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

Abstract

Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells^1-5. 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.

Fig. 1. ATP-bound DotB displays static polar localization

(a) Schematic model depicting Dot/Icm subunits tagged with sfGFP (green filling). (b) Real-time visualization with fluorescence light microscopy of DotG-sfGFP, DotL-sfGFP, DotB-sfGFP, IcmS-sfGFP and DotI-sfGFP. The abundance of Dot/Icm fusion proteins at the poles is displayed as frequency curves. Median values of the polarity scores are presented. Significance was calculated in comparison to sfGFP expressed from dotB operon. Scale bar, 3 μm. (c) Representative images of bacterial cells expressing sfGFP or the indicated Dot proteins tagged with sfGFP. Images of a cell with polar-localized DotB-sfGFP (Pole) and cytosolic DotB-sfGFP (cytosol) are shown. Images were acquired before photobleaching (−4″), immediately after photobleaching (Bleach), and 10 seconds after photobleaching (10″). Scale bar, 1 μm (d) Graphs depict the average normalized fluorescence intensity from FRAP experiments and results are presented as means ± SEM at every time point. Significance was calculated in comparison to sfGFP expressed from dotB operon. (e) Schematic model depicting DotB-sfGFP fusion protein with the locations of point mutations that were analyzed. Point mutations that reduce ATP binding are marked with black arrows. The E191K mutation conferring enhanced ATP binding without hydrolysis is marked with a red arrow. (f) Each dot represents the polarity score for individual bacteria producing sfGFP or the indicated DotB-sfGFP protein expressed from the endogenous location on the chromosome. ΔWA and ΔWB represent Walker A and Walker B motifs deletions respectively. The black horizontal lines represent the medians of the polarity scores. Significance was calculated in comparison to DotB-sfGFP. (g) Real-time visualization of native and mutant forms of DotB-sfGFP or sfGFP. Scale bar, 3 μm. Samples sizes (n), significance values (p) and the type of statistical test used to calculate the significance in panels b, d and f are shown in Supplementary information Table 2.

Fig. 2. The ATPase DotO is essential for polar recruitment of DotB

(a) Real-time visualization of DotB_E191K-sfGFP expressed in mutants deficient in the indicated Dot or Icm components. Deletion strains were grouped according to the following subcomplexes or cellular locations: core complex (i), inner membrane (ii), coupling protein complex (iii), periplasm (iv) and cytosol (v). sfGFP was expressed from dotB operon and served as a control. Scale bar, 3μm. (b) The polarity scores of DotB_E191K-sfGFP in the indicated deletion strains. The black horizontal lines represent the medians of the polarity scores. Significance was calculated in comparison to DotB_E191K-sfGFP. (c) Representative images from 4 independent experiments of DotB-sfGFP, DotB_E191K-sfGFP and DotO-sfGFP fluorescence shown at selected time points (200, 400, 600 and 800 milliseconds). Images were acquired using a motorized spinning disc confocal microscope and were restored by the NearestNeighbors deconvolution algorithm of SlideBook™. (d) Representative Kymographs from 4 independent experiments of DotB-sfGFP, DotB_E191K-sfGFP and DotO-sfGFP fluorescence over time. Samples sizes (n), significance values (p) and the type of statistical test used to calculate the significance in panel b are shown in Supplementary information Table 2.

Fig. 3. The ATPase DotO is placed above DotB

(a) Model summarizing the hierarchy of DotO and DotB recruitment to the poles. Both proteins require the Dot/Icm machine (CC and IMC) for polar recruitment. DotB requires DotO for polar recruitment. DotL is not required for recruitment of DotO or DotB to the polar complex. (b) Real-time visualization of DotO-sfGFP expressed in strains having the indicated dot genes deleted (c) The polarity scores of DotO-sfGFP in strains having the indicated dot genes deleted. The black horizontal lines represent the medians of the polarity scores. Significance was calculated in comparison to DotO-sfGFP. (d) Real-time visualization of DotB_E191K-sfGFP co-expressed with DotO-mCherry or DotL-mCherry. Scale bar, 3μm. (e) The polarity scores of DotBE191K-sfGFP co-expressed with DotO-mCherry or DotL-mCherry. The black horizontal lines represent the medians of the polarity scores. Significance was calculated in comparison to DotBE191K-sfGFP. Samples sizes (n), significance values (p) and the type of statistical test used to calculate the significance in panels c and e are shown in Supplementary information Table 2.

Fig. 4. In situ structure of the Dot/Icm type IVB secretion machine revealed by cryo-ET and sub-tomogram averaging

(a) A tomographic slice from a representative L. pneumophila cell showing multiple Dot/Icm machines embedded in the cell envelope. Scale bar, 100nm. (b) A central section through longitudinal plane of a global average structure in situ showing the components of the type IVB machine in the context of the outer membrane (OM), the peptidoglycan (PG), and the inner membrane (IM). Scale bar, 10nm. (c) Cross-section at the position indicated (B, yellow arrow) showing 13-fold symmetric features of the OMCC. Scale bar, 10nm. (d) and (e) 3-D surface rendering of the intact Dot/Icm machine showing the architecture of the OMCC, composed of a wheel-like structure incorporated in the OM, a plug, a central hollow cylinder, and a surrounding collar. (f) A top view showing the 13-fold symmetry of the wheel.

Fig. 5. The cytoplasmic complex, showing 6-fold symmetry, composed of ATPases DotO and DotB

The cytoplasmic complex boxed in (a) was locally refined (b). (c) A reconstruction from a DotB-GFP fusion mutant, with densities corresponding to DotB (yellow arrow, see also panel B) and sfGFP (green arrow). (d) A reconstruction from a ΔdotB mutant showing the absence of density at the bottom of the inverted V structures (compare with panel b). (e) A reconstruction from a ΔdotL mutant showing no major differences compared with native T4SS (compare with panel b). (f) A ΔdotO mutant lacks the entire cytoplasmic complex. Scale bar, 10nm. 3-D surface renderings of: (g) the entire Dot/Icm T4SS, (h) cytoplasmic complex in side view, (i) side view showing DotB and sfGFP, (j) DotO hexamer of dimers in side, angled and bottom views, (k) cut-away view of entire Dot/Icm T4SS, (l and m) docking of 12 pseudo DotO C-terminal structures into the map shown as side and bottom views; a hexamer of DotB (using the homolog PilT, PDB 3JVV) was fitted into the distal short cylinder and the C terminal residues were colored in red (l).