rev. Microbiol. 14, 677–691 (2016).

    Article  Google Scholar 

  5. Pym, A. S., Brodin, P., Brosch, R., Huerre, M. & Cole, S. T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46, 709–717 (2002).

    Article  CAS  Google Scholar 

  6. Stanley, S. A., Raghavan, S., Hwang, W. W. & Cox, J. S. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl Acad. Sci. USA 100, 13001–13006 (2003).

    Article  CAS  Google Scholar 

  7. Hsu, T. et al. The primary mechanism of attenuation of bacillus Calmette-Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl Acad. Sci. USA 100, 12420–12425 (2003).

    Article  CAS  Google Scholar 

  8. Ates, L. S. et al. Essential role of the ESX-5 secretion system in outer membrane permeability of pathogenic mycobacteria. PLoS Genet. 11, e1005190 (2015).

    Article  Google Scholar 

  9. Fishbein, S., van Wyk, N., Warren, R. M. & Sampson, S. L. Phylogeny to function PE/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity. Mol. Microbiol. 96, 901–916 (2015).

    Article  CAS  Google Scholar 

  10. Houben, E. N. G. et al. Composition of the type VII secretion system membrane complex. Mol. Microbiol. 86, 472–484 (2012).

    Article  CAS  Google Scholar 

  11. Sani, M. et al. Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins. PLoS Pathog. 6, e1000794 (2010).

    Article  Google Scholar 

  12. Rosenberg, O. S. et al. Substrates control multimerization and activation of the multi-domain ATPase motor of type VII secretion. Cell 161, 501–512 (2015).

    Article  CAS  Google Scholar 

  13. Daleke, M. H. et al. General secretion signal for the mycobacterial type VII secretion pathway. Proc. Natl Acad. Sci. USA 109, 11342–11347 (2012).

    Article  CAS  Google Scholar 

  14. Korotkova, N. et al. Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25–PPE41 dimer. Mol. Microbiol. 94, 367–382 (2014).

    Article  CAS  Google Scholar 

  15. Houben, E. N. G. et al. Composition of the type VII secretion system membrane complex. Mol. Microbiol. 86, 472–484 (2012).

    Article  CAS  Google Scholar 

  16. Poulsen, C., Panjikar, S., Holton, S. J., Wilmanns, M. & Song, Y.-H. WXG100 protein superfamily consists of three subfamilies and exhibits an α-helical C-terminal conserved residue pattern. PLoS ONE 9, e89313 (2014).

    Article  Google Scholar 

  17. Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinform. 47, 5.6.1–5.6.32 (2014).

    Article  Google Scholar 

  18. Zhang, X.-L. et al. Core component EccB1 of the Mycobacterium tuberculosis type VII secretion system is a periplasmic ATPase. FASEB J. 29, 4804–4814 (2015).

    Article  CAS  Google Scholar 

  19. Pintilie, G. D., Zhang, J., Goddard, T. D., Chiu, W. & Gossard, D. C. Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J. Struct. Biol. 170, 427–438 (2010).

    Article  CAS  Google Scholar 

  20. Bitter, W. et al. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog. 5, e1000507 (2009).

    Article  Google Scholar 

  21. Lou, Y., Rybniker, J., Sala, C. & Cole, S. T. EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX-1 secretion. Mol. Microbiol. 103, 1–39 (2016).

    Google Scholar 

  22. Peña, A. et al. The hexameric structure of a conjugative VirB4 protein ATPase provides new insights for a functional and phylogenetic relationship with DNA translocases. J. Biol. Chem. 287, 39925–39932 (2012).

    Article  Google Scholar 

  23. van Winden, V. J. C. et al. Mycosins are required for the stabilization of the ESX-1 and ESX-5 type VII secretion membrane complexes. mBio 7, e01471–16 (2016).

    Article  CAS  Google Scholar 

  24. Labidi, A., Mardis, E., Roe, B. A. & Wallace, R. J. Cloning and DNA sequence of the Mycobacterium fortuitum var fortuitum plasmid pAL5000. Plasmid 27, 130–140 (1992).

    Article  CAS  Google Scholar 

  25. Zhang, Y., Werling, U. & Edelmann, W. SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res. 40, e55 (2012).

    Article  CAS  Google Scholar 

  26. Noens, E. E. et al. Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain. BMC Microbiol. 11, 27 (2011).

    Article  CAS  Google Scholar 

  27. Hughes, C. S. et al. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol. 10, 757 (2014).

    Article  Google Scholar 

  28. Savitski, M. M. et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346, 1255784 (2014).

    Article  Google Scholar 

  29. Cox, J. & Mann, M. Maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  30. Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    Article  Google Scholar 

  31. Schneidman-Duhovny, D., Inbar, Y., Nussinov, R. & Wolfson, H. J. Patchdock and SymmDock servers for rigid and symmetric docking. Nucleic Acids Res. 33, W363–W367 (2005).

    Article  CAS  Google Scholar 

  32. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  33. Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003).

    Article  CAS  Google Scholar 

  34. Svergun, D. I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).

    Article  CAS  Google Scholar 

  35. Tria, G., Mertens, H. D. T., Kachala, M. & Svergun, D. I. Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering. IUCrJ. 2, 207–217 (2015).

    Article  CAS  Google Scholar 

  36. Bernado, P., Mylonas, E., Petoukhov, M. V., Blackledge, M. & Svergun, D. I. Structural characterization of flexible proteins using small-angle X-ray scattering. J. Am. Chem. Soc. 129, 5656–5664 (2007).

    Article  CAS  Google Scholar 

  37. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  Google Scholar 

  38. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  Google Scholar 

  39. Du, D. et al. Structure of the AcrAB-TolC multidrug efflux pump. Nature 509, 512–515 (2014).

    Article  CAS  Google Scholar 

  40. Schraidt, O. & Marlovits, T. C. Three-dimensional model of Salmonella's needle complex at subnanometer resolution. Science 331, 1192–1195 (2011).

    Article  CAS  Google Scholar 

  41. Low, H. H. et al. Structure of a type IV secretion system. Nature 508, 550–553 (2014).

    Article  CAS  Google Scholar 

  42. Durand, E. et al. Biogenesis and structure of a type VI secretion membrane core complex. Nature 523, 555–560 (2015).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was generously supported through the Behörde für Wissenschaft, Forschung und Gleichstellung of the city of Hamburg (to T.C.M.) and funded by a VIDI grant from the Netherlands Organization of Scientific Research (NWO) (to E.N.G.H. and C.M.B.). K.S.H.B. was funded by a postdoctoral fellowship from the EMBL Interdisciplinary Postdoc Program (EIPOD) under Marie Curie COFUND actions. The authors thank the EMBL P12 beamline and synchrotron facilities at PETRA III (DESY, Hamburg). The authors thank M. Jeske for assistance with the ATPase assays (EMBL, Heidelberg), S. Bhushan (Nanyang Technological University, Singapore) for initial EM analysis of ESX-5 particles and F. Schluenzen for DESY high-performance cluster support. Samples were recorded at the EM facility of the Vienna Biocenter Core Facilities GmbH (VBCF), Austria. The authors thank S.J. Ludtke (Baylor College of Medicine) for comments on resolution estimation and A. Jakobi and C. Sachse (EMBL, Heidelberg) for providing feedback on the manuscript.

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Author notes

  1. Katherine S. H. Beckham, Luciano Ciccarelli and Catalin M. Bunduc: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Hamburg Unit, Notkestraße 85, 22607 Hamburg, Germany

    Katherine S. H. Beckham, Haydyn D. T. Mertens, Dmitri I. Svergun, Matthias Wilmanns & Annabel H. A. Parret

  2. Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria

    Luciano Ciccarelli, Wolfgang Lugmayr, Julia Mayr & Thomas C. Marlovits

  3. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria

    Luciano Ciccarelli, Wolfgang Lugmayr, Julia Mayr & Thomas C. Marlovits

  4. University Medical Centre Hamburg-Eppendorf (UKE), Martinistraße 52, 20246 Hamburg, Germany

    Luciano Ciccarelli, Wolfgang Lugmayr, Julia Mayr, Matthias Wilmanns & Thomas C. Marlovits

  5. Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands

    Catalin M. Bunduc, Wilbert Bitter & Edith N. G. Houben

  6. VU University Medical Center, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands

    Roy Ummels & Wilbert Bitter

  7. European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany

    Mandy Rettel & Mikhail M. Savitski

  8. Centre for Structural Systems Biology (CSSB), Notkestraße 85, 22607 Hamburg, Germany

    Matthias Wilmanns & Thomas C. Marlovits

  9. Deutsches Elektronen-Synchrotron (DESY), Notkestraße 85, 22607 Hamburg, Germany

    Thomas C. Marlovits

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Contributions

A.H.A.P., E.N.G.H., W.B., T.C.M. and M.W. supervised and supported the project. K.S.H.B., A.H.A.P., E.N.G.H., C.M.B. and L.C. designed experiments. E.N.G.H., R.U., C.M.B. and K.S.H.B. generated the constructs. K.S.H.B., C.M.B. and E.N.G.H. purified the ESX-5 complexes for EM analysis. K.S.H.B. performed gold labelling and ATPase assays. C.M.B. performed secretion analysis and disulfide bond characterization. L.C. and J.M. performed electron microscopy. L.C. collected and processed the negative stain electron microscopy data together with W.L. K.S.H.B. and H.D.T.M. collected and analysed SAXS data with the support of D.I.S. M.M.S. and M.R. conducted the mass-spectrometry analysis. K.S.H.B., A.H.A.P., E.N.G.H., C.M.B., W.B., T.C.M., L.C. and M.W. wrote the paper.

Corresponding authors

Correspondence to Thomas C. Marlovits, Annabel H. A. Parret or Edith N. G. Houben.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References. (PDF 3399 kb)

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Beckham, K., Ciccarelli, L., Bunduc, C. et al. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nat Microbiol 2, 17047 (2017). https://doi.org/10.1038/nmicrobiol.2017.47

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