Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.     

The VirB/VirD4 type IV secretion system of Bartonella is essential for establishing intraerythrocytic infection

Bartonellae are pathogenic bacteria uniquely adapted to cause intraerythrocytic infection in their human or animal reservoir host(s). Experimental infection of rats by Bartonella tribocorum revealed the initial colonization of a yet unidentified niche outside of circulating blood. This primary niche periodically seeds bacteria into the bloodstream, resulting in the invasion and persistent intracellular colonisation of erythrocytes. Here, this animal model was used for a genetic analysis of the virB locus (virB2-11) and the downstream located virD4 gene, which together encode a putative type IV secretion system (T4SS). A generic method for marker-less gene replacement allowed the generation of non-polar in-frame deletions in either virB4 or virD4. Both mutants were unable to cause bacteraemia, whereas complementation with the full-length genes in trans completely restored infectivity. Segregation analysis of the complementation plasmids further denoted that VirB4 and VirD4 are required at an early stage of the infection course before the onset of intraerythrocytic bacteraemia. This analysis of defined mutants in an in vivo model identified components of the VirB/VirD4 T4SS as the first bona fide pathogenicity factors in Bartonella.     

VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram-negative bacteria. We reported previously the structure of the ADP-bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N-terminal domains exhibit a collection of rigid-body conformations. Nucleotide binding 'locks' the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide-dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Biochemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.     

DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.     

Agrobacterium tumefaciens VirB6 protein participates in formation of VirB7 and VirB9 complexes required for type IV secretion

This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28 degrees C, a temperature that favors VirB protein turnover, but not by cells grown at 20 degrees C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.     

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system (T4SS) delivers effector proteins and DNA to plant cells during infection (1, 14). The 11 VirB proteins and VirD4 substrate receptor mediate assembly of the envelope-spanning translocation channel, whereas the VirB proteins independently of VirD4 are required for polymerization of the extracellular T pilus (6, 32, 46). These T4SS subunits include the three ATPases VirD4, VirB4, and VirB11; a trans-envelope core complex comprised of VirB7, VirB9, and VirB10; subunits involved in assembly or spatial positioning of the core complex (VirB1, VirB6, and VirB8); and other structural components (VirB2 pilin, VirB3, and pilus-associated VirB5) (1, 14, 43, 48, 55, 70). The VirB/VirD4 subunits are conserved among many Gram-negative bacterial T4SSs, and recent structures of homologs of VirD4, VirB5, VirB8, VirB10, and VirB11 and a VirB7/VirB9/VirB10 machine subassembly are supplying exciting new information about T4SS machine architectures (11, 28, 29).The pilin subunit VirB2 is a component of both the secretion channel and T pilus (39, 47, 48). Its role in substrate transfer was established with a modified chromatin immunoprecipitation (ChIP) assay termed transfer DNA (T-DNA) immunoprecipitation (TrIP), wherein the pilin (but not the T pilus) was shown to form formaldehyde-cross-linkable contacts with the translocating T-DNA substrate (10). TrIP studies with virB mutant strains also supplied evidence that VirB2 occupies a distal portion of the translocation channel near or at the outer membrane (OM) (10). Complementary genetic studies identified mutations in several VirB subunits, including VirB6, VirB9, VirB10, and VirB11, that selectively block T pilus production without affecting substrate transfer (39, 40, 41, 62). These Tra⁺ Pil⁻ “uncoupling” mutations do not bypass the requirement for VirB2 production for substrate transfer, as the further deletion of virB2 from the Tra⁺ Pil⁻ mutant strains renders these strains transfer defective (39, 41, 62). Therefore, VirB2 pilin, but not an intact T pilus, is required for passage of substrates to target cells.The pathways culminating in the integration of VirB2 into the two terminal organelles, the secretion channel and T pilus, are fundamentally poorly understood. The early VirB protein-independent reactions involve insertion of the 12.3-kDa propilin into the inner membrane (IM); cleavage of a long, 47-residue signal sequence, presumably by LepB signal peptidase; and covalent joining of the N-terminal Gln48 and C-terminal Ser121 to form the mature, cyclic pilin (24). This unusual head-to-tail cyclization reaction was also shown for the VirB2 homolog, TrbC (24/51% sequence identity/similarity) of plasmid RP4 (24, 34, 44). Other VirB2 homologs, such as F plasmid TraA (19/47% identity/similarity) (67), remain linear although their N termini are modified by N acetylation (54).Prevailing models suggest that mature forms of conjugative pilins accumulate in the IM as pools for use in assembly of the channel/pilus upon receipt of an unknown morphogenetic signal(s). The IM-integrated VirB2, TraA_F, and TrbC_RP4 pilins likely adopt similar topologies, as deduced from similar predicted secondary structures and results of reporter fusion studies with periplasmically active alkaline phosphatase (PhoA) (5, 22, 56). Two hydrophobic domains are thought to orient across the IM so that a small, intervening hydrophilic loop is cytoplasmic and the hydrophilic N and C termini are periplasmic. Detailed studies confirming this overall topology are lacking, and limited information exists regarding the nature of pilin interactions with other T4SS subunits (36, 51). Furthermore, little is known about the mechanism or energetic requirements for dislocation of membrane-integrated forms of conjugative pilins during machine morphogenesis.In A. tumefaciens, mutations in the Walker A nucleoside triphosphate (NTP) binding site motifs of the VirB4 and VirB11 ATPases render cells defective for substrate transfer and pilus production, indicating that NTP energy consumption by both ATPases is essential for assembly of the two terminal organelles (6, 7, 58, 62, 68). VirB4-like subunits are signatures of all T4SSs described to date, whereas VirB11-like proteins are common but not ubiquitous among the T4SSs (1). Some T4SSs, such as the conjugation machines encoded by Escherichia coli F-like plasmids, lack VirB11 homologs, and yet their conjugative pili extend and retract dynamically by a mechanism(s) dependent on VirB4 homologs (18, 65). On the basis of these observations, it is reasonable to propose that the VirB4-like subunits catalyze early reactions associated with assembly of conjugative pili.Here, we used the scanning cysteine accessibility method (SCAM) (9) to define the IM topology of cyclized VirB2. We then assayed for contributions of VirB subunits to the pilin structural organization. We present biochemical evidence for VirB4-mediated dislocation of VirB2 pilin from the membrane and also for a contribution by VirB11 in modulating pilin tertiary or quaternary structure. We discuss our findings in the context of recent advances in our understanding of T4SS machine assembly and architecture.     

Topology of the VirB4 C terminus in the Agrobacterium tumefaciens VirB/D4 type IV secretion system

Gram-negative type IV secretion systems (T4SSs) transfer proteins and DNA to eukaryotic and/or prokaryotic recipients resulting in pathogenesis or conjugative DNA transfer. VirB4, one of the most conserved proteins in these systems, has both energetic and structural roles in substrate translocation. We previously predicted a structural model for the large C-terminal domain (residues 425-789) of VirB4 of Agrobacterium tumefaciens. Here we have defined a homology-based structural model for Agrobacterium VirB11. Both VirB4 and VirB11 models predict hexameric oligomers. Yeast two-hybrid interactions define peptides in the C terminus of VirB4 and the N terminus of VirB11 that interact with each other. These interactions were mapped onto the homology models to predict direct interactions between the hexameric interfaces of VirB4 and VirB11 such that the VirB4 C terminus stacks above VirB11 in the periplasm. In support of this, fractionation and Western blotting show that the VirB4 C terminus is localized to the membrane and periplasm rather than the cytoplasm of cells. Additional high resolution yeast two-hybrid results demonstrate interactions between the C terminus of VirB4 and the periplasmic portions of VirB1, VirB8, and VirB10. Genetic studies reveal dominant negative interactions and thus function of the VirB4 C terminus in vivo. The above data are integrated with the existing body of literature to propose a structural, periplasmic role for the C-terminal half of the Agrobacterium VirB4 protein.     

Autoinhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS.     

VirE2, a type IV secretion substrate,interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens

Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB-encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.     

Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.Bacteria use type IV secretion (T4S) to deliver macromolecules to prokaryotes and eukaryotes (12). Animal and human pathogens deliver proteins to their eukaryotic hosts to affect cellular processes causing disease. The plant-pathogenic bacterium Agrobacterium tumefaciens delivers both proteins and DNA to plants and other eukaryotes. DNA delivered by Agrobacterium directs constitutive synthesis of phytohormones in a transformed plant cell, promoting cancerous growth (56). The Ptl toxin of Bordetella pertussis modifies G proteins by ADP-ribosylation, affecting intracellular cell signaling, and CagA of Helicobacter pylori disrupts epithelial cell polarity by inhibiting PAR1 kinase activity (37, 44, 47). T4S is ancestrally related to bacterial conjugation, a mechanism used by bacteria for interbacterial plasmid transfer, enabling them to acquire novel genes for antibiotic resistance, degradation of organic molecules, toxin production, and other virulence traits (29).The VirD4/VirB family of proteins, found conserved in many alphaproteobacteria, mediates T4S (12). The Ti plasmid-encoded Agrobacterium T4S system requires VirD4 and 11 VirB proteins, VirB1 to VirB11, for efficient DNA transfer (7, 54). The membrane and membrane-associated VirB proteins assemble a macromolecular structure at the cell membrane to promote substrate transfer (12). The octopine Ti plasmid pTiA6NC-encoded VirB6 to VirB11 proteins assemble the T4S apparatus at a cell pole (34, 35, 39). The VirD4 coupling protein targets the VirE2 substrate protein to the cell pole (4). A recent study found that the nopaline Ti plasmid pTiC58 T4S system (T4SS) and its substrates form a helical array around the cell circumference (1). Structural studies using Escherichia coli conjugative plasmid pKM101-encoded VirB homologues showed that TraN (VirB7), TraO (VirB9), and TraF (VirB10) form the core complex and that TraF forms a channel at the outer membrane (11, 23). The Agrobacterium VirB proteins assemble a T-pilus, an appendage composed primarily of VirB2, with VirB5 and VirB7 as its minor constituents (38, 40, 41, 48, 50, 55). VirB3, a homolog of the pilin-like TraL protein encoded in E. coli plasmids, is postulated to function in T-pilus assembly (52). Three ATP-utilizing proteins, VirB4, VirB11, and VirD4, supply energy for substrate translocation (3, 9, 34).The membrane topology of all the VirB proteins, except for VirB3, was determined by analyses of random phoA insertion mutants, targeted phoA fusions, and targeted bla fusions (6, 14, 15, 21, 22, 31, 35, 53). phoA and bla, which encode alkaline phosphatase and β-lactamase, respectively, serve as excellent markers for periplasmic proteins, as they are enzymatically active only when targeted to the cell periplasm (8, 30). Green fluorescent protein (GFP) is an ideal cytoplasmic marker because it fluoresces only when located in the cytoplasm (19, 20). When GFP is targeted to the periplasm through fusion with a membrane-spanning domain (MSD), it fails to fold properly and does not fluoresce.The prevailing view, based on in silico analysis, is that VirB3 is a bitopic membrane protein with a periplasmic C terminus. No phoA-positive insertions in virB3, however, were identified in two random mutagenesis studies of the virB operon (6, 15). The small size of VirB3, a polypeptide of 108 amino acids (aa), could be a contributing factor to the negative findings. Yet several PhoA-positive insertions in two smaller VirB proteins, VirB2 (74-aa mature peptide) and VirB7 (41-aa mature peptide), were successfully obtained in both studies. Therefore, the negative findings may also be indicative of the presence of a small periplasmic domain in VirB3. Biochemical studies showed that the nopaline Ti plasmid pTiC58-encoded VirB3 protein (pTiC58 VirB3) associates with the bacterial outer membrane, while VirB2 associates with both the inner and outer membranes (52). The pTiC58 VirB4 protein is required for localization of VirB3 to the outer membrane (33). VirB4 is also required for VirB3 stability (33, 55). A low level of VirB3 accumulated in a nonpolar pTiC58 virB6 deletion mutant; however, addition of virB6 in trans did not restore the level of the protein, even though it restored tumorigenicity (27). VirB3 participates in the formation of protein complexes with the T-pilus proteins VirB2 and VirB5 (55).Homologues of VirB3 are found in many alphaproteobacteria with a T4SS. While most VirB3 homologues are small proteins, several recently identified homologues are fusions of VirB3 and the immediate downstream protein VirB4 (5, 10, 24). These fusion homologs, which include Actinobacillus MagB03 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAG24434","term_id":"10880920","term_text":"AAG24434"}}AAG24434), Campylobacter CmgB3/4 ({"type":"entrez-protein","attrs":{"text":"EAQ71805","term_id":"87248841","term_text":"EAQ71805"}}EAQ71805), Yersinia pseudotuberculosis TriC ({"type":"entrez-protein","attrs":{"text":"CAF25448","term_id":"51591645","term_text":"CAF25448"}}CAF25448), Citrobacter koseri PilX3-4 ({"type":"entrez-protein","attrs":{"text":"ABV12046","term_id":"157082368","term_text":"ABV12046"}}ABV12046), and Klebsiella pneumoniae PilX3-4 ({"type":"entrez-protein","attrs":{"text":"BAF49490","term_id":"134048868","term_text":"BAF49490"}}BAF49490), have VirB3 at the N terminus and VirB4 at the C terminus. Agrobacterium VirB4 is an integral membrane protein with a cytoplasmic N terminus (14). Its homologues are expected to have a similar topology. The prevailing view that pTi VirB3 has a periplasmic C terminus is inconsistent with the cytoplasmic location of the N terminus of VirB4 in the VirB3-VirB4 fusion protein homologues.In this study, we report the membrane topology of Agrobacterium VirB3 and demonstrate that the C terminus of the protein resides in the cytoplasm. We also demonstrate that VirB3 is an inner membrane protein, not an outer membrane protein as previously reported (52). The octopine Ti plasmid pTiA6NC VirB4 protein does not affect membrane localization of VirB3 but does stabilize VirB3. VirB4, however, is not sufficient for pTiA6NC VirB3 stabilization. Two additional proteins, VirB7 and VirB8, are required for the stabilization of pTiA6NC VirB3.     

ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.     

VirB1 orthologs from Brucella suis and pKM101 complement defects of the lytic transglycosylase required for efficient type IV secretion from Agrobacterium tumefaciens

Type IV secretion systems mediate conjugative plasmid transfer as well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells. The translocation apparatus consists of 9 to 12 components, and the components from different organisms are believed to have similar functions. However, orthologs to proteins of the prototypical type IV system, VirB of Agrobacterium tumefaciens, typically share only 15 to 30% identical amino acids, and functional complementation between components of different type IV secretion systems has not been achieved. We here report a heterologous complementation in the case of A. tumefaciens virB1 defects with its orthologs from Brucella suis (VirB1s) and the IncN plasmid pKM101 (TraL). In contrast, expression of the genes encoding the VirB1 orthologs from the IncF plasmid (open reading frame 169) and from the Helicobacter pylori cag pathogenicity island (HP0523) did not complement VirB1 functions. The complementation of VirB1 activity was assessed by T-pilus formation, by tumor formation on wounded plants, by IncQ plasmid transfer, and by IncQ plasmid recipient assay. Replacement of the key active-site Glu residue by Ala abolished the complementation by VirB1 from B. suis and by TraL, demonstrating that heterologous complementation requires an intact lytic transglycosylase active site. In contrast, the VirB1 active-site mutant from A. tumefaciens retained considerable residual activity in various activity assays, implying that this protein exerts additional effects during the type IV secretion process.     

Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) translocates DNA and protein substrates across the bacterial cell envelope. Six presumptive channel subunits of this T4SS (VirD4, VirBll, VirB6, VirB8, VirB2, and VirB9) form close contacts with the VirD2-T-strand transfer intermediate during export, as shown recently by a novel transfer DNA immunoprecipitation (TrIP) assay. Here, we characterize the contribution of the hydrophobic channel component VirB6 to substrate translocation. Results of reporter protein fusion and cysteine accessibility studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, five transmembrane segments, and a cytoplasmic C terminus. TrIP studies aimed at characterizing the effects of VirB6 insertion and deletion mutations on substrate translocation identified several VirB6 functional domains: (i) a central region composed of a large periplasmic loop (P2) (residues 84 to 165) mediates the interaction of VirB6 with the exiting T-strand; (ii) a multi-membrane-spanning region carboxyl-terminal to loop P2 (residues 165 to 245) is required for substrate transfer from VirB6 to the bitopic membrane subunit VirB8; and (iii) the two terminal regions (residues 1 to 64 and 245 to 290) are required for substrate transfer to the periplasmic and outer membrane-associated VirB2 and VirB9 subunits. Our findings support a model whereby the periplasmic loop P2 comprises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate passage to the cell exterior.     

Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.     

The dimer interface of Agrobacterium tumefaciens VirB8 is important for type IV secretion system function, stability, and association of VirB2 with the core complex

Type IV secretion systems are virulence factors used by many gram-negative bacteria to translocate macromolecules across the cell envelope. VirB8 is an essential inner membrane component of type IV secretion systems, and it is believed to form a homodimer. In the absence of VirB8, the levels of several other VirB proteins were reduced (VirB1, VirB3, VirB4, VirB5, VirB6, VirB7, and VirB11) in Agrobacterium tumefaciens, underlining its importance for complex stability. To assess the importance of dimerization, we changed residues at the predicted dimer interface (V97, A100, Q93, and E94) in order to strengthen or to abolish dimerization. We verified the impact of the changes on dimerization in vitro with purified V97 variants, followed by analysis of the in vivo consequences in a complemented virB8 deletion strain. Dimer formation was observed in vivo after the introduction of a cysteine residue at the predicted interface (V97C), and this variant supported DNA transfer, but the formation of elongated T pili was not detected by the standard pilus isolation technique. Variants with changes at V97 and A100 that weaken dimerization did not support type IV secretion system functions. The T-pilus component VirB2 cofractionated with high-molecular-mass core protein complexes extracted from the membranes, and the presence of VirB8 as well as its dimer interface were important for this association. We conclude that the VirB8 dimer interface is required for T4SS function, for the stabilization of many VirB proteins, and for targeting of VirB2 to the T-pilus assembly site.     

Assembly and mechanisms of bacterial type IV secretion machines

Type IV secretion occurs across a wide range of prokaryotic cell envelopes: Gram-negative, Gram-positive, cell wall-less bacteria and some archaea. This diversity is reflected in the heterogeneity of components that constitute the secretion machines. Macromolecules are secreted in an ATP-dependent process using an envelope-spanning multi-protein channel. Similar to the type III systems, this apparatus extends beyond the cell surface as a pilus structure important for direct contact and penetration of the recipient cell surface. Type IV systems are remarkably versatile in that they mobilize a broad range of substrates, including single proteins, protein complexes, DNA and nucleoprotein complexes, across the cell envelope. These machines have broad clinical significance not only for delivering bacterial toxins or effector proteins directly into targeted host cells, but also for direct involvement in phenomena such as biofilm formation and the rapid horizontal spread of antibiotic resistance genes among the microbial community.     

VirB8: a conserved type IV secretion system assembly factor and drug target.

Type IV secretion systems are used by many gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA-protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8-12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure-function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein-protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.