Structure of a type IV secretion system

Abstract

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.

Figure 1. Purification of the R388 encoded T4SS_3-10 complex

a) SDS-PAGE analysis of the T4SS_3-10 complex. * indicates minor contaminants (from top to bottom: OmpF/OmpA, Dihydrolipoyl dehydrogenase (DLD), single-stranded DNA-binding protein (SSB), and lysozyme). b) Overview negative stain EM image and representative characteristic views (class averages) of the T4SS_3-10 complex with a schematic describing the nomenclature of observed structure components. Blue arrow indicates region of high flexibility.

Figure 2. Asymmetric composite structure of the T4SS_3-10 complex

a) Front view. The map is a composite generated by merging independently processed core complex and IMC reconstructions. b) Cut-away front view. Electron density is color-coded ranging from red to blue indicating regions of strong to weak density, respectively. The IMC has pseudo 2-fold symmetry around the particle long axis. c) Side view. U, M and L tier substitute for upper, middle and lower tier, respectively.

Figure 3. Segmentation of the T4SS_3-10 complex reconstruction

a) Side, front and bottom views. Of the two barrels, only the left one is segmented. The colour scheme used is upheld in all panels. b) Zoom cut-away view of the core complex and stalk. Dotted red line delineates the border at which the separate core complex and IMC reconstructions were merged (left). Central cross section schematic of the core complex from this study (right). c) Zoom side view of the stalk. Some of the linkers between the core complex and IMC are flexible and were therefore poorly resolved. d) Cut-away top view of the stalk and arches. e) Each barrel-like density consists of three dimeric elongated segments. Cross-sections of the lower and middle tiers (right panel) show 3-fold symmetry with a trimer of dimeric densities present in the middle tier.

Figure 4. Stoichiometric analysis and localisation of various macromolecular components within the T4SS_3-10 complex reconstruction

a) ¹²⁵Iodine labelling of T4SS_3-10/His6-B6 complex constituent proteins. Left: SDS-PAGE of the T4SS_3-10/His6-B6 complex. * indicates minor contaminants (OmpF/OmpA and Lpp). Right: SDS-PAGE analysis of ¹²⁵I-labelled proteins in left lane (Coomassie) and corresponding radiograph in right lane (¹²⁵I labelling). Relative stoichiometry was calculated by integration of band intensity and is shown at right. Reported means and corresponding standard deviations are from two separate labelling experiments on four independent purifications. b) SDS-PAGE of the VirB4/TrwK and VirB3/TrwM complex stained with SYPRO Ruby. Relative stoichiometry was calculated by integration of band intensity and is shown at right. Reported means and corresponding standard deviations are from two independent purifications. c) 5 nm gold labelling of VirB4/TrwK clusters around the IMC barrels. Scale bar equals 10 nm. d) 5 nm gold labelling of VirB6/TrwI shows a similar localisation pattern to that of VirB4/TrwK (see c). Scale bar equals 10 nm. e) Fit of the VirB4 ATPase domain from T. pseudethanolicus (4AG5), the pKM101 outer membrane complex (3JQ0), and in silico model of VirB9/TraO (3ZBJ). f) Summary schematic showing the localisation of known components and position of cell membranes. For clarity only VirB nomenclature is used in the colour key.