Recombinant Brucella ovis Type IV secretion system protein virB10 (virB10)

What is the role of VirB10 in Brucella ovis pathogenesis?

VirB10 is an essential structural component of the Type IV Secretion System in Brucella species. In B. ovis, as in other Brucella species, the VirB apparatus plays a critical role in intracellular multiplication and bacterial virulence. VirB10 specifically is necessary for the correct function of this secretion apparatus, likely enabling the pathogen to establish its replicative niche within host cells . The T4SS functions as a molecular syringe that delivers effector molecules into the host cell, determining routing of Brucella to an endoplasmic reticulum-related replication compartment . Genetic studies with virB10 mutants have demonstrated their reduced ability to replicate within host cells, confirming its importance for bacterial survival and virulence .

How does VirB10 structure contribute to Type IV secretion system function?

VirB10 spans the periplasm and integrates into both the inner and outer membranes, serving as a central scaffolding protein that connects various T4SS components. Structural analyses reveal that VirB10 contains a C-terminal domain (CTD) that forms part of the outer membrane complex . The protein undergoes conformational changes in response to ATP energy, enabling the secretion apparatus to transport substrates across the bacterial cell envelope. RMSDev and RMSF analyses of VirB10 show maximum deviations in the AP loop and fluctuations in regions surrounding loops L1, L2, and L3, suggesting these are important flexible regions that accommodate structural changes during secretion . These dynamic properties are essential for the channel function of the T4SS complex.

How is recombinant VirB10 typically produced for research purposes?

Recombinant VirB10 production involves cloning and expressing the protein as a histidine-tagged fusion in E. coli expression systems. Researchers have developed methods that yield soluble protein without requiring purification from inclusion bodies or additional solubilization and refolding steps . The procedure typically includes:

• Cloning the virB10 gene into an expression vector with a histidine tag

• Transforming the construct into a suitable E. coli strain

• Optimizing culture conditions for soluble protein expression

• Purifying the protein using nickel affinity chromatography

• Confirming protein identity and integrity via SDS-PAGE and Western blot analysis

What experimental approaches can be used to study VirB10 function in Brucella ovis?

Investigating VirB10 function requires multiple complementary approaches:

• Genetic manipulation: Creating precise polar and non-polar mutations in the virB10 gene using homologous recombination or CRISPR-Cas9 techniques. Comparing the behavior of polar virB10 mutants (affecting downstream genes) with non-polar mutants (affecting only virB10) provides insights into the specific contribution of VirB10 to virulence .

• Cellular infection models: HeLa cell infection assays provide a controlled system to assess the ability of virB10 mutants to enter and replicate within host cells. Quantification of intracellular bacterial replication at different time points post-infection reveals the impact of VirB10 on establishing the replicative niche .

• Mouse infection models: In vivo studies using mice challenge provide crucial information about virulence. Bacterial loads in blood and organs (typically spleen) should be quantified using real-time qPCR assays targeting single-copy genes such as msp5 .

• Structural biology approaches: X-ray crystallography and cryo-electron microscopy of purified VirB10 or T4SS complexes can reveal critical domains and conformational changes associated with secretion function .

• Protein-protein interaction studies: Co-immunoprecipitation, bacterial two-hybrid systems, or cross-linking experiments can identify interactions between VirB10 and other T4SS components or host cell targets.

How can immune responses to recombinant VirB10 be characterized in vaccination studies?

Comprehensive characterization of immune responses to VirB10 vaccination involves:

• Antibody responses: Determine anti-VirB10 antibody titers using ELISA with the recombinant protein as coating antigen. Endpoint titers should be calculated and correlated with protection levels .

• T cell responses: Analyze CD4+ and CD8+ T cell responses using flow cytometry. Specifically, measure:

  • IFN-γ+ CD4+ T cells (indicative of Th1 responses)

  • IL-10+ CD4+ T cells (regulatory response)

  • Double-positive (IL-10+-IFN-γ+) CD4+ T cells

  • IFN-γ+ CD8+ T cells (cytotoxic response)

• Cytokine profiling: Measure cytokine production by stimulated splenocytes using ELISA or multiplex cytokine assays.

• Protection assessment: Challenge vaccinated animals with virulent B. ovis and quantify bacterial loads in blood and target organs using qPCR .

This data indicates that VirB10 vaccination specifically stimulates a Th1-type CD4+ T cell response characterized by IFN-γ production, which correlates with the observed partial protection .

What are the critical structural domains of VirB10 that should be preserved in recombinant protein design?

When designing recombinant VirB10 constructs, researchers must carefully consider preserving key functional domains:

• Transmembrane domain: The N-terminal transmembrane region anchors VirB10 to the inner membrane. For soluble recombinant production, this hydrophobic region is often excluded.

• Periplasmic region: This central portion contains a proline-rich region that provides flexibility for conformational changes.

• C-terminal domain (CTD): This region forms part of the outer membrane complex and contains the most conserved sequences. The CTD includes:

  • α-helices that form the outer membrane pore

  • β-barrel structure that provides stability

  • Antigen-presenting (AP) loop between α2 and α3 helices, which shows maximum fluctuations in molecular dynamics studies

  • Loops L1, L2, and L3 bulging from the β-barrel that contribute to structural flexibility

Recombinant constructs should maintain the integrity of these domains, particularly the CTD, which contains immunodominant epitopes. Protein modeling and structural analysis should be performed to ensure the recombinant protein adopts proper folding similar to the native protein.

How can researchers address the challenge of distinguishing between host responses to B. ovis VirB10 and cross-reactive responses to related bacterial species?

Cross-reactivity between VirB10 proteins from different bacterial species presents a significant challenge in immunological studies. To address this:

• Sequence alignment analysis: Perform detailed bioinformatic analysis of VirB10 sequences from Brucella ovis and related bacteria to identify unique epitopes specific to B. ovis.

• Epitope mapping: Use peptide arrays or phage display libraries to identify B. ovis-specific epitopes that can be used for selective detection of anti-B. ovis VirB10 antibodies.

• Absorption studies: Pre-absorb sera with recombinant VirB10 proteins from related bacteria to remove cross-reactive antibodies before testing for B. ovis-specific responses.

• Western blot analysis: Compare reactivity patterns of antibodies against VirB10 from different bacterial species to identify size differences or unique banding patterns.

• Recombinant protein design: Engineer chimeric or truncated constructs that emphasize B. ovis-specific regions while excluding conserved domains that contribute to cross-reactivity.

• T cell response assay specificity: Test T cell responses using both conserved and variable peptide regions of VirB10 to differentiate between specific and cross-reactive T cell populations.

What are the optimal conditions for expressing soluble recombinant B. ovis VirB10?

Obtaining soluble recombinant VirB10 requires careful optimization of expression conditions:

• Expression vector selection: Use vectors with tunable promoters (like pET systems) that allow careful control of expression levels. Strong promoters often lead to inclusion body formation.

• Host strain optimization: E. coli strains like BL21(DE3)pLysS or Rosetta that supply rare codons can improve expression of Brucella proteins.

• Expression temperature: Lower temperatures (16-20°C) during induction slow protein synthesis and often enhance proper folding and solubility.

• Induction conditions: Use lower IPTG concentrations (0.1-0.5 mM) and longer induction times to favor soluble expression.

• Buffer optimization: Include stabilizing agents such as glycerol (5-10%), reducing agents, and appropriate salt concentrations in lysis and purification buffers.

• Fusion tags: Consider solubility-enhancing fusion partners such as MBP (maltose-binding protein) or SUMO in addition to histidine tags for purification.

• Cell lysis methods: Gentle lysis methods like enzymatic lysis with lysozyme followed by mild sonication help maintain protein structure and solubility.

These optimized conditions have been shown to produce adequate yields of soluble recombinant VirB10 suitable for immunization and structural studies .

What challenges might researchers encounter when creating B. ovis virB10 mutants, and how can these be addressed?

Creating and studying B. ovis virB10 mutants presents several technical challenges:

• Genetic manipulation difficulties: B. ovis has a more restricted host range and growth requirements than other Brucella species. Researchers should optimize electroporation conditions specifically for B. ovis strains and use specialized media supplemented with serum or blood.

• Polar effects on operon expression: Since virB10 is part of a multi-gene operon, mutations can affect downstream gene expression. Address this by:

  • Creating non-polar mutations that don't disrupt the reading frame

  • Using complementation studies with the wild-type gene to confirm phenotype specificity

  • Conducting qRT-PCR to verify expression levels of other operon genes

• Functional redundancy: Some virB genes may have partially redundant functions. Research shows that double mutants lacking both omp31 and omp25 cannot be obtained, suggesting essential functions . Similarly, the absence of two major lipoproteins (Omp10 and Omp19) is not tolerated . Plan mutation strategies carefully based on known genetic interactions.

• Attenuated growth: virB mutants often show growth defects in vitro, complicating phenotypic analysis. Use appropriate controls and carefully standardize inocula in infection experiments.

• Biosafety considerations: Although B. ovis is non-zoonotic, it requires BSL-2 containment, and genetic manipulation experiments should include appropriate biosafety measures.

In addressing these challenges, researchers can gain valuable insights into the specific contributions of VirB10 to B. ovis virulence and host interactions.

How can researchers effectively evaluate VirB10-based vaccine candidates against B. ovis infection?

Comprehensive evaluation of VirB10-based vaccines against B. ovis requires a systematic approach:

• Immunization protocol optimization:

  • Test different adjuvant formulations to enhance immune responses

  • Compare various routes of administration (subcutaneous, intramuscular, intraperitoneal)

  • Evaluate different dosing schedules (prime-boost intervals)

  • Determine optimal antigen concentration through dose-ranging studies

• Immune response characterization:

  • Measure antibody responses using ELISA, focusing on different antibody isotypes

  • Evaluate T cell responses through flow cytometry, particularly IFN-γ+ CD4+ T cells which have been shown to correlate with protection

  • Assess cytokine profiles from stimulated splenocytes

• Challenge model development:

  • Standardize infectious dose based on colony forming units or genome equivalents

  • Determine optimal timepoints for bacterial load assessment

  • Identify target tissues for bacterial quantification (blood, spleen, reproductive organs)

• Protection assessment:

  • Quantify bacterial loads in blood and target organs using qPCR targeting single-copy genes

  • Evaluate clinical parameters and pathological changes in infected tissues

  • Determine rate of bacterial clearance over time

• Correlates of protection analysis:

  • Perform statistical analyses to identify immune parameters that correlate with protection

  • Test for correlations between antibody titers, T cell responses, and bacterial loads

Studies with related organisms have demonstrated that VirB10 vaccination can provide partial protection, characterized by significantly lower bacterial loads in vaccinated animals compared to controls . Similar approaches can be applied to B. ovis vaccine development.

How might systems biology approaches enhance our understanding of VirB10 function in B. ovis?

Systems biology offers powerful tools to comprehensively understand VirB10's role in B. ovis pathogenesis:

• Transcriptomics: RNA-seq analysis can reveal:

  • How virB10 expression changes under different environmental conditions

  • Genes co-regulated with virB10 during infection

  • Host cell transcriptional responses to wild-type versus virB10 mutant infection

• Proteomics:

  • Mass spectrometry analysis of the T4SS "secretome" can identify effector proteins transported by the virB machinery

  • Quantitative proteomics comparing wild-type and virB10 mutants can reveal differential protein abundance

  • Phosphoproteomics can identify signaling pathways affected by VirB10-dependent secretion

• Metabolomics:

  • Metabolic profiling of bacteria with and without functional VirB10 can reveal metabolic adaptations dependent on T4SS function

  • Host cell metabolic changes during infection may identify metabolic pathways targeted by T4SS effectors

• Interactomics:

  • Protein-protein interaction networks can map the complete interactome of VirB10

  • Bacterial two-hybrid or pull-down assays can identify both bacterial and host interaction partners

• Multi-omics integration:

  • Computational integration of different omics datasets can provide a systems-level understanding of VirB10 function

  • Network analysis can identify key nodes in pathogenicity networks dependent on VirB10

These approaches could identify new targets for therapeutic intervention and vaccine development against B. ovis infection.

What are the emerging strategies for enhancing recombinant VirB10 immunogenicity in vaccine development?

Recent advances suggest several promising approaches to enhance VirB10 vaccine efficacy:

• Structural vaccinology:

  • Structure-based design of VirB10 immunogens that present key epitopes in optimal conformations

  • Stabilization of VirB10 in specific conformational states that induce protective immune responses

  • Epitope-focused design targeting the most immunogenic and protective regions

• Advanced delivery systems:

  • Nanoparticle formulations that enhance antigen presentation and stability

  • Liposomal delivery systems that promote cross-presentation to CD8+ T cells

  • Virus-like particles displaying VirB10 epitopes in high density

• Adjuvant optimization:

  • TLR agonists specifically tailored to enhance Th1-type responses needed for intracellular pathogen control

  • Combination adjuvants targeting multiple innate immune pathways

  • Cytokine-adjuvanted formulations, particularly with IL-12 to enhance IFN-γ production

• Multivalent approaches:

  • Combining VirB10 with other T4SS components (such as VirB9-1 and VirB9-2) in a single formulation

  • Fusion constructs linking VirB10 to other immunogenic Brucella antigens

  • DNA prime-protein boost strategies to enhance both cellular and humoral immunity

• Immune response modulation:

  • Strategies to target specific dendritic cell subsets for optimal T cell priming

  • Approaches to overcome potential immune evasion mechanisms

  • Methods to enhance duration of protective immunity through memory T cell generation

Studies in related organisms have demonstrated that VirB10 can induce protective CD4+ Th1 immune responses characterized by IFN-γ production . These emerging strategies aim to enhance and broaden these protective responses.

What are the key considerations for researchers beginning work with B. ovis VirB10?

Researchers entering the field of B. ovis VirB10 should consider the following key points:

• Biosafety considerations: Although B. ovis is non-zoonotic, appropriate biosafety practices (BSL-2) must be followed when working with the organism.

• Genetic context: VirB10 functions as part of the larger virB operon containing 13 open reading frames. Understanding the genetic organization and regulation of this operon is essential for proper experimental design .

• Structural complexity: VirB10 spans multiple cellular compartments and undergoes conformational changes during T4SS function. Consider this structural complexity when designing recombinant constructs or planning mutagenesis experiments .

• Technical challenges: Be prepared for challenges in protein expression, as VirB10 may produce multiple fragments during recombinant production . Optimization of expression conditions is critical for obtaining functional protein.

• Interdisciplinary approach: Effective study of VirB10 requires expertise in molecular biology, immunology, structural biology, and infection models. Collaborative approaches are often most successful.

• Translational potential: While focusing on basic research questions, consider the translational potential of findings for vaccine development and diagnostic applications.