Mechanism and structure of the bacterial type IV secretion systems

Abstract

The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the 'archetypal' A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Keywords: Conjugation; DNA transfer; Pathogenesis; Pilus; Protein translocation; Traffic ATPase.

Fig. 1

Schematic depicting subunits and subassemblies of the A. tumefaciens VirB/VirD4 type IV secretion system (T4SS). Bottom: The 11 VirB subunits are synthesized from the virB operon and VirD4 from the separate virD operon. Lower: The VirB/VirD4 subunits are inserted in the inner membrane (IM) or delivered to the periplasm (P) with general topologies/locations as indicated. Upper: The subunits form a network of interactions resulting in four functional subassemblies: i) T4CP substrate receptor, ii) inner membrane translocase (IMC), iii) outer membrane complex (OMC)/core complex, and iv) extracellular pilus. The T4CP, IMC, and OMC interact to form the substrate translocation channel (red arrow). During translocation, the T-DNA forms formaldehyde-crosslinkable contacts with the 6 VirB/VirD4 subunits listed [38]. The IMC and OMC, together with the VirB1 transglycosylase, mediate assembly of the conjugative pilus.

Fig. 2

Examples of T4SS structural adaptations. Bottom: Gram-positive conjugation systems are composed mainly of homologs of the A. tumefaciens T4CP (VirD4) and IMC (VirB3, VirB4, VirB6, VirB8), plus a cell wall hydrolase presumptively for elaboration of a channel across the cell wall. These ‘minimized’ systems lack a VirB11 homolog, the OMC/core, and conjugative pili, and may or may have associated surface adhesins. The T4CP/IMC of the L. pneumophila Dot/Icm system has three channel ATPases, but the membrane channel is compositionally much more complex than that of A. tumefaciens. Middle: The OMC/core can vary in structure/composition. The E. coli F plasmid-encoded OMC consists of a core complex (TraV/K/B), as well as the complex of Tra/Trb proteins shown whose function is implicated in pilus extension and retraction. In the H. pylori Cag system, the presumptive core subunits are much larger than their A. tumefaciens counterparts and possess novel domains that extend beyond the cell surface and associate with extracellular pili. Top: T4SS-encoded surface organelles can vary in composition and structure in comparison to the A. tumefaciens VirB-encoded pilus. The Bartonella spp. Trw system elaborates antigenically-variable pili through use of different pilins, and the H. pylori Cag system elaborates pili that are composed of several different subunits. The sole substrate of the Cag T4SS, CagA, also associates with the Cag pilus. Some T4SSs do not elaborate pili, e.g., Gram-positive conjugation systems, or instead produce novel fibrous or sheathed structures or surface adhesins.

Fig. 3

Type IV substrate translocation signals and accessory factors. Most substrates are translocated through the T4CP-dependent pathway. DNA substrates are processed by Dtr processing factors through assembly of the relaxosome at the origin of transfer sequence (oriT). Lower left: Relaxases responsible for nicking at oriT can carry positively-charged C-terminal or internal translocation signals (TS’s) for docking with the T4CP receptor. A conserved sequence found in the TraI_R1 TSA motif and present in TSA and TSB motifs of MobA_R1162, TraI_R1, and TrwC_R388 is shown. Upper left: A model of the TSA motif from TraI_R1 generated from an X-ray structure [64]. The motif bears structural homologies to SF1B helicase domains. Mutations of residues shown in stick representation and color-coded disrupted TSA translocation function: red, significant impairment; pink, moderate impairment; yellow, no effect; blue, stimulation of TSA translocation function. Molecular graphics and analyses were performed with the UCSF Chimera package [158] using the PDB ID (4L0J) of the TraI_R1 TSA domain structure [64]. Lower right: DNA substrates and effector proteins can be recruited directly to the T4CP (Chaperone-independent) or via association with chaperones or spatial adaptors. Protein substrates can carry one or more of a combination of C-terminal, N-terminal or internal translocation signals; chaperones and spatial adaptors are in parantheses and the relevant species or plasmid is in parantheses (A.t., A. tumefaciens VirB/VirD4; L.p., L. pneumophila Dot/Icm; B.h., Bartonella henselae VirB/VirD4; H.p., H. pylori Cag). Upper right: In the T4CP-independent pathway, B. pertussis S1–S5 subunits are secreted across the inner membrane by the Sec system and assemble in the periplasm as the pertussis toxin (PT), which then docks by an unknown mechanism with the Ptl T4SS for extrusion across the outer membrane. Most T4SS substrates dock with the T4CP and are delivered in one step through the IMC/OMC channel.

Fig. 4

X-ray and CryoEM structures of T4SS machine subunits and subassemblies. The schematic shows the T4SS translocation channel and conjugative pilus. Available crystal structures of homologs of individual VirB and VirD4 T4CP subunits are shown. A CryoEM structure of the pKM101-encoded core complex and an X-ray structure of the distal half (outer layer) of the core complex along with a magnified view of the pore-forming antennae projection (AP) are presented. Structures were reproduced with permission as follows: T4CP (TrwB_R388), [86]; VirB11 (Cagβ_H.p.), Yeo et al. [111]; VirB4 (TrwK_R388), Pena et al. [105]; VirB5 (TraC_pKM101), Yeo et al. [159]; VirB8 (VirB8_B.s.), [121]; Core complex CryoEM (pKM101 TraN/TraO/TraF), Fronzes et al. [33]; Core complex X-ray (pKM101, O-layer) Chandran et al. [34].

Fig. 5

Postulated biogenesis pathway for the A. tumefaciens virB-encoded pilus. VirB2 pilin is inserted into the membrane and processed in several steps to yield a pool of cyclized pilins. VirB4, aided by VirB11, catalyzes dislocation of mature pilins and feeds the pilin monomers into the site of pilus assembly within the core complex. Pilus polymerization initiates either on an inner membrane platform composed of VirB6 and VirB8 or an outer membrane platform composed of the cap structure of the core complex. An alternative pathway proposes that pilin monomers are shunted through the periplasm to the core’s cap.