In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography

Abstract

Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

Keywords: Legionella pneumophila; Dot/Icm; Type IV secretion systems; electron cryotomography; subtomogram averaging.

Figure 1. In situ structure of Dot/Icm T4BSS

1. A, B

   Tomographic slices through intact Legionella pneumophila cells. Black arrows point to Dot/Icm particles. Scale bar, 100 nm.

2. C

   Enlarged view of Dot/Icm particles, outer membrane (OM), and inner membrane (IM). Scale bar, 20 nm.

3. D

   Tomographic slices showing a top view of a Dot/Icm particle (white arrow head), enlarged in the inset. Scale bar, 100 nm.

4. E

   Tomographic slice through a L. pneumophila cell lacking the dot/icm genes. Scale bar 100, nm.

5. F

   Subtomogram average of wild‐type Dot/Icm particles. The subtomogram average was generated using 386 particles. OM, outer membrane; IM, inner membrane. Scale bar, 10 nm.

6. G

   Schematic representation of the subtomogram average labeling the prominent densities.

7. H

   Local resolution of the subtomogram average calculated by ResMap 29.

Figure EV1. Western blot analysis of expression of core components

Western blot showing that the Lp02 strain expresses all the core component proteins (DotFGHCD) (left lane), the dot/icm super‐deletion strain (SΔ) does not express any of the core‐complex proteins (middle lane), and the reconstituted core‐complex strain expresses all the core‐complex proteins at comparable levels to the Lp02 strain (right lane). The unrelated cytoplasmic protein isocitrate dehydrogenase (ICDH) is used as a loading control.Source data are available online for this figure.

Figure EV2. Flexibility within Dot/Icm particles

1. When a mask is used to focus alignment on the densities near the outer membrane, little density is seen around the inner membrane.

2. Similarly, when the mask is moved near the inner‐membrane part, densities near the outer‐membrane complex are barely visible.

3. Composite model generated by juxtaposing the outer‐membrane and inner‐membrane averages.

4. Relative translations found between the outer‐membrane‐aligned and inner‐membrane‐aligned regions (green dots).

Data information: OM, outer membrane; IM, inner membrane. Dotted yellow lines indicate where the outer‐membrane average is merged with the inner‐membrane average to generate the composite model. Figs 1F and 2C, and EV2C represent same image. Scale bars, 10 nm.

Figure 2. Subtomogram average of a reconstituted subcomplex

1. Subtomogram average of a reconstituted subcomplex consisting of DotC, DotD, DotF, DotG, and DotH in the dot/icm deletion mutant. The subtomogram average was generated using 261 particles. Dotted yellow lines indicate where the outer‐membrane average is merged with the inner‐membrane average to generate the composite images.

2. Difference map between the WT complex and the reconstituted core complex. Densities missing in the reconstituted complex are colored yellow, additional densities in red.

3. Outline of the core complex (orange dotted line) superimposed on the Dot/Icm structure. The same image is presented as in Fig 1F.

Data information: OM, outer membrane; IM, inner membrane. Scale bars, 10 nm.

Figure 3. Genetic organization of T4ASSs and T4BSSs

1. Organization of the T4ASS (VirB type) and T4BSS (Dot/Icm type) genes. The T4BSS has a more elaborate and complex genetic organization. Genes colored in blue are ATPases.

2. VirB10 and DotG show clear sequence similarity with both proteins having a conserved TrbI domain in their C‐terminal region.

Figure 4. Comparison between T4ASSs and T4BSSs

1. A

   Structure of the Dot/Icm complex with the outlines of existing structures of T4ASS subcomplexes (e.g., outline of the VirB10 part from the crystal structure 3JQO (red), outline of a cryo‐EM single‐particle reconstruction of purified VirB4 ATPase (yellow), and outline of a 2D average of purified Helicobacter pylori T4ASS core complex (white)) superimposed. Double‐headed arrows indicate width of each barrel. OM, outer membrane; IM, inner membrane. Scale bar, 10 nm.

2. B

   Surface representation (left) and central cross section (right) of crystal structure 3JQO, an outer‐membrane complex of parts of VirB7, VirB9, and VirB10 from the T4ASS encoded by plasmid pKM101. Red colored part of the cross section is density for VirB10, and light‐pink color is combined density for VirB7 and VirB9. The outline of VirB10 density matches the hat density of the Dot/Icm structure (red dotted line in panel A, see also Movie EV2).

3. C

   Crystal structure 3JQO fit into the VirB_3‐10 negative‐stain single‐particle reconstruction, showing its location with respect to the outer membrane 13, 15.

4. D

   Isosurface of (left) and central cross section through (right) a single‐particle reconstruction of purified VirB4 ATPase (EMD‐5505) (Pena et al 16). Because it is a hexameric barrel‐shaped structure, its cross section is two parallel rod‐like densities similar to the IM‐associated densities found in purified VirB_3‐10 complex from the R388 plasmid (panel H) and cytoplasmic densities found in the Dot/Icm structure in situ (panel A).

5. E

   2D class average images of purified H. pylori T4ASS subcomplex comprising Cag3, CagM, CagT/VirB7, CagX/VirB9, CagY/VirB10 in top and side views (reproduced from 22).

6. F

   Same side view as in (E) but rotated and enlarged to the same scale as the Dot/Icm structure. Outline marked in white dotted line and superimposed on the Dot/Icm structure in panel (A).

7. G, H

   Schematic representations of the Legionella pneumophila Dot/Icm T4BSS (G) and the Escherichia coli R388‐encoded T4ASS (H, adapted from 15) showing dimensions, underlying structural similarities, and differences.

Data information: Scale bars for all panels except (E), 10 nm. Scale bar for panel (E), 25 nm.