An in vivo high-throughput screening approach targeting the type IV secretion system component VirB8 identified inhibitors of Brucella abortus 2308 proliferation

Abstract

As bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such "antivirulence" drugs. The VirB8 protein from the mammalian pathogen Brucella was chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibited virB gene transcription in Brucella abortus 2308, suggesting that targeting of the secretion system has complex regulatory effects in vivo. One compound strongly inhibited the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model. The results presented here show that in vivo screens with the bacterial two-hybrid assay are suited to the identification of inhibitors of Brucella type IV secretion system function.

FIG. 1.

High-throughput screening of the Canadian Compound Collection for inhibitors of VirB8 dimerization. E. coli strain BTH101 expressing VirB8 as a fusion to the T18 and T25 domains of adenylate cyclase was cultivated in 96-well microtiter plates in TGYEP medium, and the β-galactosidase activities reflecting this protein-protein interaction were determined in the absence and in the presence of 29,567 molecules from the Canadian Compound Collection. Shown is a plot of the two screening replicates reported as percent residual activity relative to the average of the high controls. Hits were identified as molecules reducing the activity to less than 50% in both replicates (shown in gray), a statistical cutoff 2 standard deviations below the high control mean.

FIG. 2.

Controls of the specificity of VirB8 interaction inhibitors. E. coli strain BTH101 expressing fusions of VirB8 or VirB10 or of interacting leucine zippers (ZIP) to the T18 and T25 domains of adenylate cyclase was cultivated in 96-well microtiter plates in TGYEP medium, and the β-galactosidase activities reflecting the protein-protein interactions were determined in the absence (C [control]) and presence of 20 inhibitors that specifically inhibited VirB8-VirB8 or VirB8-VirB8 and VirB8-VirB10, but not VirB10-VirB10 and ZIP-ZIP interactions (the identities of the molecules are given in Table 1). Averages from 8 replicates are shown, and the standard deviations are not shown for clarity.

FIG. 3.

Chemical structures of the 20 VirB8 interaction inhibitors chosen for further analysis after specificity tests.

FIG. 4.

Impacts of VirB8 interaction inhibitors on VirB protein levels in B. abortus 2308. Brucella was cultivated in a virulence gene-inducing minimal medium in the presence of small molecules, as indicated. C, DMSO control; HSL, C12-homoserine lactone; and VirB8 interaction inhibitors (B8I- plus numbers above lanes). B. abortus cells were lysed, followed by SDS-PAGE and Western blotting with specific antisera as indicated.

FIG. 5.

Impacts of B8I-2 and C12-HSL on transcription in B. abortus 2308. Brucella was cultivated in virulence gene-inducing minimal medium in the presence of small molecules as indicated. qRT-PCR was performed using primers for virB1, virB6, virB11, vjbR, gyrA, and bcsp31. The fold change was calculated over untreated Brucella. B8I-2, 10 μM; C12-HSL, 5 μM.

FIG. 6.

Effects of inhibitor B8I-2 on the intracellular replication of B. abortus 2308. (A) Replication of the Brucella wild type (wt) and virB deletion mutant (ΔvirB) inside J774 macrophages and effects when C12-homoserine lactone (5 μM) and B8I-2 at the concentrations shown were added at the end of the cocultivation period. (B) Replication of the Brucella wild type and virB deletion mutant inside J774 macrophages and effects when B8I-2 (10 μM) was added at the end of the cocultivation period, after the wash with fresh medium, or at the times shown after the elimination of extracellular bacteria with gentamicin. (C) Time course of effects of B8I-2 on intracellular survival and replication. B8I-2 was added at 10 μM at the beginning of the assay and maintained throughout the 48-h period. The data shown are compiled from two independent assays, each performed in triplicate. (D) Structure of B8I-2, a salicylidene acylhydrazide. The error bars represent the standard deviations; the asterisks indicate significance at a P value of <0.05.

FIG. 7.

Sedimentation equilibrium analysis of the dimerization of VirB8. Analytical ultracentrifugation to analyze the self-association of wild-type VirB8 alone (WT) or in the presence of B8I-2 (WT + B8I-2) and of the dimer site variant VirB8^M102R (M102R). The lower graphs show a representative fit of the experimental data to a monomer/dimer model in the case of VirB8 and to a single-species (i.e., monomer) model in the case of VirB8 plus B8I-2 and M102R. The upper graphs show the residuals of the fit. The representative fits shown here were taken from the data obtained for proteins at an A₂₈₀ of 0.5 and rotor speeds of 20,000, 24,000, and 28,000 rpm in a Beckman Coulter XL-A analytical ultracentrifuge.