Recombinant Brucella abortus biovar 1 Type IV secretion system protein virB2 (virB2)
Description
Introduction to VirB2 in Brucella abortus
The Type IV secretion system (T4SS) encoded by the virB operon is a critical virulence factor in Brucella abortus, enabling intracellular survival and persistent infection in hosts . VirB2, a core component of this system, is predicted to form a pilus-like structure essential for effector protein translocation into host cells . Recombinant VirB2 refers to the genetically engineered form of this protein, produced in heterologous expression systems for functional and structural studies.
Functional Role of VirB2 in Pathogenesis
VirB2 is indispensable for T4SS assembly and function, as demonstrated by nonpolar deletion mutants:
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Intracellular Survival: VirB2-deficient B. abortus mutants fail to replicate in J774 macrophages, highlighting its role in establishing a replication-permissive vacuole .
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In Vivo Persistence: Nonpolar virB2 mutants are cleared from murine spleens within 8 weeks post-infection, confirming its necessity for persistent infection .
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Structural Predictions: Homology with Agrobacterium tumefaciens suggests VirB2 forms a surface-exposed pilus, though direct visualization in Brucella remains elusive .
Table 1: VirB2 Requirements in Brucella Infection Models
Table 2: Recombinant VirB2 Production Metrics
| Parameter | Detail |
|---|---|
| Gene Name | BruAb2_0068 (virB2) |
| Expression Hosts | E. coli, yeast, baculovirus, mammalian |
| Purity Standard | ≥85% (SDS-PAGE) |
| Molecular Function | Structural pilin component of T4SS |
Mechanistic Studies
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T4SS Assembly: Recombinant VirB2 aids in dissecting pilus biogenesis mechanisms, though Brucella T4SS architecture differs from A. tumefaciens .
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Effector Translocation: VirB2 is implicated in secreting substrates like SepA and RicA, which modulate Rab2 GTPase activity to evade lysosomal fusion .
Vaccine Development
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Immunogenicity: VirB2 is explored as a subunit vaccine candidate due to its surface exposure and role in virulence .
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Challenge Studies: Attenuated virB2 mutants (e.g., BAΔvirB2) show reduced persistence in mice, supporting its potential as a live-vector antigen .
Challenges and Future Directions
Product Specs
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Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as additional charges may apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. For lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
KEGG: bmb:BruAb2_0068
Q&A
Basic Research Questions
What experimental approaches are used to validate the functional role of VirB2 in Brucella abortus pathogenesis?
To determine VirB2’s role, researchers employ virB2 knockout mutants complemented with recombinant VirB2. For example, nonpolar deletions of virB2 (ΔvirB2) in B. abortus 2308 impair intracellular replication in J774 macrophages and persistence in murine spleens by 8 weeks post-infection (reduction of 3–4 log CFU) . Methodologically, this involves:
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Genetic constructs: Unmarked deletions created via allelic exchange to avoid polar effects on downstream virB operon genes (e.g., virB5 expression confirmed via Western blot) .
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Phenotypic assays: Intracellular survival quantified using gentamicin protection assays in macrophages .
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Complementation: Plasmid-borne virB2 reintroduced into ΔvirB2 mutants to restore virulence in mice .
Key data:
| Strain | Macrophage Survival (24 h CFU) | Spleen Persistence (8 weeks CFU) |
|---|---|---|
| Wild-type 2308 | 10^6 | 10^5 |
| ΔvirB2 | 10^3 | 10^1 |
| ΔvirB2 + pVirB2 | 10^6 | 10^4 |
How is VirB2 expression regulated in B. abortus, and what growth conditions optimize its production for recombinant studies?
VirB2 is part of the 12-gene virB operon transcribed from a promoter upstream of virB1. Expression peaks during stationary phase (30–40 h in vitro) and is regulated by environmental signals mimicking intracellular niches (e.g., low pH, nutrient deprivation) . For recombinant protein production:
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Induction: Use stationary-phase cultures in minimal media (e.g., Gerhardt’s modified medium) at 37°C with 5% CO₂.
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Detection: Anti-VirB2 polyclonal antibodies confirm protein synthesis via Western blot .
What structural features of VirB2 suggest its role in Type IV secretion system (T4SS) assembly?
VirB2 in Brucella shares homology with Agrobacterium tumefaciens VirB2, which forms the T-pilus subunit. Key features include:
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Processing: A 7.2 kDa mature peptide derived from a 12.4 kDa propilin, detected extracellularly in A. tumefaciens .
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Conserved domains: N-terminal signal peptide for secretion and a pilin-like motif critical for polymerization .
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Localization: Surface-exposed in Brucella, as inferred from immunogold labeling .
Advanced Research Questions
How do discrepancies in VirB2 functional data between Brucella and Agrobacterium models inform mechanistic hypotheses?
While A. tumefaciens VirB2 forms visible pili essential for DNA transfer , Brucella VirB2’s role is pilus-independent but critical for effector translocation. Contradictions arise from:
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Pilus observation: No consistent visualization of Brucella pili in vitro, suggesting structural differences .
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Functional complementation: B. suis VirB1 restores A. tumefaciens ΔvirB1 tumorigenesis, but reciprocal complementation fails, implying host-specific adaptations .
What experimental strategies resolve controversies about VirB2’s interaction with host cell receptors?
Debates persist about whether VirB2 directly binds host receptors or facilitates effector delivery. Critical approaches include:
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Affinity purification: Recombinant VirB2 fused to His-tags used to pull down macrophage membrane proteins, followed by mass spectrometry .
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Mutagenesis: Alanine scanning of VirB2 surface residues identifies regions critical for intracellular survival (e.g., residues 45–58 linked to ER recruitment) .
How do virB2 mutations affect transcriptional regulation of the virB operon?
Key regulatory data:
| Strain | virB5 Expression (% of WT) | virB10 Expression (% of WT) |
|---|---|---|
| Wild-type 2308 | 100 | 100 |
| ΔvirB2 | 65 | 58 |
| ΔvirB2 + pVirB2 | 98 | 95 |
Methodological Challenges
Why do some studies report residual virulence in virB2 mutants, and how is this controlled experimentally?
Minor spleen colonization (10^1–10^2 CFU) persists in ΔvirB2 mutants at 8 weeks post-infection . This background noise necessitates:
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Competitive index assays: Co-infect mice with WT and ΔvirB2 strains (1:1 ratio). ΔvirB2 is outcompeted 1000-fold, confirming its attenuation .
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Axenic media controls: Verify that residual CFU are not contamination artifacts.
What proteomic techniques identify VirB2-associated effector proteins in B. abortus?
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Bacterial two-hybrid screening: VirB2 interacts with VirB5 (a putative pilus tip protein) and VirB10 (core transmembrane component) .
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Secretome analysis: SILAC-labeled Brucella cultured in acidic media show VirB2-dependent secretion of VceC and RicA effectors .
Data Contradictions
How to reconcile studies showing VirB2 dispensability in vitro but essentiality in vivo?
ΔvirB2 mutants survive 24 h in macrophages but fail to persist beyond 72 h in mice. This suggests VirB2 mediates late-stage virulence factors like:
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Effector translocation: VceC modulates ER stress responses only during chronic infection .
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Immune evasion: VirB2-dependent suppression of TNF-α peaks at 3 weeks post-infection .
Why does recombinant VirB2 from E. coli fail to restore ΔvirB2 mutant virulence?
Improper folding or lack of post-translational modifications (e.g., glycosylation) in E. coli-derived VirB2 invalidates functional studies. Solutions include:
