Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems.

Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili.

Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens.

The VirB/D4 type IV secretion system of Agrobacterium tumefaciens translocates virulence factors (VirE2, VirF, and the VirD2-T-DNA complex) to plant cells. The membrane-bound translocation machinery consists of 12 proteins (VirB1-11 and VirD4) required for substrate translocation. Protein-protein interactions in the membranes were analyzed after extraction with the mild detergent dodecyl-beta-d-maltoside followed by separation under native conditions. Incubation of the membranes with increasing concentrations of the detergent differentially extracted virulence proteins. Separation of the solubilized proteins by blue native electrophoresis revealed cofractionation between two classes of protein complexes containing VirB7. The first class, consisting of major T-pilus component VirB2 and associated proteins VirB5 and VirB7, comigrated in the low molecular mass portion of the gel of about 100 kDa. The second class contains putative translocation complex core components VirB8, VirB9, and VirB10 in the high molecular mass portion of the gel larger than 232 kDa, as well as VirB7. Solubilized proteins were characterized further by gel filtration chromatography. This procedure separated T-pilus-associated proteins VirB2, VirB5, and VirB7 in the low molecular mass range from the other components of the translocation machinery and the substrates VirE2 and VirD2. Fractionation of VirB7-containing complexes (VirB7-VirB7 homodimers and VirB7-VirB9 heterodimers) suggested that they may link the T-pilus components to the core of the translocation machinery. Based on previously described VirB protein interactions and biochemical analysis of C58 wild type as well as of virB5 and virB6 deletion mutants, a model of T-pilus assembly in A. tumefaciens is suggested.

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.

Production of the type IV secretion system differs among Brucella species as revealed with VirB5- and VirB8-specific antisera.

Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortus- and Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.