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A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel

Nature Microbiology volume 3, pages 678–686 (2018)Cite this article

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Abstract

Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells1,2,3,4,5. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot–Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot–Icm apparatus was solved in situ by cryo-electron tomography at 3.5 nm resolution and the cytoplasmic complex was solved at 3.0 nm resolution. These structures revealed a cell envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB–DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS. These data define distinct stages in Dot–Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.

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Fig. 1: ATP-bound DotB displays static polar localization.
Fig. 2: The ATPase DotO is essential for polar recruitment of DotB.
Fig. 3: The ATPase DotO is placed above DotB.
Fig. 4: In situ structure of the Dot–Icm type IVB secretion machine revealed by cryo-electron tomography and subtomogram averaging.
Fig. 5: The cytoplasmic complex, showing 6-fold symmetry, composed of ATPases DotO and DotB.

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Acknowledgements

B.H. and J.L were supported by the National Institutes of Health (R01AI087946 and R01GM107629) and the Welch Foundation (AU-1714). D.C. and C.R. were supported by the NIH (R37AI041699 and R21AI130671). P.C. was supported by the NIH (R01GM48476). We are grateful to S.S. Ivanov (Louisiana State University) for insightful suggestions and critique; H. Nagai (Gifu University) for the L. pneumophila ΔT4SS strain; R.R. Isberg (Tufts University) for the antibody to DotO; E.H. Rego (Yale University) for technical assistance with FRAP.

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  1. These authors contributed equally: David Chetrit, Bo Hu.

Authors and Affiliations

  1. Department of Microbial Pathogenesis, Microbial Sciences Institute and Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT, USA

    David Chetrit, Craig R. Roy & Jun Liu

  2. Department of Microbiology and Molecular Genetics, Houston, TX, USA

    Bo Hu, Peter J. Christie & Jun Liu

  3. Department of Pathology and Laboratory Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA

    Bo Hu & Jun Liu

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Contributions

B.H., C.R., D.C. and J.L. designed research. D.C. constructed the L. pneumophila expression plasmids and strains. B.H. and D.C. collected and together with C.R., J.L. and P.C. analysed the data. B.H., C.R., D.C., J.L. and P.C. wrote the paper.

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Correspondence to Craig R. Roy or Jun Liu.

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Supplementary information

Supplementary Information

Supplementary Figures 1–8, and Supplementary Tables 1 and 2.

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Supplementary Video 1

Real-time visualization of DotB–sfGFP, sfGFP, and DotBE191K–sfGFP expressed from dot dotB operon in Legionella pneumophila.

Supplementary Video 2

3D visualization of a tomographic reconstruction and the intact T4SS machine

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Chetrit, D., Hu, B., Christie, P.J. et al. A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel. Nat Microbiol 3, 678–686 (2018). https://doi.org/10.1038/s41564-018-0165-z

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