3.A.7 The Type IV (Conjugal DNA-Protein Transfer or VirB) Secretory Pathway (IVSP) Family
Protein complexes of the IVSP family consist of multiple subunits that span the two membranes of the Gram-negative bacterial cell envelope and export proteins or DNA-protein complexes out of the cell and into the cytoplasm of a recipient cell (Saier, 2007). These systems are very promiscuous, being capable of transporting DNA-protein complexes into other bacteria, yeast and plants. The VirB system of agrobacterial species is specifically designed to transfer T-DNA into plant cells, but it can also transfer the IncQ plasmid RSF1010 into plant cells and into other agrobacterial cells. The various Inc systems are designed to mediate plasmid transfer from the donor bacterium to a recipient bacterium. While additional proteins may be required for the complete transfer process, proteins of the VirB system (VirB2-11 and VirD4) appear to be the primary ones involved in export from the cytoplasm across the two membranes of the agrobacterial envelope. VirB1 is a lytic transglycosylase involved in the local remodeling of the peptidoglycan. A type IV secretion system transport signal is present at the C terminus of VirD2 and it is necessary for virulence (van Kregten et al., 2009). This suggests a role for VirD2 as a pilot protein driving translocation of the T-strand.
T4SSs have undergone extensive modular evolution through recombination, and the resulting mosaicism complicates phylogeny-based classification. Alvarez-Martinez and Christie (2009) classify T4SSs on the basis of function as conjugation machines, effector translocators, or DNA release/uptake systems, but this approach has its limitations, because conjugation systems also translocate protein substrates independently of DNA, and some effector translocator systems also conjugatively transfer DNA to target cells. Recent work suggests that all prokaryotic T4SSs possess several common mechanistic features, and many have also acquired novel properties for specialized purposes (Alvarez-Martinez and Christie, 2009).
VirB1 consists of two domains. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell. Both domains are required for wild-type virulence. VirB1* is essential for the formation of the T pilus, a subassembly of the IVSP complex, composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit) (Zupan et al., 2007). Thus, VirB1 is a bifunctional protein required for IVSP assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the IVSP complex while the C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.
The export process appears to be driven by ATP hydrolysis. Substrates transferred to the plant cell cytoplasm include VirE2, VirF and the VirD2-T-DNA complex. Two ATPases, VirB4, and VirB11, localized to the cytoplasmic side of the inner membrane, may energize the process. VirB4 together with VirB6 and VirB10 may comprise the integral membrane transport pore. VirB8, VirB9 and VirB10 may also span the cytoplasmic membrane as well as the periplasm and the outer membrane. The latter three proteins may connect the inner membrane complex with the outer membrane complex consisting of disulfide linked VirB7 and VirB9. Thus, a minimum of six proteins comprise the structural and catalytic elements of the bimembrane channel complex. Proteins covalently linked to the DNA may be recognized as a prelude to nucleoprotein complex transport. The VirE2 and VirF proteins of Agrobacterium tumefaciens have been shown to be transformed with the DNA to plants. VirB4, B8, B5 and B2 have been shown to interact. VirB4 interacts and stabilizes VirB8 and mediates incorporation of VirB5 and VirB2 into external pili (Yuan et al., 2005). VirB6 may function in T-pilus biogenesis and as a component of the IV SP apparatus. It interacts with VirB3 and VirB9 but not VirB4, 5 or 11. (Judd et al., 2005).
The twelve proteins of the vir-encoded T4SS can be divided into several subgroups: (1) the transfer pilus (T pilus) composed of VirB2 (the major pilin subunit), VirB5 (a minor pilin subunit), and VirB7 (a lipoprotein that may help anchor the pilus to the outer membrane); (2) the trans envelope proteins VirB6-VirB10; (3) three inner membrane-associated proteins with nucleotide binding domains (VirD4, VirB4, and VirB11), which likely energize transporter assembly or substrate translocation; and (4) proteins with additional specific or unknown functions such as (a) VirB1 (a two-domain protein with two proposed functions, peptidoglycan lysis to assemble the trans envelope core structure and plant interaction via the secreted VirB1 fragment VirB1*), (b) VirD4 (thought to couple the DNA substrate to its cognate T4SS transporter), and (c) VirB3, a pilin-like protein required for the assembly of the T-pilus and for T-DNA secretion. VirB3 has 2 TMSs with both ends in the cytoplasm (Mossey et al., 2010). Stabilization of VirB3 requires VirB4, VirB7 and VirB8 (Mossey et al., 2010).
The Agrobacterium tumefaciens VirB/D4 T4SS transfers oncogenic T-DNA to plant cells, transferred as a nucleoprotein T-complex with VirD2 as the pilot protein. As a derivative of plasmid conjugation systems, the VirB/D4 T4SS can also transfer certain mobilizable plasmids and bacterial proteins like VirE2 and VirF. A cytoplasmic VirD2-binding protein (VBP) is involved in the recruitment of the T-complex to the energizing components of the T4SS, including VirD4, VirB4, and VirB11 (Guo et al., 2007). VBP is also important for the recruitment of a conjugative plasmid to a different transfer system independent of VirB/D4. Thus, VBP functions as a recruiting protein that helps couple nucleoprotein substrates to the appropriate transport sites for conjugative DNA transfers (Guo et al., 2007).
Draper et al., 2006 have studied the VirB4 ATPase and its protein interactions. Both VirB4 and VirB11 are probably hexamers, and the C-terminus of VirB4 interacts with the N-terminus of VirB11 such that the VirB4 C-terminus stacks above VirB11 in the periplasm. VirB4 localizes to the membrane and periplasm, not cytoplasm. The C-terminus of VirB4 also interacts with VirB1, B8 and B10. These observations and others allowed Draper et al., 2006 to propose a structural model for the complex. Interestingly, VirB3-VirB6 and VirB8-VirB11, but not VirB7, are essential for mediating persistence of Brucella in the reticuloendothelial system (den Hartigh et al., 2008).
Agrobacterium tumefaciens VirB10 couples ATP consumption to substrate transfer through the VirB/D4 type IV secretion channel (TVSP) and also mediates biogenesis of the virB-encoded T pilus. Jakubowski et al., 2009 determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain.Their results indicated that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. Distinct domains of VirB10 regulate formation of the secretion channel or the T pilus (Jakubowski et al., 2009 ).
The VirB system of A. tumefaciens is related to (1) a natural competence (CAG; ComB) system in Helicobacter pylori, which may also be involved in transfer of virulence factors including the CagA antigen into host animal cells by a type IV secretion system, (2) the TraS/TraB system of the Pseudomonas aeruginosa conjugative plasmid, RP1, (3) the Ptl system, involved in secretion of pertussis toxin of Bordetella pertussis, (4) the trb system from plasmid pTiC58, one of three loci required for conjugal transfer of this Ti plasmid, (5) the Tra system of plasmid F in E. coli, and the Dot conjugative transfer/virulence system of Legionella pneumophila. Although members of the type IV secretion family share many characteristics, not all systems contain the same sets of genes. Thus, the VirB system of Ti plasmids and the trb system of RP4 have only six genes in common. The distantly related CAG system of H. pylori contains only 4 trb homologues, and the Dot system of L. pneumophila contains only 2 recognizable VirB homologues. Homologues of only one VirB protein, VirB10 (TrbI), are present in all known type IV secretion systems characterized.
Distant homologues of many components of the VirB type IV protein secretion system of Agrobacteria are involved in natural competence in Helicobacter pylori (Hofreuter et al., 2003). These proteins are the ComB2-B4 and ComB6-B10 proteins, distantly related to VirB2-B4 and VirB6-B10 of Agrobacteria. Other VirB constituents of Agrobacteria have not yet been identified or shown to be involved in competence (Karnholz et al., 2006).
Helicobacter pylori use a type IV secretion system to deliver CagA into eukaryotic cells to induce interleukin-8 secretion. This system has a filamentous surface organelle with a rigid needle covered by CagY, a VirB10 homologue. CagY also clusters in the outer membrane while a VirB7 homologue is found at the base of the organelle (Rohde et al., 2003). The structure resembles the needle-like structures of type III secretion systems (TC #3.A.6).
VirD4 of Agrobacteria and TrwB of the E. coli R388 conjugative system are known to play a role in DNA transfer. TrwB is an integral membrane protein (507 aas) with two N-terminal TMSs and a large C-terminal cytoplasmic domain of about 450 residues. It forms a hexameric structure with six equivalent monomers forming an almost spherical quaternary structure like the F1-ATPase (TC# 3.A.2) and a central channel, 20 Å in diameter (Gomis-Rüth et al., 2001). It has two domains, an α/β nucleotide-binding domain reminiscent of RecA and DNA ring helicases, and an all-α domain. It resembles the SpoIIIE family FtsK protein (TC# 3.A.12). Like these proteins, TrwB and VirD4 might energize (or contribute to the energization of transfer by 'pushing' the DNA through the conjugation pore). These proteins are very distantly related to the VirB4 ATPase of Agrobacteria.
The TraG (RP4), TraG or VirD4 (A. tumefaciens Ti plasmid), TraD(F) and HP0524 (H. pylori) components of type IV protein secretion systems have been shown to form oligomers and bind DNA without sequence specificity (Schröder et al., 2002). Although they exhibit NTP binding motifs, attempts to demonstrate NTP hydrolysis activity have been unsuccessful. TraG and the TraI relaxase of plasmid RP4 have been shown to interact. TraG of RP4 is a 650 residue transmembrane protein with 4 N-terminal TMSs in the first 150 residues of the protein and possibly as many as two additional TMSs in the large C-terminal, hydrophilic domain. The N- and C-termini are in the cytoplasm. Schröder et al. (2002) propose that these proteins form a multimeric transmembrane pore to which the relaxosome binds via TraG-DNA and TraG-TraI interactions. The complete system consists of sub-complexes (Krall et al., 2002; Ward et al., 2002).
The two pathogenic γ-proteobacteria, Legionella pneumophila and Coxiello burnetii, use a functionally homologous secretion system for pathogenesis. The prototype of this so-called type IVB secretion system is the L. pneumophila Icm/Dot system. The IncI plasmid Tra/Trb system (such as that of plasmid R64) is also of this IVB type. They are very sequence divergent from the type IVA system (Lammertyn and Anne, 2004; Segal et al., 2005). Twenty-five constituents of the Icm/Dot system have been identified although not all have been shown to be important for secretion.
The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Vincent et al., (2006) have reported the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. A critical five-protein subassembly spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a conservation of the core subassembly in these evolutionarily distant type IV secretion machines.
Conjugative plasmids use a type IV ssDNA-protein translocase coupled to nucleoprotein complex (the relaxosome). TrwB is a membrane-bound homohexamer with a central pore. It hydrolyzes ATP with positive cooperativity with respect to ATP concentration and is a DNA-dependent ATPase (Tato et al., 2005). It may be the DNA-translocating motor.
Neisseria gonorrhoeae secretes chromosomal DNA via a modified type IV secretion system to the growth medium without a migration partner (Hamilton et al., 2005). The process of DNA donation for natural transformation of bacteria has been assumed to involve bacterial cell death. However, in Neisseria gonorrhoeae, mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. The GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. This model island could be integrated in strains not carrying the GGI and spontaneously excises from that site. The GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. Mutations in six putative type IV secretion system genes were assayed for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Thus, N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance (Hamilton et al., 2005).
As noted in the previous paragraphs, the Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA for natural transformation. Salgado-Pabon et al. showed that Tyr93 in TraI is required for DNA secretion while Asp120 is required for wild-type levels of DNA secretion. The TraI N-terminal region promotes membrane interaction. Possibly Tyr93 initiates DNA processing. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion (Salgado-Pabon et al., 2007). The neisserial DNA binding components are PilQ and PilG (Lang et al., 2009).
Fronzes et al. (2009) reported the 15Å resolution cryo-electon microscopy structure of the core complex of a T4SS (IVSP) system. The core complex is composed of three proteins, VirB7, VirB9 and VirB10, each present in 14 copies and forming an approximately 1.1-megadalton two-chambered, double membrane-spanning channel. The structure is doubled-walled, with each compenent apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and compostion from the other known double membrane-spanning secretion system that has been structurally characterized.
The I and O layers insert in the inner and outer membrane, respectively. Chandran et al., 2009 solved the crystal structure of an approximately 0.6 MDa outer-membrane complex containing the entire O layer, the largest determined for an outer-membrane channel. VirB10 is the outer-membrane channel with a hydrophobic double-helical transmembrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-electron microscopy (cryo-EM) and crystallographic structures points to conformational changes regulating channel opening and closing (Chandran et al., 2009).
The overall transport reaction catalyzed by IVSP family complexes is thus believed to be:
Protein or nucleoprotein (donor cytoplasm) + ATP (in) → Protein or nucleoprotein
(recipient cytoplasm or medium) + ADP (in) + Pi (in).
