Recombinant Brucella suis biovar 1 Type IV secretion system protein virB2 (virB2)

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Description

Key Features

PropertyDetail
Protein LengthFull-length mature protein (residues 37–105)
AA SequenceNGGLDKVNTSMQKVLDLLSGVSITIVTIAIIWSGYKMAFRHARFMDVVPVLGGALVVGAA AEIASYLLR
Purity>90% (SDS-PAGE)
Storage BufferTris/PBS-based buffer with 6% trehalose, pH 8.0
StabilityLyophilized powder; avoid repeated freeze-thaw cycles
UniProt IDQ7CEG0

Biological Role of virB2 in Brucella Pathogenesis

virB2 is critical for the assembly and function of the Brucella T4SS, a macromolecular complex essential for intracellular survival and persistent infection .

Functional Contributions

  • T4SS Architecture:

    • Forms the pilus-like extracellular "needle" structure, mediating effector protein translocation into host cells .

    • Interacts with other T4SS components (e.g., VirB5, VirB8, VirB11) to stabilize the secretion machinery .

  • Virulence Mechanism:

    • Enables Brucella to subvert host pathways, avoiding lysosomal degradation and establishing replication niches in the endoplasmic reticulum .

    • Nonpolar virB2 mutants show defects in intracellular replication and fail to persist in murine models .

Comparative Analysis of virB2 in Brucella Strains

FeatureBrucella suis virB2Agrobacterium tumefaciens virB2
FunctionHost cell invasion, effector translocationPlant tumor formation, DNA transfer
LocalizationBacterial surface, pilus tipBacterial surface, pilus tip
Key InteractionsVirB5, VirB8, VirB11VirB5, VirB7, VirB9

Applications

  1. Structural Studies:

    • Used to analyze T4SS assembly dynamics and protein-protein interactions via cross-linking and immunofluorescence .

  2. Diagnostic Development:

    • Evaluated as a potential antigen for serological tests, though cross-reactivity with non-Brucella pathogens may limit specificity .

  3. Vaccine Research:

    • Serves as a candidate for subunit vaccines, leveraging its immunogenicity to elicit protective host responses .

Experimental Insights

  • Expression in A. tumefaciens:

    • Heterologous expression of Brucella virB2 in A. tumefaciens enhances conjugation efficiency, suggesting conserved T4SS mechanisms across species .

  • Mutagenesis Studies:

    • Nonpolar ΔvirB2 mutants in B. abortus exhibit impaired intracellular replication in macrophages and mice, confirming virB2’s essential role in pathogenesis .

Regulation and Expression

virB2 expression is tightly regulated during Brucella infection:

  • Environmental Triggers: Induced upon host cell entry, driven by pH changes and nutrient availability .

  • Regulatory Factors:

    • HutC: Activates virB operon transcription via histidine utilization.

    • VjbR: Modulates quorum sensing and VirB expression during early infection stages .

Product Specs

Form
Lyophilized powder
Note: We prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate them in your order. We will accommodate your request whenever possible.
Lead Time
Delivery time may vary depending on the purchase method and location. Please consult your local distributor for specific delivery timelines.
Note: Our proteins are typically shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional charges will apply.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend centrifuging the vial briefly before opening to ensure all contents settle at the bottom. Please reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. We suggest adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our default glycerol concentration is 50% and can be used as a reference.
Shelf Life
Shelf life is influenced by various factors, including storage conditions, buffer composition, temperature, and protein stability. Generally, the shelf life of liquid forms is 6 months at -20°C/-80°C, while lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Storage Condition
Store at -20°C/-80°C upon receipt. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type is determined during the manufacturing process.
The tag type is established during production. If you have specific tag type requirements, please inform us, and we will prioritize development accordingly.
Synonyms
virB2; BRA0068; BS1330_II0068; Type IV secretion system protein virB2
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
37-105
Protein Length
Full Length of Mature Protein
Species
Brucella suis biovar 1 (strain 1330)
Target Names
virB2
Target Protein Sequence
NGGLDKVNTSMQKVLDLLSGVSITIVTIAIIWSGYKMAFRHARFMDVVPVLGGALVVGAA AEIASYLLR
Uniprot No.

Target Background

Function
The VirB system may be essential for the establishment of the replication niche within the host.
Database Links

KEGG: bms:BRA0068

Protein Families
PtlA family
Subcellular Location
Cell membrane; Multi-pass membrane protein.

Q&A

What is the primary role of VirB2 in the Brucella Type IV secretion system?

VirB2 functions as the major subunit of the extracellular pilus structure in the Type IV secretion system (T4SS) of Brucella species. Research has demonstrated that VirB2 is essential for the proper assembly and function of the T4SS apparatus, which is required for intracellular replication and persistent infection in both cell culture and animal models . Unlike some other T4SS components, VirB2 appears to have functions beyond merely forming the structural pilus, as evidenced by mutational studies showing that certain VirB2 variants can maintain virulence even without detectable pili formation . The protein is predicted to be localized at the bacterial surface, where it potentially mediates direct interactions with host cell components during infection establishment .

How does the T4SS architecture incorporate VirB2 into the secretion machinery?

The T4SS typically consists of 12 components organized into ATP-powered, double-membrane-spanning complexes that facilitate substrate translocation across bacterial membranes . Within this architecture, VirB2 serves as the major pilin subunit that polymerizes to form the extracellular pilus structure. The T4SS includes three ATPases (VirB4, VirB11, and VirD4) that power substrate secretion and may assist in system assembly . Recent structural studies have revealed that VirB2 assembles with phosphatidylglycerol (PG) phospholipids in a 1:1 stoichiometric ratio to form a hollow tube-like structure . The inner membrane channel typically comprises the polytopic membrane protein VirB6 and the bitopic membrane proteins VirB8 and VirB10, while VirB9 in complex with the lipoprotein VirB7 may contribute to outer membrane pore formation .

What distinguishes Brucella T4SS from other bacterial T4SSs?

The Brucella T4SS shares structural homology with other bacterial T4SSs but exhibits distinct functional characteristics related to its role in intracellular pathogenesis. While conjugative T4SSs primarily transfer DNA between bacteria, the Brucella T4SS has evolved to deliver effector proteins that modulate host cell functions, enabling bacteria to establish an intracellular replicative niche associated with the endoplasmic reticulum . Unlike the Agrobacterium tumefaciens T4SS, which induces plant tumors, the Brucella T4SS facilitates chronic infection in mammalian hosts. Comparative studies between B. abortus and A. tumefaciens have shown that despite functional similarities, there are species-specific differences in T4SS component requirements, as evidenced by the dispensability of VirB1 for persistent infection in Brucella despite its importance in other bacterial T4SSs .

How do mutations in VirB2 affect T4SS function and bacterial virulence?

Mutational analyses of VirB2 have provided significant insights into structure-function relationships within the T4SS. Research has demonstrated that nonpolar deletion mutations of virB2 render Brucella incapable of intracellular replication in J774 macrophages and persistent infection in mouse models, confirming the essential role of this protein in virulence . Specific point mutations in VirB2, particularly those affecting the luminal loop region, have differential effects on protein stability and function. For instance, R91E and R91A mutations destabilize the VirB2 protein, resulting in undetectable protein levels by Western blot analysis and complete loss of virulence in plant infection assays . In contrast, the S93A mutation produces a stable protein that accumulates at lower levels and exhibits reduced but not abolished virulence . These findings indicate that specific amino acid residues in VirB2, particularly those involved in electrostatic interactions within the pilus lumen, are critical for protein stability and T4SS functionality.

What is the significance of the VirB2 luminal loop for T4SS function?

The luminal loop of VirB2 plays a crucial role in pilus assembly and T4SS function. Structural studies have revealed that arginine 91 (R91) in this loop forms essential electrostatic interactions with phosphate groups of phosphatidylglycerol molecules incorporated into the pilus structure . These interactions create a network within the lumen with no net charge, suggesting that the precisely balanced electrostatic environment is critical for pilus stability . Experimental evidence supports this hypothesis, as mutations R91E and R91A abolish pilus formation and virulence, indicating that this residue's positive charge is indispensable . The serine 93 residue appears to play a supporting but less critical role, as the S93A mutation reduces but does not eliminate virulence . These findings suggest that the luminal loop contributes to both the structural integrity of the T4SS apparatus and potentially to substrate translocation through the pilus channel.

How does phospholipid incorporation affect VirB2 function in the T4SS pilus?

Recent structural studies have revealed that the T-pilus incorporates phosphatidylglycerol (PG) molecules in a 1:1 stoichiometric ratio with VirB2 protein subunits . This lipid incorporation appears to be a conserved feature among conjugative pili, including those from Salmonella Typhi (pED208), E. coli (F-pilus), and Klebsiella pneumoniae (pKpQIL) . The phospholipid molecules likely contribute to the structural stability of the pilus through electrostatic interactions with specific amino acid residues in VirB2, particularly R91 in the luminal loop region . The co-induction of PG-maturation genes alongside T4SS components during virulence gene expression suggests a coordinated regulation of lipid availability for pilus assembly . The requirement for specific phospholipid interactions may explain why certain mutations that disrupt these interactions lead to defects in pilus formation and T4SS function, highlighting the importance of protein-lipid interactions in bacterial secretion system assembly and function.

What techniques are most effective for studying VirB2 structure-function relationships?

Investigating VirB2 structure-function relationships requires a multi-faceted approach combining genetic, biochemical, and structural methodologies. Site-directed mutagenesis represents a powerful technique for generating specific amino acid substitutions, as demonstrated by studies creating R91E, R91A, and S93A mutations to examine the importance of the luminal loop region . These mutations should target conserved residues or domains predicted to be functionally important through sequence analysis and structural modeling. Expression of mutant proteins can be verified using Western blot analysis with anti-VirB2 specific antibodies, which also provides information about protein stability . Functionally, mutant complementation studies in virB2 deletion backgrounds allow assessment of whether specific mutations affect T4SS function. Virulence can be evaluated using established infection models such as J774 macrophage intracellular replication assays or the Kalanchoe daigremontiana leaf infection assay for Agrobacterium . For structural characterization, cryo-electron microscopy has proven valuable for elucidating T-pilus architecture and the arrangement of VirB2 subunits within the secretion apparatus .

How can researchers generate and verify nonpolar mutations in virB2?

Creating nonpolar mutations in virB2, which avoid disrupting the expression of downstream genes in the virB operon, requires careful molecular genetic approaches. The methodology involves designing deletion constructs that preserve the reading frame and regulatory elements for downstream genes while specifically targeting the virB2 coding sequence . Researchers can employ homologous recombination-based techniques, where DNA fragments flanking the virB2 gene are amplified and joined to an antibiotic resistance marker, followed by allelic exchange in Brucella . To verify the nonpolar nature of these mutations, expression analysis of downstream genes such as virB5 must be performed, typically using Western blot analysis with specific antibodies or reverse transcription-PCR to detect mRNA transcripts . In studies of B. abortus, researchers demonstrated nonpolarity by showing that virB5 was expressed during stationary phase in the ΔvirB2 mutant at levels comparable to the wild type strain . This verification is crucial because polar effects on downstream genes would confound interpretation of phenotypes specifically attributed to virB2 deletion.

What infection models are most appropriate for evaluating VirB2 function in virulence?

Several infection models have been validated for assessing VirB2 contribution to Brucella virulence, each providing complementary information about T4SS function. In vitro, the J774 macrophage cell line offers a well-established model for studying intracellular replication, with wild-type Brucella strains showing significant multiplication over 48 hours while virB2 mutants fail to replicate . For in vivo studies, the mouse model of brucellosis provides valuable insights into persistent infection capabilities. Typically, groups of mice are infected with wild-type or mutant strains, and bacterial loads in the spleen are enumerated at multiple time points (1, 3, and 8 weeks post-infection) to assess both initial colonization and long-term persistence . For related bacterial systems like Agrobacterium, the Kalanchoe daigremontiana leaf infection assay serves as a functional assay to monitor Vir protein function through tumor formation . When selecting an appropriate model, researchers should consider the specific aspect of virulence being studied—initial invasion, intracellular trafficking, replication niche establishment, or long-term persistence—as VirB2 may contribute differently to each of these processes.

How can researchers differentiate between VirB2's structural and functional roles in the T4SS?

Differentiating between VirB2's structural and functional roles requires experimental strategies that can separate pilus formation from effector translocation. One approach involves generating and characterizing "uncoupling mutants" that maintain one function while losing the other . For example, certain single-amino acid substitutions in VirB2 have resulted in a Vir+, Pil- phenotype, where bacteria remain virulent despite lacking detectable pili, suggesting that VirB2 has roles beyond merely forming the structural pilus . Researchers can employ transmission electron microscopy to visualize pili formation while simultaneously assessing virulence in appropriate models . Another strategy involves using translocation assays with reporter-tagged effector proteins to directly measure T4SS substrate delivery independent of pilus detection. Structural studies using cryo-electron microscopy can provide insights into how specific mutations affect pilus architecture without necessarily disrupting the protein's contribution to substrate translocation machinery . By correlating structural perturbations with functional outcomes across multiple mutants, researchers can build a comprehensive model of how VirB2 contributes both architecturally to the T4SS apparatus and functionally to its secretion capabilities.

What are the key considerations when analyzing VirB2 interactions with phospholipids in the T4SS?

When analyzing VirB2 interactions with phospholipids in the T4SS, researchers must address several critical considerations. First, appropriate lipid extraction and identification methods are essential, with shotgun lipidomics spectrometry being effective for characterizing the phosphatidylglycerol (PG) components incorporated into the pilus structure . Second, researchers should consider the specificity of protein-lipid interactions, as the 1:1 stoichiometric assembly of VirB2 and PG suggests precise structural requirements rather than random association . Mutation studies targeting potential lipid-interacting residues, such as those in the luminal loop region, can provide insights into the importance of these interactions for pilus stability and function . The coordination between lipid availability and T4SS assembly should also be examined, particularly since PG-maturation genes are co-induced with virulence genes during infection . Finally, researchers should consider species-specific differences in membrane composition and how these might affect T4SS assembly across bacterial pathogens. Integrating structural biology approaches with functional assays will provide the most comprehensive understanding of how protein-lipid interactions contribute to T4SS biology and bacterial virulence.

What are the most promising approaches for identifying VirB2-interacting host cell factors?

Identifying host cell factors that interact with VirB2 represents a crucial frontier in understanding T4SS-mediated pathogenesis. Several complementary approaches can advance this research area. Proximity-dependent biotin identification (BioID) or APEX2-based proximity labeling, where VirB2 is fused to a biotin ligase that biotinylates nearby proteins, could identify host proteins in close proximity during infection . Yeast two-hybrid screening using VirB2 as bait against human or animal cell cDNA libraries might identify direct protein-protein interactions, though this approach requires careful consideration of VirB2's membrane topology . Pull-down assays using purified VirB2 (potentially with attached phospholipids to maintain native conformation) followed by mass spectrometry could identify binding partners from host cell lysates . The generation of VirB2 variants with single amino acid substitutions in surface-exposed regions, followed by virulence phenotyping, might identify residues critical for host interactions without disrupting pilus structure . Finally, CRISPR-Cas9 screens in host cells followed by infection with Brucella could identify host factors required for T4SS-dependent invasion or intracellular trafficking. Integration of these approaches would provide a comprehensive map of VirB2-host interactions during infection.

How might advances in structural biology techniques further our understanding of VirB2 function?

Recent advances in structural biology techniques offer unprecedented opportunities to elucidate VirB2 function within the T4SS complex. Cryo-electron microscopy has already provided valuable insights into T-pilus architecture, revealing the 1:1 stoichiometric assembly of VirB2 and phosphatidylglycerol . Further application of this technique to visualize the entire T4SS apparatus, particularly in different functional states (assembly, secretion, disassembly), would illuminate how VirB2 contributes to the dynamic processes of substrate translocation . Single-particle cryo-electron tomography could capture T4SS complexes in situ within bacterial membranes during host cell interaction, providing contextual information about VirB2's role in establishing infection . Integrative structural approaches combining X-ray crystallography of individual domains with cryo-EM of larger assemblies and molecular dynamics simulations could generate comprehensive models of VirB2 conformational changes during pilus assembly and substrate secretion . Hydrogen-deuterium exchange mass spectrometry could identify regions of VirB2 that undergo conformational changes upon interaction with other T4SS components or host factors. These structural insights would guide rational design of targeted mutations and potential inhibitors of T4SS function for therapeutic applications.

What potential exists for developing T4SS inhibitors targeting VirB2 as antimicrobial agents?

The essential role of VirB2 in Brucella virulence makes it an attractive target for developing novel antimicrobial strategies against brucellosis and potentially other infections dependent on T4SS. Structure-based drug design approaches, informed by recent structural characterizations of the T-pilus, could identify small molecules that disrupt critical protein-protein or protein-lipid interactions . Particularly promising targets include the luminal loop region containing R91, which forms essential electrostatic interactions with phospholipids, and interfaces between VirB2 subunits within the pilus structure . High-throughput screening assays could identify compounds that inhibit pilus assembly or destabilize formed pili, using either purified components or whole-cell assays monitoring T4SS-dependent phenotypes. Peptide inhibitors mimicking key regions of VirB2 could potentially interfere with protein-protein interactions essential for T4SS assembly. Targeting the VirB2-phospholipid interaction represents another innovative approach, as compounds that compete with phosphatidylglycerol binding could disrupt pilus formation . Since virB2 mutations affecting pilus formation render bacteria avirulent, such inhibitors would not kill bacteria directly but would effectively disarm their virulence machinery, potentially reducing selective pressure for resistance development compared to conventional antibiotics .

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