Recombinant Brucella abortus Type IV secretion system protein virB8 (virB8)

What is the Brucella abortus VirB8 protein and what role does it play in bacterial virulence?

VirB8 is a critical component of the Type IV secretion system in Brucella abortus, which comprises 12 protein complexes named VirB1 to VirB12, encoded by the VirB regions . It functions as a bitopic inner membrane protein that undergoes multiple interactions with other T4S components via its periplasmic C-terminal domain, serving as an essential assembly factor for the T4SS .

Studies using nonpolar deletion mutants have conclusively demonstrated that virB8 is essential for the ability of B. abortus to survive in cultured macrophages and to persist in organs of mice, confirming its critical role in virulence . The deletion of virB8, along with virB3, virB4, virB5, virB6, virB9, virB10, and virB11, markedly reduces the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection .

How does VirB8 interact with other components of the Type IV secretion system?

VirB8 participates in multiple protein-protein interactions within the T4SS complex. Research has shown that VirB8 is an essential assembly factor, interacting with various other VirB proteins to form the functional secretion apparatus . These interactions are critical for T4SS assembly and function, making VirB8 an attractive target for inhibiting Brucella virulence.

The bacterial two-hybrid system has been successfully adapted to assay VirB8 interactions and to identify small-molecule inhibitors that specifically target these interactions . When these interactions are disrupted, there is a reduction in the levels of VirB8 and other VirB proteins, demonstrating the interconnected nature of the T4SS components .

What is the importance of VirB8 dimerization for its functionality?

X-ray crystallographic studies and structure-function analyses suggest that dimerization is important for VirB8 functionality . This understanding has informed the design of cell-based assays aimed at isolating small molecular inhibitors of VirB8 interactions, with the goal of developing T4SS inhibitors as antivirulence agents .

The dimerization of VirB8 is likely crucial for its role as an assembly factor in the T4SS, potentially providing a platform for the recruitment and organization of other T4SS components. Targeting this dimerization process has proven to be an effective strategy, as inhibitors of VirB8 interactions can strongly reduce the intracellular proliferation of B. abortus 2308 in macrophage infection models .

What structural features distinguish VirB8 proteins from Gram-negative bacteria like B. abortus from VirB8-like proteins in Gram-positive bacteria?

The structural comparison of 17 representative VirB8 and VirB8-like proteins has revealed variations in the length of secondary structures (especially beta strands), their relative orientation, the curvature of the beta sheet, and additional structural elements inserted within the minimal fold . These subtle differences contribute to the distinct structural signatures of VirB8 proteins from different bacterial sources.

How does inhibition of VirB8 affect the expression of other virB genes and the function of the T4SS?

Research has demonstrated that VirB8 interaction inhibitors not only disrupt VirB8 functionality but also reduce the levels of VirB8 and other VirB proteins . Many of these inhibitors also inhibit virB gene transcription in Brucella abortus 2308, suggesting that targeting the secretion system has complex regulatory effects in vivo .

These findings indicate that VirB8 may play a role in regulating the expression of other T4SS components, either directly or indirectly. The inhibition of VirB8 can therefore have cascading effects on the entire T4SS apparatus, making it an effective target for antivirulence strategies. Significantly, one compound that inhibits VirB8 interactions has been shown to strongly inhibit the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model .

What is the relationship between HutC and virB8 gene expression in B. abortus?

HutC, the transcriptional repressor of the histidine utilization (hut) genes, has been identified as a protein that binds specifically to the virB promoter (PvirB) in B. abortus . This suggests that B. abortus has coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes .

This relationship provides insight into the metabolic control of virulence genes in B. abortus, demonstrating how the bacterium integrates metabolic pathways with virulence factor expression. The 27-kDa HutC protein was isolated using different procedures and found to bind specifically to the virB promoter, indicating its direct role in regulating virB gene expression .

What are the most effective methods for expressing and purifying recombinant VirB8 for structural studies?

For expressing and purifying recombinant VirB8 for structural studies, researchers typically employ the following methodology:

• Gene Identification and Amplification: Through techniques such as TMT proteomics, highly expressed VirB proteins in wild-type Brucella strains can be identified . The virB8 gene is then amplified from B. abortus genomic DNA using PCR with specific primers.

• Expression System Selection: The gene is cloned into an appropriate expression vector to create a fusion protein with an affinity tag, which facilitates purification.

• Protein Expression: The construct is transformed into E. coli expression strains and protein expression is induced under optimized conditions.

• Purification Strategy: The expressed protein is purified using a combination of chromatographic techniques, typically beginning with affinity chromatography followed by size-exclusion chromatography to obtain highly pure protein.

• Crystallization: For structural studies, the purified protein is concentrated and subjected to crystallization trials using various precipitants and conditions .

• Data Collection and Structure Determination: X-ray diffraction data are collected from protein crystals, and the structure is determined using methods such as molecular replacement or experimental phasing .

This approach has been successfully used to determine the structure of VirB8-like proteins, enabling detailed structural comparisons and insights into functional mechanisms .

How can high-throughput screening approaches be utilized to identify inhibitors of VirB8 protein interactions?

High-throughput screening for VirB8 interaction inhibitors can be implemented using a bacterial two-hybrid system through the following methodology:

• Assay Development: The bacterial two-hybrid system is adapted to specifically assay VirB8 interactions, creating a cell-based assay for the isolation of small molecular inhibitors .

• Screening Strategy: Compounds from small-molecule libraries are systematically tested for their ability to disrupt VirB8 protein-protein interactions in the bacterial two-hybrid system .

• Hit Identification: Specific inhibitors of VirB8 interactions are identified from the screening process .

• Secondary Validation: Hit compounds are validated through secondary assays that assess:

  • Effects on VirB8 and other VirB protein levels in B. abortus

  • Impact on virB gene transcription

  • Ability to inhibit intracellular proliferation of B. abortus in macrophage infection models

This approach has successfully identified specific small-molecule inhibitors that not only disrupt VirB8 interactions but also inhibit the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model, demonstrating the feasibility of developing antivirulence drugs that disarm Brucella .

What models are most appropriate for evaluating the role of VirB8 in B. abortus virulence?

Two primary models have proven valuable for evaluating the role of virB8 in B. abortus virulence:

• Macrophage Infection Model:

  • J774A.1 mouse macrophage-like cells are commonly used to assess the ability of B. abortus (wild-type and virB8 mutants) to survive within professional phagocytes .

  • This model provides a controlled environment for studying intracellular survival mechanisms and for initial screening of potential inhibitors .

  • All nonpolar deletions of virB genes (including virB8) reduced the ability of B. abortus to survive in these cells to a degree similar to that caused by a deletion of the entire virB locus .

• Mouse Infection Model:

  • This involves monitoring bacterial persistence in the spleens of mice over time (typically up to 8 weeks post-infection) .

  • Deletion of virB8 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection .

  • Interestingly, while virB8 is essential for virulence in this model, virB7 is not required for persistence in mouse spleens, highlighting the specificity of different T4SS components .

These complementary models allow researchers to comprehensively evaluate the role of virB8 in both acute intracellular survival and establishment of chronic infection.

How should researchers interpret discrepancies in VirB8 function between different experimental models?

When interpreting discrepancies in VirB8 function between different experimental models, researchers should consider:

• Model-Specific Factors:

  • Cell culture models assess short-term intracellular survival, while animal models evaluate long-term persistence and immune evasion.

  • Different models may reveal distinct aspects of VirB8 function in various environments.

• Functional Context:

  • The deletion of virB8 reduces both the ability of B. abortus to survive in J774A.1 cells and to persist in mouse spleens .

  • In contrast, deletion of virB7 reduces survival in macrophages but does not affect persistence in mouse spleens, highlighting how different T4SS components may have context-dependent functions .

• Experimental Parameters:

  • Infection dose, route, and timing of assessment can all affect outcomes.

  • Differences in experimental design may account for apparent discrepancies between studies.

• Mutation Strategy:

  • The type of mutation (polar versus nonpolar) can significantly impact the observed phenotype.

  • Studies have demonstrated that nonpolar deletions of virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11 all markedly reduce virulence in mice, while virB1, virB7, and virB12 are not required for persistence in this model .

What bioinformatic approaches are valuable for analyzing the conservation and evolution of VirB8 across Brucella species?

Several bioinformatic approaches have proven valuable for analyzing VirB8 conservation and evolution:

• Sequence Analysis and Phylogenetics:

  • Protein sequences can be retrieved from NCBI and compared using tools like ClustalW from MEGA7 .

  • Phylogenetic trees can be built using methods such as the Maximum Likelihood method based on the Tamura-Nei model .

  • These analyses reveal evolutionary relationships between VirB8 proteins from different species.

• Structural Prediction and Analysis:

  • TMD prediction using tools like CCTOP

  • Secondary structure prediction using PSIPRED

  • Subdomain structure predictions using HHPRED

  • Analysis of coiled-coil domains using tools such as COILS, MULTICOIL, and DEEPCOIL

• Comparative Structural Biology:

  • Structure comparison using DALI and PDBefold to fetch and analyze VirB8 structures

  • The mTM-align algorithm has been identified as particularly well-suited for superimposing VirB8 structures despite variations in secondary structure length and orientation .

  • Conservation analysis using the Consurf server to map sequence conservation onto three-dimensional structures .

These approaches have revealed important insights, such as the clear separation of Gram-positive and Gram-negative VirB8 structures in structure-based phylogenetic trees, despite sharing the core NTF2-like fold .

What are the key considerations when designing vaccine candidates based on recombinant VirB8?

When designing vaccine candidates based on recombinant VirB8, researchers should consider:

• Epitope Identification:

  • Immunoinformatics approaches can be used to identify antigenic epitopes of VirB8, including cytotoxic T lymphocyte epitopes, helper T lymphocyte epitopes, and B cell epitopes .

  • Studies have successfully screened and identified two cytotoxic T lymphocyte epitopes, nine helper T lymphocyte epitopes, six linear B cell epitopes, and six conformational B cell epitopes for constructing a multi-epitope vaccine .

• Immune Response Assessment:

  • VirB8 can induce specific humoral and cellular immune responses .

  • It has been shown to reduce the bacterial load of B. abortus S19 in mice and provide varying degrees of protection .

• Combination Strategies:

  • Combining VirB8 with other proteins to create recombinant vaccines has successfully induced immune responses .

  • Multiple studies have confirmed that such combination approaches can enhance protective efficacy.

• Delivery and Formulation:

  • The method of antigen presentation and adjuvant selection can significantly impact vaccine efficacy.

  • Both DNA and protein-based vaccine formulations have shown promise.

• Cross-Protection Potential:

  • VirB8-based vaccines should be evaluated for their ability to protect against various Brucella species and strains.

How can VirB8 be utilized in serological diagnosis of brucellosis?

VirB8 has potential value for serological diagnosis of brucellosis, with several important considerations:

• Recombinant Antigen Production:

  • Highly expressed VirB proteins identified through TMT proteomics can be prepared as recombinant proteins for diagnostic applications .

  • Purification and quality control methods must ensure antigen integrity and consistency.

• Diagnostic Performance:

  • Research has shown potential value of various VirB proteins, including VirB5, VirB10, and VirB12, for serological diagnosis of brucellosis .

  • VirB8 may similarly serve as a valuable diagnostic antigen, either alone or in combination with other VirB proteins.

• Assay Development:

  • ELISA, lateral flow, and other immunoassay formats can be developed using recombinant VirB8.

  • Optimization of assay conditions is necessary to maximize sensitivity and specificity.

• Comparative Evaluation:

  • The diagnostic performance of VirB8-based assays should be compared with existing diagnostic methods.

  • Systematic analysis is needed to fully evaluate the potential of VirB8 for serological diagnosis of brucellosis, as existing studies have mainly focused on individual VirB proteins .

What approaches are most effective for developing inhibitors targeting VirB8 as antivirulence agents?

Development of inhibitors targeting VirB8 as antivirulence agents can follow these effective approaches:

• Target Selection Rationale:

  • VirB8 is an ideal target because it is an essential type IV secretion system component, participates in multiple protein-protein interactions, and is essential for the assembly of the translocation machinery .

  • Inhibiting VirB8 interactions can disrupt T4SS functionality without directly killing the bacteria, reducing selection pressure for resistance.

• Screening Methodology:

  • The bacterial two-hybrid system can be adapted to assay VirB8 interactions for high-throughput screening .

  • Small-molecule libraries can be systematically screened to identify specific inhibitors of VirB8 interactions .

• Mechanism of Action Analysis:

  • VirB8 interaction inhibitors can reduce levels of VirB8 and other VirB proteins .

  • Many inhibitors also affect virB gene transcription in Brucella abortus 2308, suggesting complex regulatory effects in vivo .

• Efficacy Evaluation:

  • The ability of compounds to inhibit intracellular proliferation of B. abortus 2308 in macrophage infection models provides a critical efficacy endpoint .

  • One identified compound strongly inhibited intracellular proliferation, demonstrating the feasibility of this approach .

• Lead Optimization:

  • Hit molecules (specific VirB8 interaction inhibitors) can be further developed into leads for antivirulence drugs that effectively disarm Brucella .

  • Structure-activity relationship studies can guide the optimization of inhibitor potency and selectivity.