Abstract

To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and eukaryotic cells. The T6SS comprises two functional subcomplexes: a bacteriophage-related tail structure anchored to the cell envelope by a membrane complex. As in other contractile injection systems, the tail is composed of an inner tube wrapped by a sheath and built on the baseplate. In the T6SS, the baseplate is not only the tail assembly platform, but also docks the tail to the membrane complex and hence serves as an evolutionary adaptor. Here we define the biogenesis pathway and report the cryo-electron microscopy (cryo-EM) structure of the wedge protein complex of the T6SS from enteroaggregative Escherichia coli (EAEC). Using an integrative approach, we unveil the molecular architecture of the whole T6SS baseplate and its interaction with the tail sheath, offering detailed insights into its biogenesis and function. We discuss architectural and mechanistic similarities but also reveal key differences with the T4 phage and Mu phage baseplates.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The cryo-EM structures of the full complex TssKFG, TssK and TssFGE have been deposited in the Electron Microscopy Data Bank under ID codes EMD-0008, EMD-0010 and EMD-0009. The TssKFG, TssK and TssFGE models have been deposited in the PDB under ID codes PDB 6GIY, 6GJ3 and 6GJ1. Raw cryo-EM data are available on request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

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

  2. 2.

    Durand, E., Cambillau, C., Cascales, E. & Journet, L. VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors. Trends Microbiol. 22, 498–507 (2014).

  3. 3.

    Alcoforado Diniz, J., Liu, Y.-C. & Coulthurst, S. J. Molecular weaponry: diverse effectors delivered by the type VI secretion system. Cell Microbiol. 17, 1742–1751 (2015).

  4. 4.

    Russell, A. B., Peterson, S. B. & Mougous, J. D. Type VI secretion system effectors: poisons with a purpose. Nat. Rev. Microbiol. 12, 137–148 (2014).

  5. 5.

    Chassaing, B. & Cascales, E. Antibacterial weapons: targeted destruction in the microbiota. Trends Microbiol. 26, 329–338 (2018).

  6. 6.

    Sana, T. G., Lugo, K. A. & Monack, D. M. T6SS: The bacterial ‘fight club’ in the host gut. PLoS Pathog. 13, e1006325 (2017).

  7. 7.

    Pukatzki, S., Ma, A. T., Revel, A. T., Sturtevant, D. & Mekalanos, J. J. 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).

  8. 8.

    Durand, E. et al. Crystal structure of the VgrG1 actin cross-linking domain of the Vibrio cholerae type VI secretion system. J. Biol. Chem. 287, 38190–38199 (2012).

  9. 9.

    Sana, T. G. et al. Internalization of Pseudomonas aeruginosa strain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network. mBio 6, e00712 (2015).

  10. 10.

    Ma, A. T., McAuley, S., Pukatzki, S. & Mekalanos, J. J. Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells. Cell Host Microbe 5, 234–243 (2009).

  11. 11.

    Leiman, P. G. & Shneider, M. M. Contractile tail machines of bacteriophages. Adv. Exp. Med. Biol. 726, 93–114 (2012).

  12. 12.

    Ge, P. et al. Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states. Nat. Struct. Mol. Biol. 22, 377–382 (2015).

  13. 13.

    Böck, D. et al. In situ architecture, function, and evolution of a contractile injection system. Science 357, 713–717 (2017).

  14. 14.

    Heymann, J. B. et al. Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila. J. Biol. Chem. 288, 25276–25284 (2013).

  15. 15.

    Shikuma, N. J. et al. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343, 529–533 (2014).

  16. 16.

    Cascales, E. Microbiology: and amoebophilus invented the machine gun! Curr. Biol. 27, R1170–R1173 (2017).

  17. 17.

    Brackmann, M., Wang, J. & Basler, M. Type VI secretion system sheath inter‐subunit interactions modulate its contraction. EMBO Rep. 19, 225–233 (2018).

  18. 18.

    Basler, M., Pilhofer, M., Henderson, G. P., Jensen, G. J. & Mekalanos, J. J. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182–186 (2012).

  19. 19.

    Ballister, E. R., Lai, A. H., Zuckermann, R. N., Cheng, Y. & Mougous, J. D. In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc. Natl Acad. Sci. USA 105, 3733–3738 (2008).

  20. 20.

    Brunet, Y. R., Henin, J., Celia, H. & Cascales, E. Type VI secretion and bacteriophage tail tubes share a common assembly pathway. EMBO Rep. 15, 315–321 (2014).

  21. 21.

    Leiman, P. G. et al. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc. Natl Acad. Sci. USA 106, 4154–4159 (2009).

  22. 22.

    Basler, M., Ho, B. T. & Mekalanos, J. J. Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152, 884–894 (2013).

  23. 23.

    Ho, B. T., Basler, M. & Mekalanos, J. J. Type 6 secretion system-mediated immunity to type 4 secretion system-mediated gene transfer. Science 342, 250–253 (2013).

  24. 24.

    LeRoux, M. et al. Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa. eLife 4, e05701 (2015).

  25. 25.

    Vettiger, A. & Basler, M. Type VI secretion system substrates are transferred and reused among sister cells. Cell 167, 99–110 (2016).

  26. 26.

    Zoued, A. 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).

  27. 27.

    Brunet, Y. R., Zoued, A., Boyer, F., Douzi, B. & Cascales, E. The type VI secretion TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via multiple contacts and serves as assembly platform for tail tube/sheath polymerization. PLoS Genet. 11, e1005545 (2015).

  28. 28.

    Nguyen, V. S. et al. Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex. Nat. Microbiol. 2, 17103 (2017).

  29. 29.

    Aschtgen, M.-S., Gavioli, M., Dessen, A., Lloubès, R. & Cascales, E. The SciZ protein anchors the enteroaggregative Escherichia coli type VI secretion system to the cell wall. Mol. Microbiol. 75, 886–899 (2010).

  30. 30.

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

  31. 31.

    Flaugnatti, N. et al. A phospholipase A 1 antibacterial type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery. Mol. Microbiol. 99, 1099–1118 (2016).

  32. 32.

    Yap, M. L., Mio, K., Leiman, P. G., Kanamaru, S. & Arisaka, F. The baseplate wedges of bacteriophage T4 spontaneously assemble into hubless baseplate-like structure in vitro. J. Mol. Biol. 395, 349–360 (2010).

  33. 33.

    Leiman, P. G. et al. Morphogenesis of the T4 tail and tail fibers. Virol. J. 7, 355 (2010).

  34. 34.

    Bingle, L. E., Bailey, C. M. & Pallen, M. J. Type VI secretion: a beginner’s guide. Curr. Opin. Microbiol. 11, 3–8 (2008).

  35. 35.

    Nazarov, S. et al. Cryo-EM reconstruction of type VI secretion system baseplate and sheath distal end. EMBO J. 37, e97103 (2018).

  36. 36.

    Taylor, N. M. I. et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature 533, 346–352 (2016).

  37. 37.

    English, G., Byron, O., Cianfanelli, F. R., Prescott, A. R. & Coulthurst, S. J. Biochemical analysis of TssK, a core component of the bacterial type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex. Biochem. J. 461, 291–304 (2014).

  38. 38.

    Logger, L., Aschtgen, M. S., Guérin, M., Cascales, E. & Durand, E. Molecular dissection of the interface between the type VI secretion TssM cytoplasmic domain and the TssG baseplate component. J. Mol. Biol. 428, 4424–4437 (2016).

  39. 39.

    Zoued, A. et al. Structure–function analysis of the TssL cytoplasmic domain reveals a new interaction between the type VI secretion baseplate and membrane complexes. J. Mol. Biol. 428, 4413–4423 (2016).

  40. 40.

    Yap, M. L. et al. Sequential assembly of the wedge of the baseplate of phage T4 in the presence and absence of gp11 as monitored by analytical ultracentrifugation. Macromol. Biosci. 10, 808–813 (2010).

  41. 41.

    Arisaka, F., Yap, M. L., Kanamaru, S. & Rossmann, M. G. Molecular assembly and structure of the bacteriophage T4 tail. Biophys. Rev. 8, 385–396 (2016).

  42. 42.

    Wang, S., Sun, S., Li, Z., Zhang, R. & Xu, J. Accurate de novo prediction of protein contact map by ultra-deep learning model. PLoS Comput. Biol. 13, e1005324 (2017).

  43. 43.

    Onoue, Y. et al. Construction of functional fragments of the cytoplasmic loop with the C-terminal region of PomA, a stator component of the Vibrio Na+ driven flagellar motor. J. Biochem. 155, 207–216 (2014).

  44. 44.

    Rowe, H. M. et al. Modification of the CpsA protein reveals a role in alteration of the Streptococcus agalactiae cell envelope. Infect. Immun. 83, 1497–1506 (2015).

  45. 45.

    Hopf, T. A. et al. Sequence co-evolution gives 3D contacts and structures of protein complexes. eLife 3, e03430 (2014).

  46. 46.

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

  47. 47.

    Chang, Y., Rettberg, L. A., Ortega, D. R. & Jensen, G. J. In vivo structures of an intact type VI secretion system revealed by electron cryotomography. EMBO Rep. 18, 1090–1099 (2017).

  48. 48.

    Douzi, B. et al. Structure–function analysis of the C-Terminal domain of the type VI secretion TssB tail sheath subunit. J. Mol. Biol. 430, 297–309 (2018).

  49. 49.

    Kostyuchenko, V. A. et al. Three-dimensional structure of bacteriophage T4 baseplate. Nat. Struct. Biol. 10, 688–693 (2003).

  50. 50.

    Kostyuchenko, V. A. et al. The tail structure of bacteriophage T4 and its mechanism of contraction. Nat. Struct. Mol. Biol. 12, 810–813 (2005).

  51. 51.

    Leiman, P. G., Chipman, P. R., Kostyuchenko, V. A., Mesyanzhinov, V. V. & Rossmann, M. G. Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host. Cell 118, 419–429 (2004).

  52. 52.

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

  53. 53.

    Brunet, Y. R., Bernard, C. S., Gavioli, M., Lloubès, R. & Cascales, E. An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster. PLoS Genet. 7, e1002205 (2011).

  54. 54.

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

  55. 55.

    Aschtgen, M.-S., Bernard, C. S., De Bentzmann, S., Lloubes, R. & Cascales, E. SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli. J. Bacteriol. 190, 7523–7531 (2008).

  56. 56.

    Chaveroche, M. K., Ghigo, J. M. & d’Enfert, C. A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 28, E97 (2000).

  57. 57.

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

  58. 58.

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

  59. 59.

    Brunet, Y. R., Espinosa, L., Harchouni, S., Mignot, T. & Cascales, E. Imaging type VI secretion-mediated bacterial killing. Cell Rep. 3, 36–41 (2013).

  60. 60.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

  61. 61.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

  62. 62.

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

  63. 63.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

  64. 64.

    Asarnow, D. pyem (GitHub, 2016); https://github.com/asarnow/pyem

  65. 65.

    Sanchez, R. M. Recentering and Subboxing of Particles (REP) (GitHub, 2017); https://github.com/rkms86/REP

  66. 66.

    Terwilliger, T. C., Sobolev, O., Afonine, P. V. & Adams, P. D. Automated map sharpening by maximization of detail and connectivity. Acta Crystallogr. Sect. D Struct. Biol. 74, 545–559 (2018).

  67. 67.

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

  68. 68.

    Simkovic, F., Ovchinnikov, S., Baker, D. & Rigden, D. J. Applications of contact predictions to structural biology. IUCrJ 4, 291–300 (2017).

  69. 69.

    Marks, D. S. et al. Protein 3D structure computed from evolutionary sequence variation. PLoS ONE 6, e28766 (2011).

  70. 70.

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

  71. 71.

    Leaver-Fay, A. et al. Rosetta3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).

  72. 72.

    Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. D Struct. Biol. 74, 531–544 (2018).

  73. 73.

    McGuffin, L. J., Bryson, K. & Jones, D. T. The PSIPRED protein structure prediction server. Bioinformatics 16, 404–405 (2000).

  74. 74.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

  75. 75.

    Clarke, O. B. Coot Trimmings (GitHub, 2017); https://github.com/olibclarke/coot-trimmings.

  76. 76.

    Bouvier, G., Bardiaux, B. & Nilges, M. Automatic building of protein atomic models from cryo-EM maps. Biophys. J. 114, 190a–191a (2018).

  77. 77.

    Ovchinnikov, S. et al. Protein structure determination using metagenome sequence data. Science 355, 294–298 (2017).

  78. 78.

    Rieping, W., Bardiaux, B., Bernard, A., Malliavin, T. E. & Nilges, M. ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23, 381–382 (2007).

  79. 79.

    Wang, S., Peng, J., Ma, J. & Xu, J. Protein secondary structure prediction using deep convolutional neural fields. Sci. Rep. 6, 18962 (2016).

  80. 80.

    Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

  81. 81.

    Mareuil, F., Malliavin, T. E., Nilges, M. & Bardiaux, B. Improved reliability, accuracy and quality in automated NMR structure calculation with ARIA. J. Biomol. NMR 62, 425–438 (2015).

  82. 82.

    Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improving the quality of NMR and crystallographic protein structures by means of a conformational database potential derived from structure databases. Protein Sci. 5, 1067–1080 (2008).

  83. 83.

    Wang, R. Y. R. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016).

  84. 84.

    Barad, B. A. et al. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

  85. 85.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 12–21 (2010).

  86. 86.

    Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. Sect. D Biol. Crystallogr. 71, 136–153 (2015).

  87. 87.

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

  88. 88.

    Williams, C. Using C-Alpha Geometry to Describe Protein Secondary Structure and Motif PhD thesis, Duke Univ. (2016).

Download references

Acknowledgements

We thank members of the E.C. and R.F. research groups, and J. Sturgis for helpful discussions and support, M. Weigt and C. Feinauer for useful discussion on evolutionary covariance, T. Huynh and G. Canet for assistance with the computer clusters at the Institut Pasteur and Institut Européen de Chimie et Biologie (IECB), respectively, A. Kosta (Plateforme de microscopie, Institut de Microbiologie de la Méditerranée (IMM), Marseille, France) for providing access to the IMM EM facility and for checking the quality of samples by negative stain EM, A. Bezault for support at the cryo-EM facility at IECB and T. Doan and L. Espinosa for their helpful support with the fluorescence microscopy device and analysis. We thank M. Nilges (Institut Pasteur) for access to the computer cluster of the ERC project 'BayCellS'. This work was supported by the CNRS, the Aix-Marseille Université, the Institut Pasteur and the INSERM, and by grants from the Agence Nationale de la Recherche (grants no. ANR-14-CE14-0006 and ANR-17-CE11-0039 to E.C., and no. ANR-11-EQPX-008 to J.C.R.). Work of Y.C. was supported by an 'Ecole Doctorale' PhD fellowship from the 'Fondation pour la Recherche Médicale' (grant no. FRM-ECO20160736014). R.F. and C.R. were supported by IDEX Bordeaux through a 'chaire d’excellence' to R.F. We acknowledge the European Synchrotron Radiation Facility for provision of beamtime on CM01 and thank G. Effantin and E. Kandiah for assistance. F.A. thanks the French Institute of Bioinformatics (IFB; grant no. ANR-11-INBS-0013) for financial support.

Author information

Author notes

  1. These authors contributed equally: Yassine Cherrak, Chiara Rapisarda.

Affiliations

  1. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, UMR7255, Aix-Marseille Université – CNRS, Marseille, France

    • Yassine Cherrak
    • , Eric Cascales
    •  & Eric Durand
  2. Institut Européen de Chimie et Biologie, University of Bordeaux, Pessac, France

    • Chiara Rapisarda
    •  & Rémi Fronzes
  3. CNRS UMR 5234 Microbiologie Fondamentale et Pathogénicité, Paris, France

    • Chiara Rapisarda
    •  & Rémi Fronzes
  4. Institut Pasteur, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, CNRS UMR 3528, C3BI USR 3756, Paris, France

    • Riccardo Pellarin
    • , Guillaume Bouvier
    • , Benjamin Bardiaux
    •  & Fabrice Allain
  5. USR 2000, CNRS, Institut Pasteur, Paris, France

    • Christian Malosse
    • , Martial Rey
    •  & Julia Chamot-Rooke
  6. Mass Spectrometry for Biology Unit, Institut Pasteur, Paris, France

    • Christian Malosse
    • , Martial Rey
    •  & Julia Chamot-Rooke
  7. Laboratoire d’Ingénierie des Systèmes Macromoléculaires , Institut de Microbiologie de la Méditerranée, UMR7255, INSERM, Marseille, France

    • Eric Durand

Authors

  1. Search for Yassine Cherrak in:

  2. Search for Chiara Rapisarda in:

  3. Search for Riccardo Pellarin in:

  4. Search for Guillaume Bouvier in:

  5. Search for Benjamin Bardiaux in:

  6. Search for Fabrice Allain in:

  7. Search for Christian Malosse in:

  8. Search for Martial Rey in:

  9. Search for Julia Chamot-Rooke in:

  10. Search for Eric Cascales in:

  11. Search for Rémi Fronzes in:

  12. Search for Eric Durand in:

Contributions

E.D., R.F. and E.C. designed the research, assembled results and wrote the paper with input from all authors. E.D. and Y.C. performed the biochemical experiments and the initial negative stain EM observations. C.R. performed the sample preparation for cryo-EM, de novo reconstruction and structure refinement. C.R. and R.F. analysed the cryo-EM data and the final structure. C.M. and M.R. performed the mass spectrometry experiments. Y.C. performed all in vivo experiments. R.P. and G.B. carried out the method development and structural modelling. B.B. and F.A. carried out the structural modelling. J.C.R. performed the design and analysis of the mass spectrometry experiments.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Rémi Fronzes or Eric Durand.

Supplementary information

  1. Supplementary Information

    Supplementary Results, Supplementary Tables 13, Supplementary References, Supplementary Figures 1–19.

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41564-018-0260-1

Further reading