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Mechanism of loading and translocation of type VI secretion system effector Tse6

Nature Microbiologyvolume 3pages11421152 (2018) | Download Citation


The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular machine secretes antibacterial effector proteins by undergoing cycles of extension and contraction; however, how effectors are loaded into the T6SS and subsequently delivered to target bacteria remains poorly understood. Here, using electron cryomicroscopy, we analysed the structures of the Pseudomonas aeruginosa effector Tse6 loaded onto the T6SS spike protein VgrG1 in solution and embedded in lipid nanodiscs. In the absence of membranes, Tse6 stability requires the chaperone EagT6, two dimers of which interact with the hydrophobic transmembrane domains of Tse6. EagT6 is not directly involved in Tse6 delivery but is crucial for its loading onto VgrG1. VgrG1-loaded Tse6 spontaneously enters membranes and its toxin domain translocates across a lipid bilayer, indicating that effector delivery by the T6SS does not require puncturing of the target cell inner membrane by VgrG1. Eag chaperone family members from diverse Proteobacteria are often encoded adjacent to putative toxins with predicted transmembrane domains and we therefore anticipate that our findings will be generalizable to numerous T6SS-exported membrane-associated effectors.

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Data availability

The cryo-EM density maps of the PFC and the VgrG1-Tse6-EF-Tu complex in nanodisc are deposited into the Electron Microscopy Data Bank with the accession numbers EMD-0135 and EMD-0136, respectively. The refined models of VgrG1 in the PFC and the VgrG1-Tse6-EF-Tu complex in nanodisc have the PDB entry IDs 6H3L and 6H3N, respectively. Relevant data and details of plasmids and strains are available from the corresponding author upon request.

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


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We thank O. Hofnagel and D. Prumbaum for their valuable assistance in electron microscopy and C. Gatsogiannis, M. Stabrin, as well as T. Moriya, for the lively exchange regarding image processing. We thank the SPHIRE developer team, in particular P.A. Penczek, for developing the cryo-EM image processing software used in this study. D.Q. is a fellow of Fonds der Chemischen Industrie. This work was supported by the Max Planck Society (to S.R.), the European Council under the European Union’s Seventh Framework Programme (FP7/ 2007–2013) (grant no. 615984 to S.R.), the National Institutes of Health (R01-AI080609 to J.D.M.), the Howard Hughes Medical Institute (to J.D.M.), start-up funds from McMaster University (to J.C.W.), a project grant from the Canadian Institutes of Health Research (PJT-156129 to J.C.W.) and equipment provided by the Michael DeGroote Institute for Infectious Disease Research (to J.C.W.).

Author information


  1. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    • Dennis Quentin
    •  & Stefan Raunser
  2. Michael DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    • Shehryar Ahmad
    • , Premy Shanthamoorthy
    •  & John C. Whitney
  3. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    • Shehryar Ahmad
    • , Premy Shanthamoorthy
    •  & John C. Whitney
  4. Department of Microbiology, University of Washington, Seattle, WA, USA

    • Joseph D. Mougous
  5. Howard Hughes Medical Institute, Seattle, WA, USA

    • Joseph D. Mougous


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S.R., J.C.W. and J.D.M. designed the project. J.C.W. and J.D.M. provided protein complexes. D.Q. prepared specimens, recorded, analysed and processed the EM data, performed the liposome-based in vitro assay and prepared figures. S.R. managed the project. S.A. and J.C.W. performed the biochemical and cellular in vivo experiments. P.S. introduced point mutations into EagT6. D.Q., J.C.W. and S.R. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to John C. Whitney or Stefan Raunser.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–12, Supplementary Tables 1–3

  2. Reporting Summary

  3. Supplementary Video 1

    Flexibility of Tse6tox–EF-Tu–Tsi6 subcomplex. This video highlights the flexibility of the Tse6tox–EF-Tu–Tsi6 subcomplex

  4. Supplementary Video 2

    Architecture of VgrG1–Tse6–EF-Tu complex in lipid nanodiscs. This movie describes relevant structural features of the VgrG1–Tse6–EF-Tu complex in lipid nanodiscs

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