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

Abstract

VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein-protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure-function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein-protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein-protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105, T201, R230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context.

Fig. 1.

Amino acids changed in this study shown in the B. suis VirB8sp x-ray structure. Ribbon representation showing a model of the VirB8sp structure in different orientations. Dimer interface residues E214, M102, and Y105 are shown in magenta; active-site groove residues K182, L151, and Q144 are shown in red; β-sheet's solvent-exposed face residues R230 and T201 are shown in green; very high-identity patch Y126, D152, and W119 are shown in blue; and I112 is shown in brown. The model was generated with macpymol (pymol.sourceforge.net) based on the VirB8sp Protein Data Bank file, www.pdb.org (PDB ID code 2BHM).

Fig. 2.

Crosslinking, gel filtration, and analytical ultracentrifugation to assess dimer formation of VirB8sp. (A) StrepIIVirB8sp was incubated in the presence of increasing amounts of DSS before SDS/PAGE and Western blotting with VirB8sp-specific antisera. Arrowheads indicate the formation of higher molecular mass complexes, and molecular masses of reference proteins are shown on the right (in kDa). (B) Chromatogram showing elution of different concentrations of VirB8sp from a Superdex S75 column, 128 μM (black line), and 2 μM (gray line). The corresponding absorbance scales are indicated (mAU, milliabsorbance units at 280 nm). (C) Plot of the calculated molecular masses of StrepIIVirB8sp (rhombus), M102R (triangle), Y105R (square), and E214R (circle) obtained by size-exclusion chromatography as a function of the applied concentration. (D and E) Sedimentation equilibrium analysis of StrepIIVirB8sp and M102R, respectively. The lower graphs show a representative fit of the experimental data to a monomer/dimer model in case of StrepIIVirB8sp and to a single species (= monomer) model in case of M102R. The upper graphs show the residuals of the fit. The representative fits shown here were taken from the data obtained for proteins at concentration of A₂₈₀ 0.3 and rotor speeds of 20,000, 24,000, and 28,000 rpm in a Beckman Coulter XL-A analytical ultracentrifuge.

Fig. 3.

BNE to analyze the interaction of VirB8sp and its variants with VirB4s. StrepIIVirB4s-enriched extract was mixed with StrepIIVirB8sp and variants, followed by BNE and Western blotting with VirB8sp-specific (A) and StrepII-specific (B) antisera for detection of StrepIIVirB4. The migration of high-molecular-mass StrepIIVirB4s-StrepIIVirB8sp complexes and of the unbound R230D variant in the low-molecular-mass portion of the gel are indicated. (C) Results of SDS/PAGE and Western blotting of samples from the same experiment as protein-loading controls. Molecular masses of reference proteins are shown on the right (in kDa).

Fig. 4.

Analysis of interaction between StrepIIVirB8sp variants and StrepIIVirB10sp by crosslinking. (A) StrepIIVirB8sp and variants were mixed with StrepIIVirB10sp and incubated with 0.1 mM DSS followed by SDS/PAGE and Western blotting with VirB8s-specific antiserum. Crosslinked products of StrepIIVirB8sp in the absence of StrepIIVirB10sp are indicated by ∗, the occurrence of a novel product in the presence of StrepIIVirB10sp is indicated by an arrow, and arrowheads indicate reduced amounts of this presumptive heterodimer in case of two variants as compared with WT (representative results of three repetitions are shown). (B) Western blots of SDS gels from crosslinked samples as loading controls. An additional VirB8sp-specific signal of lower molecular mass (indicated by a triangle) was observed in the presence of DSS that may correspond to an internal crosslink causing the change of migration. Molecular masses of reference proteins are shown on the right (in kDa).

Fig. 5.

Functionality of VirB8sp variants in B. suis and in the heterologous T4SS assembly assay in A. tumefaciens. (A) J774 macrophages were infected with B. suis 1330 (wt), BS1008 (ΔvirB8), and BS1008 carrying plasmids for expression of VirB8s and its variants, and intracellular growth was quantified 48 h after infection (logarithmic scale, geometric mean ± standard deviation from 3–12 independent repetitions). Complementation by pIN38 (VirB8s wt) is indicated by the dashed line. (B) The recipient UIA143 carrying pTrcB1+3–12 (wt), pTrcB1+3–12^ΔvirB8 (ΔvirB8), and strains complemented with pTrcPVirB8s and variants were cocultivated with the donor A348 pLS1. The transconjugants (TC) were identified by growth on selective agar, and the pLS1 transfer efficiency (transconjugants per recipient, TC/R) into UIA143 carrying pTrcB1+3–12^ΔvirB8, and pTrcPVirB8s was set to 100% (indicated by dashed line) (error bars show standard deviation from four repetitions, and bacterial titer is given in a linear scale).