Recent advances in the structural and molecular biology of type IV secretion systems

Abstract

Bacteria use type IV secretion (T4S) systems to deliver DNA and protein substrates to a diverse range of prokaryotic and eukaryotic target cells. T4S systems have great impact on human health, as they are a major source of antibiotic resistance spread among bacteria and are central to infection processes of many pathogens. Therefore, deciphering the structure and underlying translocation mechanism of T4S systems is crucial to facilitate development of new drugs. The last five years have witnessed considerable progress in unraveling the structure of T4S system subassemblies, notably that of the T4S system core complex, a large 1 MegaDalton (MDa) structure embedded in the double membrane of Gram-negative bacteria and made of 3 of the 12 T4S system components. However, the recent determination of the structure of -3MDa assembly of 8 of these components has revolutionized our views of T4S system architecture and opened up new avenues of research, which are discussed in this review.

Figure 1

Schematic of the T4S system. Subunits at the right, identified with the A. tumefaciens VirB/VirD4 nomenclature, assemble as the T4S apparatus/pilus across the Gram-negative cell envelope. Hexameric ATPases establish contacts with the integral inner membrane (IM) subunits to form an inner membrane complex. VirB7, VirB9, and VirB10 form a core complex extending from the IM, periplasm, and outer membrane (OM). A domain of unspecified composition (grey bullet structure) and the pilus assemble within the central chamber of the core complex.

Figure 2

EM reconstructions showing the structure of the T4SS_3–10 complex and the core complex. (a) Front view (left) and cut-away front view (right) of the T4SS_3–10 complex (EMD-2567) comprising the core/outer membrane complex (core/OMC, green), the stalk (grey) and the inner membrane complex (IMC, blue). U-tier, M-tier and L-tier stand for upper, middle and lower tier, respectively. The inner (IM) and outer (OM) membranes are indicated. (b) pKM101 core complex (EMD-2232) (top) and truncated core complex lacking the N-terminal part of VirB10 (EMD-2233) (bottom): side view (left) and cut-away side view (right). The bottom right panel shows the superposition of the difference map (between the full-length and the truncated core complex cryo-EM maps) in green, and the cryo-EM structure of the truncated core complex in orange (as in bottom left). The VirB10 N-terminus forms the inner wall of the I-layer and the base. (c) T4SS_3–10 complex with fitted crystal structures of the VirB4 C-terminal domain from Thermoanaerobacter pseudethanolicus (PDB: 4AG5) and the pKM101 outer membrane complex (PDB: 3JQO) and in silico model of the N-terminal domain of VirB9 from pKM101 (PDB: 3ZBJ).

Figure 3

Crystal structures of the T4S system subunits and subassemblies. (a) pKM101 outer layer complex (PDB: 3JQO); (b) VirB8 periplasmic domain of Brucella suis (PDB: 2BHM); (c) VirB5 from pKM101 plasmid (1R8I); (d) cytoplasmic domain of VirD4 from R388 plasmid (PDB: 1GKI); (e) VirB11 homologue from Helicobacter pylori (PDB: 2PT7); (f) VirB4 C-terminal domain from Thermoanaerobacter pseudethanolicus (PDB: 4AG5).

Figure 4

Schematic of the T4S system showing potential substrate translocation pathways: two-step mechanism (a) and one-step mechanism (b). Only components of the A. tumefaciens VirB/D4 system that make contacts with T-DNA are indicated. See main text for details.