The Brucella suis Type IV Secretion System Assembles in the Cell Envelope of the Heterologous Host Agrobacterium tumefaciens and Increases IncQ Plasmid pLS1 Recipient Competence

Abstract

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...

Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity

“…Apart from the in vitro assays for VirB protein interactions, one of the main advances of our recent work was the development of a heterologous assay of B. suis T4SS assembly in A. tumefaciens (29). This assay exploits the ability of a subset of B. suis T4SS components (VirB1 and VirB3-VirB12) to increase the recipient competence of A. tumefaciens for T4SS-mediated plasmid transfer, and similar findings were reported for the Agrobacterium VirB T4SS (30).…”

“…Escherichia coli strains JM109 (32) and GJ1158 (33) were used as hosts for cloning and protein overproduction, respectively. A. tumefaciens strain A348 was used as pLS1 donor and strain UIA143 as host for the heterologous expression of the B. suis T4SS, plasmid propagation, and cell cultivation, which were conducted as described (29). For detection of VirBs protein production, B. suis strains were cultivated in minimal medium (34), and J774 murine macrophage-like cells were infected with a standard gentamicin protection assay (28).…”

“…We showed recently that the production of a subset of the B. suis VirB proteins (VirB1 and VirB3-12) in A. tumefaciens increased the recipient competence for IncQ plasmid pLS1 in conjugation experiments (29). This system serves to analyze B. suis T4SS assembly without the need for work under biosafety level 3 conditions, and it was here applied to measure complementation.…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…Intriguingly, the VirB Bs proteins also stimulate DNA uptake when synthesized in agrobacterial recipient cells. Although the underlying mechanism for enhanced DNA uptake is unknown, this assay enables further structure-function studies of the Brucella VirB machinery in the A. tumefaciens surrogate host (45). Additionally, although most Brucella subunits are not interchangeable with homologs of other T4SS, VirB1 Bs functionally substitutes for VirB1 At (133).…”

Swapping of Periplasmic Domains between Brucella suis VirB8 and a pSB102 VirB8 Homologue Allows Heterologous Complementation

“…We have previously shown that VirB proteins are expressed at a very low level by B. suis in rich or minimal medium at neutral pH, whereas transcription of the B. suis virB operon is strongly induced in acid minimal medium, conditions thought to mimic the environment encountered in the phago-some (2, 13). We used Western blotting with antibodies raised against recombinant B. suis VirB5, VirB8, VirB9, VirB10, and VirB12 to compare expression of these proteins by wild-type B. suis and the BS1008 mutant cultured in virB operon-inducing medium at either pH 7.0 or pH 4.5 as described previously (3,14). Low-level expression of the five VirB proteins was detected in wild-type B. suis after incubation at neutral pH, and very strong induction was observed in acid minimal medium (Fig.…”