Abstract

Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine–glycine repeat protein G (VgrG) spike and punctures the prey’s cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)—TssJ, TssM and TssL—and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct–TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.

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Primary accessions

Electron Microscopy Data Bank

Data deposits

The EM structure of the TssJLM complex has been deposited in the Electron Microscopy Data Bank (EMDB) under accession number emd-2927. The crystal structures of the TssM32Ct–nb25 complex, and of the TssM26Ct fragment and TssM26Ct–TssJ complexes, have been deposited in the Protein Data Bank under accession numbers 4Y7M, 4Y7L and 4Y7O, respectively.

References

  1. 1.

    , & Evolutionary explanations for cooperation. Curr. Biol. 17, 661–672 (2007)

  2. 2.

    & Bacterial landlines: contact-dependent signaling in bacterial populations. Curr. Opin. Microbiol. 12, 177–181 (2009)

  3. 3.

    , & Type VI secretion system effectors: poisons with a purpose. Nature Rev. Microbiol. 12, 137–148 (2014)

  4. 4.

    , , & Structure and regulation of the type VI secretion system. Annu. Rev. Microbiol. 66, 453–472 (2012)

  5. 5.

    , , , & Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc. Natl Acad. Sci. USA 104, 15508–15513 (2007)

  6. 6.

    et al. Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475, 343–347 (2011)

  7. 7.

    et al. Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 496, 508–512 (2013)

  8. 8.

    , , , & Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe 16, 94–104 (2014)

  9. 9.

    , , , & Tae, Tle, and beyond: the versatile arsenal of Type VI secretion effectors. Trends Microbiol. 22, 498–507 (2014)

  10. 10.

    , , & Bacterial evolution. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science 347, 63–67 (2015)

  11. 11.

    The type VI secretion toolkit. EMBO Rep. 9, 735–741 (2008)

  12. 12.

    , , , & Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J. 28, 315–325 (2009)

  13. 13.

    , , , & Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182–186 (2012)

  14. 14.

    et al. Structure of the type VI secretion system contractile sheath. Cell 160, 952–962 (2015)

  15. 15.

    The type VI secretion system – a widespread and versatile cell targeting system. Res. Microbiol. 164, 640–654 (2013)

  16. 16.

    , & A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15, 9–21 (2014)

  17. 17.

    et al. Architecture and assembly of the type VI secretion system. Biochim. Biophys. Acta 1843, 1664–1673 (2014)

  18. 18.

    , & Tubules and donuts: a type VI secretion story. Mol. Microbiol. 76, 815–821 (2010)

  19. 19.

    , , & Type VI secretion and bacteriophage tail tubes share a common assembly pathway. EMBO Rep. 15, 315–321 (2014)

  20. 20.

    et al. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500, 350–353 (2013)

  21. 21.

    , , , & Imaging type VI secretion-mediated bacterial killing. Cell Rep. 3, 36–41 (2013)

  22. 22.

    , , , & The SciZ protein anchors the enteroaggregative Escherichia coli type VI secretion system to the cell wall. Mol. Microbiol. 75, 886–899 (2010)

  23. 23.

    , , , & SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli. J. Bacteriol. 190, 7523–7531 (2008)

  24. 24.

    , & An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens. J. Bacteriol. 191, 4316–4329 (2009)

  25. 25.

    et al. Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. PLoS Pathog. 7, e1002386 (2011)

  26. 26.

    , & IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion. J. Biol. Chem. 287, 15610–15621 (2012)

  27. 27.

    , , , & The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 type VI secretion system, is inserted by YidC. MicrobiologyOpen 1, 71–82 (2012)

  28. 28.

    et al. Structural characterization and oligomerization of the TssL protein, a component shared by bacterial type VI and type IVb secretion systems. J. Biol. Chem. 287, 14157–14168 (2012)

  29. 29.

    & Crystal structure of the bacterial type VI secretion system component TssL from Vibrio cholerae. J. Microbiol. 53, 32–37 (2015)

  30. 30.

    , & Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

  31. 31.

    et al. Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid antibody. Acta Crystallogr F. 71, 266–271 (2015)

  32. 32.

    et al. Inhibition of type VI secretion by an anti-TssM llama nanobody. PLoS ONE 10, e0122187 (2015)

  33. 33.

    et al. Deciphering the assembly of the Yersinia type III secretion injectisome. EMBO J. 29, 1928–1940 (2010)

  34. 34.

    , & Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus. Proc. Natl Acad. Sci. USA 102, 11498–11503 (2005)

  35. 35.

    , & Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J. 15, 978–988 (1996)

  36. 36.

    , & PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer. Mol. Microbiol. 23, 657–668 (1997)

  37. 37.

    et al. Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica. J. Bacteriol. 186, 5366–5375 (2004)

  38. 38.

    & Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization. Mol. Microbiol. 30, 47–56 (1998)

  39. 39.

    & The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG. Mol. Microbiol. 28, 1367–1380 (1998)

  40. 40.

    , , , & An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster. PLoS Genet. 7, e1002205 (2011)

  41. 41.

    & One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

  42. 42.

    , & A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 28, e97 (2000)

  43. 43.

    & RF cloning: a restriction-free method for inserting target genes into plasmids. J. Biochem. Biophys. Methods 67, 67–74 (2006)

  44. 44.

    & Promoter swapping unveils the role of the Citrobacter rodentium CTS1 type VI secretion system in interbacterial competition. Appl. Environ. Microbiol. 79, 32–38 (2013)

  45. 45.

    et al. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nature Methods 3, 623–628 (2006)

  46. 46.

    et al. TssK is a trimeric cytoplasmic protein interacting with components of both phage-like and membrane anchoring complexes of the type VI secretion system. J. Biol. Chem. 288, 27031–27041 (2013)

  47. 47.

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

  48. 48.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  49. 49.

    Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015)

  50. 50.

    et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

  51. 51.

    , & Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

  52. 52.

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

  53. 53.

    , , , & PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003)

  54. 54.

    , , & ATSAS 2.1, a program package for small-angle scattering data analysis. J. Appl. Crystallogr. 39, 277–286 (2006)

  55. 55.

    La diffraction des rayons X aux très petits angles; application à l’étude de phénomènes ultramicroscopiques. Ann. Phys. (Paris) 12, 161–237 (1939)

  56. 56.

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

  57. 57.

    & DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342–346 (2009)

  58. 58.

    & Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 (2003)

  59. 59.

    & Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34, 33–41 (2001)

  60. 60.

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

  61. 61.

    & Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

  62. 62.

    et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

  63. 63.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  64. 64.

    , & Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003)

  65. 65.

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

  66. 66.

    & Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

  67. 67.

    The PyMOL Molecular Graphics System. v.1.5.0.4 (Schrödinger, LLC, 2014)

  68. 68.

    , & Atomic modeling of cryo-electron microscopy reconstructions – joint refinement of model and imaging parameters. J. Struct. Biol. 182, 10–21 (2013)

  69. 69.

    & Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)

  70. 70.

    Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

  71. 71.

    , , & Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAMTM): application to lipid-specific membrane protein topogenesis. Methods 36, 148–171 (2005)

  72. 72.

    , , & Movements of the TolR C-terminal domain depend on TolQR ionizable key residues and regulate activity of the Tol complex. J. Biol. Chem. 282, 17749–17757 (2007)

  73. 73.

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

  74. 74.

    et al. Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nature Struct. Mol. Biol. 16, 477–485 (2009)

  75. 75.

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

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Acknowledgements

This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060). We thank O. Francetic for providing anti-DglA and anti-OmpF antibodies. We thank E. Marza, P. Violinova Krasteva and H. Remaut for comments on the manuscript, and T. Mignot, M. Guzzo and L. Espinosa for advice about the fluorescence microscopy experiments and the statistical analyses. We also thank the members of the R.F. and E.C. research groups for discussions and suggestions, and R. Lloubès, J. Sturgis and A. Galinier for encouragement. We thank the ERSF and Soleil Synchrotron radiation facilities for beamline allocation.

Author information

Author notes

    • Eric Durand
    • , Van Son Nguyen
    •  & Abdelrahim Zoued

    These authors contributed equally to this work.

Affiliations

  1. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Aix-Marseille Université - CNRS, UMR 7255, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

    • Eric Durand
    • , Abdelrahim Zoued
    • , Laureen Logger
    • , Marie-Stéphanie Aschtgen
    •  & Eric Cascales
  2. Architecture et Fonction des Macromolécules Biologiques, CNRS, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    • Eric Durand
    • , Van Son Nguyen
    • , Silvia Spinelli
    • , Aline Desmyter
    • , Alain Roussel
    •  & Christian Cambillau
  3. G5 Biologie structurale de la sécrétion bactérienne, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Eric Durand
    • , Annick Dujeancourt
    •  & Rémi Fronzes
  4. UMR 3528, CNRS, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Eric Durand
    • , Gérard Péhau-Arnaudet
    • , Benjamin Bardiaux
    • , Annick Dujeancourt
    •  & Rémi Fronzes
  5. AFMB, Aix-Marseille Université, IHU Méditerranée Infection, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    • Eric Durand
    • , Van Son Nguyen
    • , Silvia Spinelli
    • , Aline Desmyter
    • , Alain Roussel
    •  & Christian Cambillau
  6. Unité de Bioinformatique Structurale, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Benjamin Bardiaux

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Contributions

E.D., A.Z., C.C., E.C. and R.F. designed the experiments. A.Z. constructed the EAEC mutant and fluorescent strains and performed the fluorescence microscopy experiments and statistical analyses. L.L. and M.S.A. constructed the TssM cysteine derivatives and performed the accessibility experiments. E.D. assisted by An.D. purified the TssJLM complex and performed its biochemical characterization. E.D. and G.P.A. collected the EM data. E.D. and R.F. obtained the 3D reconstruction of the TssJLM complex. V.S.N., S.S., A.R. and C.C. purified, crystallized and solved the X-ray structures. Al.D. generated the nanobody. B.B. obtained the energy-minimized models of the closed and open states of the TssM26Ct–TssJ complex.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Christian Cambillau or Eric Cascales or Rémi Fronzes.

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https://doi.org/10.1038/nature14667

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