Recombinant Brucella suis biovar 1 Type IV secretion system protein virB8 (virB8)

What is the structural organization of Brucella suis VirB8?

VirB8 is a bitopic inner membrane protein with a distinct structural organization. The amino terminus encompasses the first 67 amino acids and contains a short cytoplasmic tail followed by a single hydrophobic transmembrane domain. The carboxy-terminal moiety comprises approximately 172 amino acids and is believed to be entirely periplasmic . Three-dimensional structures of the periplasmic domain have been determined for both Brucella suis and Agrobacterium tumefaciens VirB8 proteins through X-ray crystallography . The periplasmic domain possesses distinct interaction surfaces that mediate crucial protein-protein interactions necessary for Type IV secretion system (T4SS) assembly and function .

How does VirB8 contribute to the assembly of the Type IV secretion system?

VirB8 plays a critical role as a nucleation center during T4SS assembly. Research has demonstrated that VirB8:

• Acts as a recruitment factor required to gather VirB9 and VirB10 into clusters in the outer membrane

• Facilitates the localization of VirB proteins at the bacterial cell pole

• Undergoes multiple protein-protein interactions to mediate assembly of the translocation machinery

• Functions as an essential assembly factor for the multiprotein T4SS complex

The importance of VirB8 in T4SS assembly is evidenced by the fact that Brucella suis mutants with deletions in the virB8 gene show attenuated virulence in macrophage infection models, which can be restored through complementation with functional VirB8 .

What experimental evidence supports VirB8's role in Brucella virulence?

Several lines of experimental evidence confirm VirB8's essential role in Brucella virulence:

• A B. suis strain with a nonpolar deletion in virB8 (BS1008) showed severely impaired intracellular growth in J774 macrophages, similar to that observed with polar virB mutants

• Complementation of the virB8 deletion mutant with plasmid-encoded VirB8 restored intracellular replication to near wild-type levels

• Structure-function analyses have identified specific amino acid residues critical for VirB8 function in vivo, such as Y105, T201, and R230, which when mutated impair the ability of B. suis to replicate intracellularly

• The overexpression of a FLAG-tagged VirB8 protein not only reduced complementation efficiency but also exhibited a dominant negative effect on wild-type strain virulence, suggesting the importance of proper VirB8 stoichiometry and conformation

Which specific residues are critical for VirB8 dimerization and interactions?

Structure-function analysis of the periplasmic domain of Brucella suis VirB8 has identified several key amino acid residues involved in protein-protein interactions:

These residues represent potential targets for structure-based drug design efforts aimed at disrupting T4SS function .

How can researchers study VirB8 protein-protein interactions in vitro?

Several methodological approaches have been successfully employed to study VirB8 interactions:

• Analytical ultracentrifugation: This technique was used to quantitatively assess the self-association (dimerization) of purified VirB8 periplasmic domain variants .

• Gel filtration chromatography: Complementary to ultracentrifugation, this approach helped characterize changes in the oligomeric state of VirB8 variants with mutations at the dimer interface (M102, Y105, and E214) .

• Bacterial two-hybrid system: This system was adapted to assay VirB8 interactions and was successfully used in a high-throughput screen to identify small-molecule inhibitors of VirB8 interactions .

• Structure-function analysis: By engineering site-directed mutations in residues identified by X-ray crystallography and testing their effects on protein-protein interactions, researchers have mapped the interaction surfaces of VirB8 .

• In vitro binding assays: These assays can be used to assess the direct interaction between purified VirB8 variants and other T4SS components like VirB4 and VirB10 .

What are the implications of VirB8-homolog interactions for T4SS assembly?

Comparative analysis of VirB8 with its homologs provides insights into T4SS assembly:

• TraJ, a VirB8 homologue from plasmid pSB102, shares more than 50% identity with VirB8 but cannot functionally replace VirB8 in B. suis

• The cytoplasmic and transmembrane domains of TraJ display only 39% identity with the corresponding part of VirB8, whereas the periplasmic domain exhibits more than 63% identity

• A chimeric protein containing the N-terminus of B. suis VirB8 and the C-terminal periplasmic domain of TraJ partially restored virulence in a virB8 deletion mutant

These findings suggest that:

• The specific N-terminal domain of VirB8 is critical for proper membrane localization and function in Brucella

• Despite high sequence conservation in periplasmic domains, subtle structural differences may determine functional specificity

• Homology-based approaches can inform the design of chimeric proteins to dissect domain-specific functions in T4SS assembly

How can researchers generate and characterize a virB8 deletion mutant?

Based on the methods described in the search results, researchers can follow this protocol to generate and validate a virB8 deletion mutant:

• Construction of deletion mutant:

  • Engineer an in-frame deletion of virB8 into the chromosome of the wild-type strain B. suis 1330 following established procedures

  • Confirm the deletion by PCR and sequencing

• Phenotypic characterization:

  • Assess intracellular growth in J774 macrophage cell cultures by monitoring bacterial counts at different time points post-infection (e.g., 24h, 48h)

  • Compare intracellular growth kinetics with wild-type strain to quantify attenuation

  • Analyze expression of other VirB proteins to assess potential polar effects on the virB operon

• Complementation analysis:

  • Clone the virB8 gene into a broad-host-range vector under control of the B. suis virB promoter

  • Introduce the complementation plasmid into the deletion mutant via electroporation

  • Verify restoration of intracellular growth to confirm the phenotype is specifically due to the absence of VirB8

What approaches can be used to assess VirB8 interaction with other T4SS components?

Several experimental approaches can be employed to study VirB8 interactions with other T4SS components:

• Site-directed mutagenesis:

  • Based on crystal structure, identify residues potentially involved in protein-protein interactions

  • Generate specific point mutations in VirB8 (e.g., T201A/Y for VirB10 interaction, R230D for VirB4 interaction)

  • Purify the VirB8 variants and characterize their interactions in vitro

• Heterologous assay systems:

  • Use Agrobacterium tumefaciens as a surrogate system to assess T4SS assembly

  • This approach allows monitoring of T4SS component interactions in a related but more tractable bacterial system

• Chimeric protein analysis:

  • Create fusion proteins between VirB8 and its homologs (e.g., TraJ)

  • Analyze which domains are critical for specific interactions and functions

  • Express these chimeras in the virB8 deletion strain to assess complementation efficiency

• Protein stability and expression analysis:

  • Monitor expression levels of VirB8 variants under virulence gene-inducing conditions (e.g., minimal medium at acidic pH)

  • Use Western blotting with anti-VirB8 antibodies to verify that observed phenotypes are not due to protein instability

What are the optimal conditions for expressing and purifying recombinant VirB8?

While the search results don't provide explicit protocols for VirB8 purification, based on the described research approaches, the following strategy can be inferred:

• Expression construct design:

  • Clone the periplasmic domain of VirB8 (residues ~68-240) for soluble expression

  • Consider His-tag or other affinity tags for purification

  • Avoid modifying the C-terminus with FLAG tags, as this has been shown to interfere with function

• Expression conditions:

  • Express in E. coli under conditions that favor proper protein folding

  • Consider reduced temperature (e.g., 18-25°C) during induction to enhance solubility

• Purification approach:

  • Use affinity chromatography as the initial purification step

  • Follow with size exclusion chromatography to assess and separate different oligomeric states

  • Analytical ultracentrifugation can be used to characterize the purified protein's oligomerization state

• Functional validation:

  • Verify proper folding through circular dichroism or thermal shift assays

  • Assess ability to dimerize and interact with VirB4/VirB10 fragments in vitro

  • For structural studies, screen crystallization conditions similar to those previously successful

What strategies can be employed to identify inhibitors of VirB8 function?

The search results highlight several approaches for identifying VirB8 inhibitors:

• Bacterial two-hybrid (B2H) screen:

  • Adapt the B2H system to assay VirB8 interactions, particularly dimerization

  • Perform high-throughput screening of small-molecule libraries to identify compounds that disrupt these interactions

  • This approach has successfully identified specific small-molecule inhibitors of VirB8 interactions

• Structure-based drug design:

  • Utilize the crystal structure of VirB8 to identify potential binding pockets at interaction interfaces

  • Target key residues involved in dimerization (M102, Y105, E214) or interactions with VirB4 (R230) and VirB10 (T201)

  • Perform in silico screening followed by biochemical validation

• Functional screening:

  • Screen compounds for their ability to inhibit intracellular growth of Brucella in macrophage infection models

  • Validate that growth inhibition correlates with disruption of VirB8 function

How can identified VirB8 inhibitors be validated in experimental models?

A sequential validation approach can be implemented:

• Biochemical validation:

  • Confirm that potential inhibitors directly interfere with VirB8 dimerization or interaction with VirB4/VirB10 using purified proteins

  • Determine binding affinities and structure-activity relationships

• Cellular assays:

  • Test effects on VirB gene expression and protein levels in Brucella

  • Assess impact on T4SS assembly using fluorescently tagged components

• Infection models:

  • Evaluate inhibitor efficacy in J774 macrophage infection assays

  • Monitor reduction in intracellular bacterial proliferation

  • Compare results with virB8 mutant strains to confirm the mechanism of action

• Specificity testing:

  • Assess effects on other bacterial species with related T4SS

  • Verify lack of toxicity toward host cells

  • Determine whether inhibition is specific to VirB8 or affects multiple T4SS components

What is the rationale for targeting VirB8 as an antivirulence strategy against Brucella?

The search results present several compelling reasons for targeting VirB8:

• Essential virulence factor: VirB8 is required for T4SS function and Brucella intracellular survival

• Therapeutic need:

  • Brucella causes long-lasting severe infections requiring treatment with two antibiotics over 4-6 weeks

  • Despite aggressive antibiotic therapies, relapses are frequent due to Brucella's intracellular lifestyle

  • Brucella is considered a potential bioterror threat due to easy aerosol transmission

• Antivirulence approach advantages:

  • Targeting virulence factors rather than essential genes may reduce selective pressure for resistance

  • T4SS inhibitors would constitute alternatives to or enhancements of current antibiotic treatment regimens

  • Disarming the pathogen by depriving it of its essential virulence factor offers a novel therapeutic strategy

• Structural and functional data support:

  • Detailed structural information is available for VirB8

  • Key interaction sites have been mapped and validated

  • Proof-of-concept studies have identified VirB8 inhibitors that reduce intracellular proliferation of Brucella

How does VirB8 stoichiometry affect T4SS assembly and function?

The search results suggest several important considerations regarding VirB8 stoichiometry:

• Expression level effects:

  • Complementation of a virB8 deletion with plasmid-encoded VirB8 (10-20 copies) results in much higher expression than in the wild-type strain

  • This overexpression slightly reduced growth during the first 24h post-infection but reached wild-type levels by 48h

  • High levels of VirB8 expression did not affect the levels of other VirB proteins in the cells

• Dominant negative effects:

  • Expression of FLAG-tagged VirB8 not only showed reduced complementation efficiency but also reduced the virulence of wild-type B. suis, suggesting interference with T4SS assembly or function

  • This indicates that proper stoichiometry and conformation of VirB8 are critical for T4SS function

• Protein stability considerations:

  • FLAG-tagged VirB8 showed decreased protein levels when bacteria were cultured in minimal medium at acidic pH compared to neutral pH

  • This suggests that modification of the C-terminus may disrupt protein-protein interactions and/or target VirB8 dimers for proteolysis

These findings highlight the need for careful control of VirB8 expression levels in experimental systems and suggest that both insufficient and excessive VirB8 can impair T4SS function.

What are the molecular mechanisms of VirB8-mediated T4SS assembly?

Based on the search results, the following molecular mechanisms can be proposed:

• Sequential assembly model:

  • VirB8 functions as a nucleation center for T4SS assembly

  • It recruits VirB9 and VirB10 into clusters in the outer membrane

  • It also promotes localization of VirB proteins at the cell pole

• Critical interactions:

  • VirB8 dimerization appears to be essential for functionality

  • Interaction with VirB4 (an ATPase) may couple energy transduction to assembly processes

  • Interaction with VirB10 links inner membrane components to outer membrane complexes

• Domain-specific functions:

  • The N-terminal domain, including the transmembrane segment, provides proper membrane anchoring and species-specific functionality

  • The periplasmic C-terminal domain mediates multiple protein-protein interactions essential for assembly

• Regulatory effects:

  • VirB8 interaction inhibitors reduced the levels of VirB8 and other VirB proteins and inhibited virB gene transcription in B. abortus, suggesting VirB8 may also have regulatory roles

What insights can comparative analysis of VirB8 homologs provide?

Comparative analysis of VirB8 and its homologs reveals:

This comparative approach offers opportunities to:

• Identify conserved features critical for general T4SS function

• Distinguish species-specific adaptations that might be targeted for selective intervention

• Design chimeric proteins to investigate domain-specific functions across different T4SS