Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gramnegative 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.

VirB1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type IV secretion systems via localized lysis of the peptidoglycan. This paper presents the biochemical analysis of interactions of purified Brucella suis VirB1 with core components of the type IV secretion system. Genes encoding VirB1, VirB8, VirB9, VirB10 and VirB11 were cloned into expression vectors; the affinity-tagged proteins were purified from Escherichia coli, and analyses by gel filtration chromatography showed that they form monomers or homo-multimers. Analysis of protein–protein interactions by affinity precipitation revealed that VirB1 bound to VirB9 and VirB11. The results of bicistron expression experiments followed by gel filtration further supported the VirB1–VirB9 interaction. Peptide array mapping identified regions of VirB1 that interact with VirB8, VirB9 and VirB11 and underscored the importance of the C-terminus, especially for the VirB1–VirB9 interaction. The binding sites were localized on a structure model of VirB1, suggesting that different portions of VirB1 may interact with other VirB proteins during assembly of the type IV secretion machinery.

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Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related ␣ 2 -proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.Brucella species are pathogens of mammals, which cause severe infections and abortions in animals and long-lasting febrile diseases in humans (65). They impact agriculture by causing zoonotic diseases of cattle (Brucella abortus), sheep (B. melitensis), and swine (B. suis), which cause substantial economic losses, and they pose a threat for those handling the animals (8, 28). The eradication of Brucella from livestock has succeeded in some parts of the world, but expensive control and surveillance systems are necessary due to the possibility of reinfection of livestock from wildlife. In addition to its threat to commercial agriculture, Brucella is considered as a potential category B bioterror agent (32). Brucella infections are very long-lasting, and current treatment regimens require 6 to 8 weeks of therapy with two antibiotics (61). Several live attenuated vaccines are effective for animals, but safe vaccines for humans are currently not available (28). The threat posed by Brucella infections gives research on the molecular basis of virulence and persistence in the mammalian body a high priority.Brucella species survive and multiply inside mammalian cells, including cells of the immune system such as macrophages (12, 51). They inhibit apoptosis of infected cells and apparently evade the immune response of their hosts, causing long-lasting infections (48). After entering macrophages via lipid rafts, the Brucella-containing vacuole (BCV) does not fuse with the lysosomes, thus avoiding rapid cell destruction (13). Instead, the BCV follows a novel intracellular trafficking pathway, which interacts with the endoplasmic reti...

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