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

Abstract

A Brucella suis mutant with a nonpolar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Expression of TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.Type IV secretion systems (T4SS) are used by gram-negative bacteria to transport macromolecules across the bacterial envelope into a recipient cell (4, 6). Transported substrates include nucleoprotein complexes, transported during bacterial conjugation or during transfer of transferred DNA from Agrobacterium tumefaciens, as well as protein virulence factors translocated into eukaryotic host cells by pathogens.The T4SS is a complex multiprotein structure spanning the bacterial envelope. Most T4SS are composed of up to 11 individual protein building blocks plus, in many cases, a 12th "coupling protein," thought to bring the macromolecular substrates to the secretion system. Biochemical analysis of the A. tumefaciens VirB system, the T4SS paradigm, is beginning to show how the individual components assemble in the bacterial envelope and how the substrates are secreted (5). The VirB1 protein is a murein-digesting lytic transglycosylase that locally digests the peptidoglycan, allowing the assembly of the T4SS. The VirB3, VirB6, and VirB7 to VirB10 proteins are believed to form a channel-like structure spanning both bacterial membranes; the VirB4 and VirB11 proteins, with the VirD4 coupling protein, are ATPases associated with the cytoplasmic face of the inner membrane; and the VirB2 and VirB5 proteins form a pilus-like structure on the bacterial surface.The VirB8 protein has been shown to play a key role in the assembly of the T4SS machinery. Using microscopic, biochemical, and genetic approaches to study function in A. tumefaciens, it was discovered that VirB8 acts as a nucleation center that is required to recruit VirB9 and VirB10 into clusters in the outer membrane (10) and to localize VirB proteins at the cell pole (8). The amino terminus of VirB8, encompassing the first 67 amino acids, contains a short cytoplasmic tail followed by a single hydrophobic transmembrane domain. The carboxy-terminal moiety of the protein of 172 amino acids is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domain of VirB8 from both Brucella suis and A. tumefaciens have been determined (1, 18). Using site-directed mutagenesis to change selected residues of the B.suis VirB8 protein, we have shown that changes that inhibit VirB8 dimerization or that inhibit the interactions with VirB4 or VirB10 also affect T4SS assembly and virulence (12). Here, we describe the construction and characterization of the B. suis strain with a nonpolar deletion of virB8 used in that study and show, for the first time, complementation with a chimeric pr...

Interactions betweenBrucella suisVirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

“…The TraJ protein from pSB102 shares more than 50% identity with B. suis VirB8 at the amino acid level, and this percentage increases to more than 60% when only the periplasmic domain is considered. In a previous study, we have taken advantage of this close similarity between VirB8 and TraJ to examine the possibility of a functional heterologous complementation of VirB8 by TraJ in BS1008, a B. suis mutant carrying an in-frame deletion of the virB8 gene (38). From our results, it appeared that the protein TraJ was unable to complement BS1008.…”

“…We examined the effects on the virulence of the wildtype strain of B. suis of overexpressing one of these proteins from a multicopy plasmid. The six different strains of B. suis we used were wild-type strain 1330 (S1) without any plasmid or the same strain containing the empty vector (pIN34) or a plasmid expressing one of the full-length proteins described previously (pIN38, pIN54, pIN56, and pIN57) (38). Intracellular survival and multiplication were assessed at different times following the beginning of the infection (Fig.…”

“…All of the other plasmids used in the BACTH studies were derived from pIN73 and pIN94 by inserting DNA fragments obtained by PCR between the unique BamHI and EcoRI sites. The construction of some derivatives of pBBR1-MCS used in infection studies has already been described (38). Other plasmids were designed specifically for this study from plasmids used for BACTH analysis.…”

“…All experiments included a positive control (plasmids encoding fusion proteins with a leucine zipper from Saccharomyces cerevisiae GCN4) and a negative control (empty plasmids). Wild-type strain B. suis 1330 was transformed by electroporation as previously described (38). All of the resulting Brucella strains were used to infect J774 murine macrophage-like cells in a standard gentamicin protection assay as described previously (38).…”

“…Wild-type strain B. suis 1330 was transformed by electroporation as previously described (38). All of the resulting Brucella strains were used to infect J774 murine macrophage-like cells in a standard gentamicin protection assay as described previously (38).…”