Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS
Abstract
Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.
Additional Information
© 2019 The Author(s), under exclusive licence to Springer Nature Limited. Received 13 July 2018. Accepted 12 March 2019. Published 22 April 2019. We thank R. Isberg (Tufts University, Medford, MA, USA) for antibodies that recognize DotF and DotH, E. Buford for technical assistance and P. Levin (Washington University, St Louis, MO, USA) for assistance with deconvolution microscopy. 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 C. Oikonomou for the creation of the domain maps and for help structuring and revising the text. We also recognize E. Darwin for key suggestions and critical appraisal of this manuscript. This work was funded by the NIH grant R01AI127401 to G.J.J. and the NIH grant R01AI48052 to J.P.V. These authors contributed equally: Debnath Ghosal, Kwangcheol C. Jeong. Author Contributions: D.G., K.C.C.J., J.P.V. and G.J.J. conceived the project. K.C.C.J., J.P.V. and J.G. constructed and characterized the L. pneumophila expression plasmids and strains. K.C.J. and J.P.V. collected the immunofluorescence data. D.G. collected the tomography data. D.G., K.C.C.J., J.P.V., G.J.J., Y.-W.C. and L.T. analysed the data. A.G. made Supplementary Video 1. D.G., J.P.V., K.C.C.J. and G.J.J. wrote the manuscript with input from other authors. Data availability: The subtomogram average of the Dot/Icm (DotF-sfGFP) complex that supports the findings of this study has been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code: EMD-0566. All other density maps are available from the corresponding authors on request. The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information documents. The authors declare no competing interests.Attached Files
Accepted Version - nihms-1523869.pdf
Submitted - 312009.full.pdf
Supplemental Material - 312009-1.pdf
Supplemental Material - 41564_2019_427_MOESM1_ESM.pdf
Supplemental Material - 41564_2019_427_MOESM2_ESM.pdf
Supplemental Material - 41564_2019_427_MOESM3_ESM.mov
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Additional details
- Alternative title
- Molecular architecture of the Legionella Dot/Icm type IV secretion system
- PMCID
- PMC6588468
- Eprint ID
- 90527
- Resolver ID
- CaltechAUTHORS:20181030-153432354
- NIH
- R01AI127401
- NIH
- R01AI48052
- Howard Hughes Medical Institute (HHMI)
- Created
-
2018-10-30Created from EPrint's datestamp field
- Updated
-
2022-02-17Created from EPrint's last_modified field