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Cryo-EM structure of the extended type VI secretion system sheath–tube complex

Nature Microbiology volume 2, pages 1507–1512 (2017)Cite this article

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Abstract

The bacterial type VI secretion system (T6SS) uses contraction of a long sheath to quickly thrust a tube with associated effectors across membranes of eukaryotic and bacterial cells1,2,3,4,5. Only limited structural information is available about the inherently unstable precontraction state of the T6SS. Here, we obtain a 3.7 Å resolution structure of a non-contractile sheath–tube complex using cryo-electron microscopy and show that it resembles the extended T6SS inside Vibrio cholerae cells. We build a pseudo-atomic model of the complete sheath–tube assembly, which provides a mechanistic understanding of coupling sheath contraction with pushing and rotating the inner tube for efficient target membrane penetration. Our data further show that sheath contraction exposes a buried recognition domain to specifically trigger the disassembly and recycling of the T6SS sheath by the cognate ATP-dependent unfoldase ClpV.

The type VI secretion system (T6SS) is a cytosolic phage tail-like structure connected to a transmembrane complex1,6,7. The phage tail-like components include a baseplate complex and a tube of Hcp hexamers surrounded by a contractile sheath assembled from VipA/VipB (TssB/TssC) heterodimers. The current model suggests that the energy released during sheath contraction is converted into movement of the inner Hcp tube through the baseplate, which results in physical puncturing of the target membrane in close contact8,9,10. A similar mechanism is used by many other nanomachines to breach membranes during various biological processes11: (1) R-type pyocins punch holes into bacterial membranes12,13, (2) contractile phages translocate proteins and DNA14,15,16 and (3) certain nanomachines deliver toxins and signalling molecules to eukaryotic cells17,18. Although the T6SS sheath is evolutionarily related to the sheaths of contractile phage tails and R-type pyocins12,15,19,20,21,22,23, it contains a distinct surface-exposed Domain 3 important for sheath recycling. Upon contraction, the VipB N terminus within Domain 3 is recognized by the cognate ATPase ClpV, which unfolds the contracted sheath to allow assembly of new extended sheaths1,24,25,26,27,28.

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Fig. 1: Cryo-EM structure of the VipA-N3 sheath–tube complex closely resembles the intracellular wild-type extended sheath–tube complex.
Fig. 2: Model of T6SS sheath contraction.
Fig. 3: Domain 3 of the VipA-N3 sheath is inaccessible to ClpV.
Fig. 4: Structure of the Hcp tube and its interaction with the sheath.

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Acknowledgements

The work was supported by Swiss National Science Foundation (SNSF) grant 31003A_159525 and the University of Basel. H.S. acknowledges support from the SNSF NCCR TransCure. Calculations were performed at sciCORE (http://scicore.unibas.ch/) scientific computing core facility at the University of Basel. We acknowledge S. Ursich for the help in sample preparation for cryo-ET.

Author information

Authors and Affiliations

  1. Focal Area Infection Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056, Basel, Switzerland

    Jing Wang, Maximilian Brackmann & Marek Basler

  2. Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland

    Daniel Castaño-Díez, Kenneth N. Goldie & Henning Stahlberg

  3. BioEM Lab, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland

    Daniel Castaño-Díez

  4. Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt am Main, Germany

    Mikhail Kudryashev

  5. Buchmann Institute for Molecular Life Sciences, Max-von-Laue Str. 17, 60438, Frankfurt am Main, Germany

    Mikhail Kudryashev

  6. Focal Area Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056, Basel, Switzerland

    Timm Maier & Henning Stahlberg

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Contributions

J.W. collected cryo-electron microscopy data, performed image processing and generated atomic models. M.Br. isolated and purified the sheaths. M.K. performed some initial electron microscopy data collection and data analysis. D.C.-D. provided support and contributed to data analysis. K.N.G. and H.S. provided support and supervised data collection. T.M. contributed to and advised on atomic model building. M.Ba. conceived the project and analysed the data. M.Br., J.W. and M.Ba. wrote the manuscript. All authors read the manuscript.

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Correspondence to Marek Basler.

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Life Sciences Reporting Summary

Supplementary Figures 1–5, Supplementary Tables 1–6, Supplementary Video legends.

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Supplementary Video 1

Details of tomography reconstruction of the wild-type extended sheath, VipA-N3 sheath structure and Hcp tube.

Supplementary Video 2

Proposed mechanism of T6SS assembly and contraction.

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Wang, J., Brackmann, M., Castaño-Díez, D. et al. Cryo-EM structure of the extended type VI secretion system sheath–tube complex. Nat Microbiol 2, 1507–1512 (2017). https://doi.org/10.1038/s41564-017-0020-7

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