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.
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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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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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.
Supplementary Figures 1–32, Supplementary Video Legend, Supplementary Tables 1 and 2, and Supplementary References.
3D representation of the Dot/Icm complex showing a windowed secretion chamber (salmon, DotH; grey, DotD; green, DotK; and cyan, DotC), wings (yellow, DotF), a secretion channel (red, DotG) and the top-view of the complex. Cytoplasmic components are not shown. In this 3D representation, IcmF, IcmX and DotA are not visible. The blue structure below the secretion channel represents the UF seed that initiates polar Dot/Icm complex assembly.