In vivo structure of the Legionella type II secretion system by electron cryotomography

A Publisher Correction to this article was published on 19 February 2020

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Abstract

The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane of many Gram-negative bacteria1,2,3. Here, using electron cryotomography and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily related type IV pilus (T4P) system4,5, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including, for example, that the T2SS–ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our electron cryotomography map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope and the interactions between its different subcomplexes.

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Fig. 1: Visualization of the T2SS in frozen-hydrated L. pneumophila cells.
Fig. 2: Individual T2SS subcomplexes.
Fig. 3: Architectural model of the T2SS.

Data availability

The subtomogram averages of the L. pneumophila T2SS have been deposited in the Electron Microscopy Data Bank under the following accession codes: EMD-20713 (wild type, aligned on the OM part) and EMD-20712 (wild type, aligned on the IM part). All additional data/information are available from the authors upon request. The authors declare that all data supporting the findings of this study, including source data for Fig. 1, are available within the paper and its Supplementary Information.

Change history

  • 19 February 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported by National Institutes of Health grants AI127401 to G.J.J. and AI043987 to N.P.C. ECT data were recorded at the Beckman Institute Resource Center for Transmission Electron Microcopy at Caltech and the cryo-EM facility at Janelia Research Campus. We thank D. Ortega, Y.-W. Chang and R.C. White for helpful discussions. M.K. is supported by a postdoctoral Rubicon fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek.

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Contributions

D.G. and G.J.J. conceived the project. D.G. prepared samples and recorded and processed tomography data. D.G., K.W.K. and G.J.J. analysed data. H.Z., H.K.T., A.E.L., I.E.M., N.P.C. and J.P.V. generated and characterized L. pneumophila mutants. M.K. helped in model building. D.G., K.W.K., N.P.C. and G.J.J. wrote the manuscript with input from all the authors.

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Correspondence to Grant J. Jensen.

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Extended data

Extended Data Fig. 1

Comparison between the T2SS and related molecular machines T4aP and T4bP systems.

Extended Data Fig. 2 T2SS flexibility.

Subtomogram averages of all particles aligned on (a) the OM-associated complex and (b) the IM-associated complex. The distribution of the green dots in (b) indicates the translations imposed on the OM complexes to align the IM complexes. (c) A composite average using the upper and lower halves of (a) and (b), respectively. d) Local resolution of (c) calculated by Resmap. (e) Focused alignment near the base of the secretin channel revealed the presence of a plug-like structure. 20% of the particles with highest cross-correlation showed this distinct density. In the rest of the particles, the plug density is either not present or so dynamic that including them makes the plug almost invisible. (f) Previously reported in situ averages of the T4aP (WT, piliated) and T4bP (WT, piliated and ∆tcpR mutant) machines in states with cytoplasmic dome, ring and disks for comparison33,34. White arrows indicate cytoplasmic disks. Scale bars, 10 nm (ac), (e), (f). Panel reproduced from: f, ref. 33, Springer Nature Ltd; ref. 34, AAAS.

Extended Data Fig. 3 Position of the T2SS secretin with respect to the OM.

(a, b) Atomic models of the V. cholerae (PDB ID: 5WQ8) and E. coli (PDB ID: 5WQ7) T2SS secretins superimposed on our subtomogram average based on the position of the gate. (c) Positions of the OM on these structures as suggested in earlier publications13,15. The widths of the suggested OM spanning regions were only ~ 1.8 nm, but real membranes are known to be 5–7 nm wide. In all reported atomic models, the secretin channel is suggested to extend beyond the OM13,9. However, when we overlaid the secretin atomic models on our subtomogram average, it only reached through the inner leaflet of the OM. Scale bars, 10 nm. (d) Tomographic slices of mutant L. pneumophila cells lacking all major and minor pilins (ΔlspGHIJK). Showing representative individual T2SS particles. No pseudopilus or lower-periplasmic ring is visible. A similar result was obtained when we examined a L. pneumophila ΔlspHIJK mutant. Scale bar, 10 nm (d). For each strain, number of tomograms recorded and number of particles found are listed in the SI Table-1.

Extended Data Fig. 4 Position of the T2SS with respect to the PG layer in L. pneumophila.

(a, b) Tomographic slices through L. pneumophila cells showing T2SS particles (red arrowheads) and the peptidoglycan layer (PG, yellow arrowheads). (c, d) Tomographic slices through L. pneumophila cells showing T4BSS particles (red arrowheads) and the peptidoglycan layer (PG, yellow arrowheads). (e) Tomographic slice through a L. pneumophila cell showing both T4SS and T2SS particles (red arrowheads) and the peptidoglycan (PG, yellow arrowheads) in the same cell. (f, g) Subtomogram averages of the T4BSS and T2SS, respectively. DotK (shown as green arrow) in the T4BSS is known to interact with the PG layer confirming its location just a few nm below the OM (f). We therefore conclude that the PG layer surrounds the T2SS at approximately the level of the gate (g). For each strain, number of tomograms recorded and number of particles found are listed in the SI Table-1. Scale bars, 100 nm (ae), 10 nm (f, g). Panel f reproduced from: b, ref. 27, Springer Nature Ltd.

Extended Data Fig. 5

Cryo-EM data collection, refinement and validation statistics.

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Source Data Fig. 2

Unprocessed Western Blots

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Ghosal, D., Kim, K.W., Zheng, H. et al. In vivo structure of the Legionella type II secretion system by electron cryotomography. Nat Microbiol 4, 2101–2108 (2019). https://doi.org/10.1038/s41564-019-0603-6

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