Structural and functional diversity of type IV secretion systems

Abstract

Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.

Fig. 1 |. The functional versatility of type IV secretion systems.

Various pathogenic bacteria and symbionts deploy type IV secretion systems (T4SSs) to deliver effector proteins, DNA–protein complexes or other macromolecules into eukaryotic or protozoan host cells. a, The T4SS establishes contact-dependent interkingdom interactions by injecting effectors directly into eukaryotic cells to promote bacterial intracellular survival and symbiosis. b, Many bacterial species and a few Archaea deploy a contact-dependent T4SS for the delivery of DNA and toxins to other bacteria or Archaea. Various species in the Xanthomonadales instead deploy T4SSs for the contact-dependent delivery of protein toxins to kill other bacteria for niche establishment. c, Some bacteria can deploy T4SSs for the contact-independent uptake or release of DNA. ssDNA, single-stranded DNA.

Fig. 2 |. Structure of minimal type IV secretion system and pilus biogenesis mechanism model.

a, Global organization to atomic details of the minimized R388-encoded type IV secretion system (T4SS). The entire cryo-electron microscopy (cryo-EM) structure of the R388 T4SS is shown with half-left in cryo-EM density coloured by sub-complexes (Electron Microscopy Data Bank (EMDB) entry 12707, EMDB 12708, EMDB 12709, EMDB 13767 and EMDB 12933) and half-right in ribbon and surface semi-transparent representation coloured by proteins (Protein Data Bank (PDB) identifier (ID) 7O3J, PDB ID 7O3T, PDB ID 7O3V, PDB ID 7Q1V and PDB ID 7OIU). In the top-left corner, the cryo-electron tomography (cryo-ET) of pKM101 T4SS density (EMDB 24098 and 24100) coloured by sub-complexes is displayed to show that the structure of purified R388 T4SS is similar to the in situ T4SS structure. For each sub-complex, structure details, symmetry and membrane localization are indicated. Black dashed lines demarcate the boundaries of the outer membrane and inner membrane. b, Model of pilus biogenesis mechanism. The T4SS is schematically represented in slice view and coloured by protein. Four states are shown: (1) T4SS in similar state to that observed by cryo-EM and shown in part a. (2) The pilus biogenesis state with VirB11 bound at the bottom of VirB4; VirB2 is extracted from the inner membrane and recruited to VirB6 through the coordinated actions of the VirB4-VirB11 ATPases. (3) As layers of VirB2 are recruited, the pilus grows from the VirB6 assembly sites and VirB5 remains at the pilus tip. (4) As the pilus grows, the O-layer of the outer membrane core complex (OMCC) opens up and the pilus with VirB5 at the tip extends into the extracellular milieu to establish contact with potential recipient cells. IMC, inner membrane complex. Part a adapted from ref. , CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).

Fig. 3 |. Structural organization of expanded type IV secretion system.

a, Three expanded type IV secretion system (T4SS) outer membrane core complex (OMCC) structures are shown. The OMCCs of the F plasmid (Protein Data Bank (PDB) identifier (ID) 7OKN and PDB ID 7OKO), Legionella pneumophila (PDB ID 7MUS) and Helicobacter pylori (PDB ID 6X6S and 6X6J) are shown in surface representation and coloured in dark red, blue and green for the VirB7-like, VirB9-like and VirB10-like proteins, respectively, and in grey for other components. Notably, L. pneumophila and H. pylori OMCCs contain an outer membrane cap (OMC) and a periplasmic ring (PR). b, Cryo-electron tomography (cryo-ET) maps of the F plasmid with and without pilus. The maps (Electron Microscopy Data Bank (EMDB) entry 9344 and 9347) are coloured by sub-complexes (that is, green for the OMCC, red for the stalk, yellow for the arches, blue for the inner membrane complex (IMC) and grey for the pilus). The junction of the pilus and stalk is not well defined. c, Cryo-ET map of the L. pneumophila T4SS (EMDB entry 7611 and 7612) coloured as in part b, in front and slice view. d, Cryo-ET of the H. pylori T4SS (EMDB entry 0634 and 0635) coloured as in part b, in front and slice view.

Fig. 4 |. Examples of type IV secretion system subunit adaptations for functional diversification.

The R388-encoded type IV secretion system (T4SS) is shown on the left for reference. VirB5 subunits are deployed for binding of target-cell receptors; these subunits can localize at the tips of conjugative pili or on the bacterial cell surface. Some bacteria encode several copies of VirB2 or VirB5 subunits whose variable sequences are postulated to bind different target-cell receptors or contribute to evasion of the host immune system. Extended VirB6 subunits carry large hydrophilic domains, several of which have been shown or are implicated in localizing at the cell surface to promote adhesion or immunomodulation, or blocking redundant plasmid transfer. F systems elaborate F pili that dynamically extend and retract to establish contacts with potential recipient cells at a distance. F systems also code for TraN subunits, whose extracellular domains interact with outer membrane proteins (OMPs) on recipient cells to promote F plasmid transfer and specify plasmid host range. Several T4SSs possess variant forms of the VirB7–VirB9–VirB10 core complex subunits, as exemplified for CagY in the Helicobacter pylori Cag system. The H. pylori system elaborates a conjugative pilus, which is decorated by other Cag subunits and the CagA secretion substrate. Various T4SSs functionally interact with other surface adhesins, such as pKM101-encoded Pep or H. pylori OMPs, to promote target-cell binding.

Fig. 5 |. Structure of VirD4-like and VirB11-like ATPases.

a, Side view (left) and top view (right) of two VirD4-like ATPase structures: hexameric TrwB from R388 plasmid (Protein Data Bank (PDB) identifier (ID) 1GKI) and hexameric Cagβ from Helicobacter pylori (PDB ID 8DOL). Structures are shown in ribbon representation and coloured by monomer. b, Side view (left) and top view (right) of two VirB11-like ATPase structures; hexameric DotB from Legionella pneumophila (PDB ID 6GEF) and hexameric Cagα from H. pylori (PDB ID 1NLZ). Structures are shown in ribbon representation and coloured by monomer. c, Organization and structure of type IV coupling complex (T4CC) from L. pneumophila. The monomeric cryo-electron microscopy (cryo-EM) structure of the T4CC (PDB ID 6SZ9) is shown in surface representation and coloured by protein: VirD4-like DotL in red, DotM in cyan, DotN in blue, DotZ in yellow, DotY in green, IcmS in pink and IcmW in purple. The module made up of the extreme C terminus of (DotL_C-ter) and IcmSW is flexible, and its motion area is represented in mesh. Crystal structure of the DotL_C-ter–IcmSW module in the presence of LvgA adaptor (orange) and VpdB substrate (black) is shown (PDB ID 7BWK). On the right, a model of the hexameric T4CC structure is shown in top view using the same colour coding as in part c.

Fig. 6 |. Models for substrate recruitment and transport through the type IV secretion system.

a, Conjugative type IV secretion system (T4SS) recruitment and secretion mechanism model. Silhouette of F plasmid T4SS is shown in blue. First, the DNA is processed by a relaxosomal complex made of TraM (dark green), TraY (turquoise), IHF (purple and dark blue) and TraI (light green). The relaxosome is recruited by TraD_VirD4 ATPase, which energizes the secretion of the TraI–single-stranded DNA (ssDNA) through the T4SS apparatus into the host. The relative position of TraD_VirD4 and the global organization of the inner membrane complex during DNA secretion are unknown. b, T4SS effector recruitment and secretion mechanism model. Silhouette of Legionella pneumophila Dot/Icm T4SS is shown in blue. The type IV coupling complex (T4CC) acts as an effector recruitment platform and is schematically represented and positioned beside the complex formed by the hexameric dimers of DotO and DotB, although its precise localization is unknown. Effector proteins are captured by the T4CC at different binding sites and DotL_VirD4 energizes substrate translocation via one of two possible routes across the inner membrane. Route 1: the T4CC feeds substrates into the DotO–DotB energy centre at the base of the T4SS channel for transit in one step across the entire cell envelope. Route 2: the T4CC feeds substrates into the lumen of the DotL hexamer for delivery across the inner membrane. In a second translocation step, substrates are recruited from the periplasm by the T4SS channel for passage to the cell surface and into target cells. Part b adapted with permission from ref. , Microbiology Society.

Fig. 7 |. Structure comparison between minimal, expanded and archaea pilus.

a, Side view of all known pilus structures. Pilus structures (Protein Data Bank (PDB) identifier (ID) 8EXH Agrobacterium tumefaciens T pilus, PDB ID 8CW4 Escherichia coli N pilus, PDB ID 5LER E. coli F pilus, PDB ID 5LEG Salmonella enterica subsp. enterica serovar Typhimurium pED208, PDB ID 7JSV Klebsiella pneumoniae pKpQIL, PDB ID 8DFU Aeropyrum pernix CedA1 and PDB ID 8DFT Pyrobaculum calidifontis TedC) are in surface representation with one strand coloured in grey. b, Top view of pilus structures. Diameter and lumen sizes are indicated. c, For each pilus, one monomer of VirB2 with its lipid is shown in ribbon representation. The arrows in the minimized type IV secretion systems (T4SSs) highlight the presence of a ‘kink’, which is characteristic of this group. Parts a and b adapted from ref. , CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).