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. 2013 Nov 28:3:3369.
doi: 10.1038/srep03369.

Common and distinct structural features of Salmonella injectisome and flagellar basal body

Akihiro Kawamoto  1 , Yusuke V Morimoto, Tomoko Miyata, Tohru Minamino, Kelly T Hughes, Takayuki Kato, Keiichi Namba
Affiliations

Common and distinct structural features of Salmonella injectisome and flagellar basal body

Akihiro Kawamoto et al. Sci Rep. .

Abstract

Bacterial pathogens use an injectisome to deliver virulence proteins into eukaryotic host cells. The bacterial flagellum and injectisome export their component proteins for self-assembly. These two systems show high structural similarities and are classified as the type III secretion system, but it remains elusive how similar they are in situ because the structures of these complexes isolated from cells and visualized by electron cryomicroscopy have shown only the export channel and housing for the export apparatus. Here we report in situ structures of Salmonella injectisome and flagellum by electron cryotomography. The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems. The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.

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Figures

Figure 1
Figure 1. Schematic representation of the structures of Salmonella injectisome (NC, left) and flagellar hook basal body (FBB, right).
Different colors indicate structures of different categories: pale green, structures of isolated complexes; blue, additional structures present in the in situ complexes; red, putative structures not yet visible in the in situ complexes. Insets are the axial sections of isolated NC and FBB obtained by cryoEM image analysis. OR: outer membrane ring, IR: inner membrane ring, OM: outer membrane, PG: peptidoglycan layer, CM: cytoplasmic membrane.
Figure 2
Figure 2. Characteristics of Salmonella mini-cell produced by the overproduction of FtsZ.
(a) Negatively stained EM image of a Salmonella wild-type cell with a diameter of ∼1.0 μm and a mini-cell with a diameter of ∼0.5 μm. Scale bar, 500 nm. (b) Bright-field and fluorescence microscopy images of wild-type cells and mini-cells are merged to show cell bodies and the flagellar filaments. The filaments were labeled with the Alexa fluorescent dye (red). Mini-cells are highlighted by square box of dashed or solid line, and the one in the solid-line square is magnified. Scale bar, 4 μm.
Figure 3
Figure 3. NC and FBB in the tomogram of Salmonella mini-cell.
(a) NC and (b) FBB are highlighted with a box on an 11 nm-thick slice of each tomogram. Scale bar, 100 nm. Magnified images of NC and FBB are shown in the insets. The NC and FBB are indicated by red and white arrowheads, respectively. The two small white arrows indicate the C ring of FBB. Scale bar, 50 nm.
Figure 4
Figure 4. Comparison of in situ structures of NC and FBB.
The central sections of NC (a) and FBB (b) structures in situ after subtomogram average. (c), (d) The axial sections of isolated NC and FBB obtained by cryoEM image analysis are colored yellow and superimposed on their in situ structures, respectively. The cytoplasmic densities designated as the wing-like disc and the globular density in the text are indicated by red and blue arrows and are assigned to InvAC and InvC ATPase in NC and FlhAC and FliI ATPase in FBB, respectively. The white arrow indicates the connection between the wing-like disc and a putative structure of the sorting platform composed of SpaO, OrgA and OrgB in NC and that between the disc and the inner lobe of the C ring in FBB. The C ring conformation shows significant differences in the orientations of the cylindrical wall and the inner lobe between the isolated and in situ structures. OM: outer membrane, CM: cytoplasmic membrane. Scale bar, 10 nm.
Figure 5
Figure 5. Superposition of cryoEM density maps of isolated NC and FBB on and docking of the atomic models of MxiAC, FlhAC and FliI into the density maps of in situ NC and FBB.
Magnified views of the membrane and cytoplasmic portions of NC (a,c) and FBB (b,d). Side views of half-cut section (a,b) and bottom views (c,d) of in situ (grey) and isolated (beige) structures are shown in solid surface representation. The ribbon models in purple are the nonameric rings of Shigella MxiAC (PDB ID 4A5P; ref. 16) (A, C) and FlhAC (b,d) fitted into the wing-like disc densities, and those in cyan are the hexameric ring model of FliI ATPase fitted into the globular densities (a,b).
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