Recombinant Brucella melitensis biotype 1 Type IV secretion system protein virB10 (virB10)

What is the VirB10 protein in Brucella melitensis and what is its role in the Type IV Secretion System?

VirB10 is a critical structural component of the Type IV Secretion System (T4SS) in Brucella melitensis. The T4SS is encoded by the virB operon and functions as a major virulence factor for Brucella species. This secretion apparatus disrupts cellular pathways, induces host immune responses by secreting effector molecules, promotes bacterial replication within host cells, and facilitates persistent infection .

Structurally, VirB10 is a bilayer protein inserted into the bacterial endosome with a proline-rich region that plays a key role in core complex assembly and substrate secretion . It functions as part of a transenvelope "core" structure essential for the assembly of the secretion channel . VirB10 has a unique architecture that allows it to extend across the periplasm via its proline-rich region (PRR) while its β-barrel domain interacts with the VirB7-VirB9 channel complex .

Functionally, VirB10 exhibits dynamic properties, coupling inner membrane energy to the stable assembly of the core complex. Upon sensing ATP energy consumption by the inner membrane VirD4 and VirB11 ATPases, VirB10 undergoes a structural transition necessary for forming a stable VirB7–VirB9–VirB10 complex, which ultimately enables the passage of DNA substrates through the translocation channel .

What methodologies are effective for expressing and purifying recombinant VirB10 protein?

Successful expression and purification of recombinant VirB10 protein typically involves molecular cloning techniques followed by protein expression in bacterial systems and subsequent purification under appropriate conditions. Based on research evidence, the following methodological approach is recommended:

• Gene Amplification and Cloning: Amplify the virB10 gene from Brucella genomic DNA using PCR with specific primers containing appropriate restriction sites. Clone the amplified gene into a suitable expression vector containing a histidine tag for easier purification .

• Expression System Selection: Utilize E. coli BL21(DE3) or similar expression strains for protein production. Induction is typically performed using IPTG (Isopropyl β-D-1-thiogalactopyranoside) at optimized concentrations and temperatures .

• Protein Purification Approach: As demonstrated in previous studies, purification under denaturing conditions has proven effective for VirB10. This involves:

  • Cell lysis using sonication in denaturing buffer containing urea

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Refolding through gradual dialysis to remove denaturants

  • Final purification steps such as size exclusion chromatography if needed

Purified recombinant VirB10 should be validated by SDS-PAGE analysis and Western blotting using anti-His antibodies or specific anti-VirB10 antibodies. Protein functionality can be assessed through its ability to interact with other T4SS components such as VirB9 .

How can researchers evaluate VirB10's potential as a diagnostic marker for brucellosis?

VirB10 has shown promising potential as a serological marker for Brucella infection, offering advantages over traditional lipopolysaccharide (LPS)-based diagnostic methods. To evaluate VirB10's diagnostic capabilities, researchers should consider the following methodological approaches:

• Indirect ELISA Development:

  • Coat ELISA plates with purified recombinant VirB10 protein at optimized concentrations

  • Test against well-characterized serum panels (positive and negative controls)

  • Determine optimal cutoff values using ROC curve analysis

  • Calculate sensitivity, specificity, and other performance metrics

• Cross-reactivity Assessment:

  • Test the recombinant VirB10-based ELISA against sera from patients with other febrile diseases

  • Include sera from individuals infected with other gram-negative bacteria, particularly those sharing antigenic features with Brucella

  • Recent studies have shown that rVirB10 exhibits minimal cross-reactivity in serum samples from febrile patients without brucellosis, suggesting high specificity for diagnostic applications

• Comparative Evaluation:

  • Compare performance against gold standard tests including culture methods and conventional serological tests like Rose Bengal Test and standard LPS-based ELISAs

  • A systematic comparison reveals that VirB10-based diagnostics may show slightly lower sensitivity than traditional LPS antigens but potentially higher specificity

• Immunoreactivity Testing:

  • Assess immunoreactivity using experimental infection models (e.g., Balb/C mice infected with B. melitensis)

  • Test against clinical isolates and field samples

  • Evaluate antibody response kinetics during different stages of infection

These approaches help establish whether VirB10 can serve as a reliable diagnostic marker with advantages over current methods, particularly in terms of specificity and reduced cross-reactivity with other gram-negative bacteria.

What protein-protein interactions does VirB10 engage in within the T4SS complex?

VirB10 engages in multiple critical protein-protein interactions that are essential for T4SS assembly and function. Understanding these interactions is important for both basic research and applied studies focused on diagnostics and vaccine development.

The key interactions of VirB10 include:

• VirB10-VirB9 Interactions:

  • VirB10 directly interacts with VirB9 components to form a stable complex

  • Specifically, interactions between VirB10 and VirB9-1 as well as VirB9-2 have been confirmed through both far-Western blotting and immunoprecipitation assays

  • These interactions contribute to the formation of the outer membrane complex of the T4SS

• VirB10-VirB7 Interactions:

  • VirB10 interacts with VirB7 to form a VirB7-VirB9-VirB10 complex

  • This tripartite complex serves as the "core" structure for assembly of the secretion channel

  • The complex forms in an ATP-dependent manner, requiring energy input from the inner membrane ATPases VirD4 and VirB11

• Energy-Sensing Interactions:

  • VirB10 interacts with the ATPases VirD4 and VirB11

  • Upon ATP consumption by these ATPases, VirB10 undergoes a structural transition

  • This energy-dependent conformational change is necessary for stable complex formation and substrate translocation

These interactions highlight VirB10's central role as a dynamic coordinator in T4SS, linking energy consumption at the inner membrane to productive assembly of the secretion machinery. The multiple interaction sites within VirB10 make it a promising candidate for targeted interventions, whether for vaccine development or novel antimicrobial strategies.

What structural domains of VirB10 are critical for its function in T4SS assembly and operation?

VirB10 contains several distinct structural domains that contribute to its versatile functions in T4SS assembly and operation. Understanding these domains is essential for structure-function analyses and targeted modifications for research applications:

• N-terminal Transmembrane Domain:

  • Anchors VirB10 to the inner membrane

  • Essential for proper localization within the bacterial cell envelope

  • Mutational studies indicate this domain is required for stable integration into the membrane

• Proline-Rich Region (PRR):

  • Extends across the periplasmic space

  • Acts as a flexible linker that accommodates changes in periplasmic width

  • Plays a key role in core complex assembly and substrate secretion

  • The proline-rich composition provides structural flexibility while maintaining sufficient rigidity

• β-Barrel Domain:

  • Interacts with the VirB7-VirB9 channel complex at the outer membrane

  • Critical for forming the stable VirB7-VirB9-VirB10 core complex

  • Mutations in this domain can selectively affect T pilus biogenesis without disrupting substrate transfer, suggesting a regulatory role in channel/pilus assembly

• ATP-Sensing Region:

  • Responds to ATP consumption by the inner membrane ATPases (VirD4 and VirB11)

  • Undergoes conformational changes that activate the secretion machinery

  • Essential for coupling energy utilization to secretion channel assembly

Mutational analyses have revealed that VirB10 can be modified to "uncouple" its dual functions, with certain mutations selectively blocking T pilus biogenesis while preserving substrate transfer capabilities. This suggests that VirB10 plays a regulatory role in determining whether the T4SS assembles a secretion channel or an extracellular pilus .

How can researchers design multi-epitope vaccine candidates incorporating VirB10 against Brucella infections?

Designing effective multi-epitope vaccine (MEV) candidates incorporating VirB10 requires a systematic reverse vaccinology approach. The following methodological framework is recommended based on recent research:

• Epitope Prediction and Selection:

  • Obtain the amino acid sequence of VirB10 from databases such as UniProt

  • Predict B-cell epitopes using tools like BepiPred, ABCpred, and DiscoTope

  • Identify both linear and conformational B-cell epitopes

  • Predict T-cell epitopes (both cytotoxic T lymphocyte and helper T lymphocyte epitopes) using tools like NetMHC, IEDB, and SYFPEITHI

  • Select epitopes based on antigenicity, immunogenicity, and population coverage

  Recent studies identified one cytotoxic T lymphocyte (CTL) epitope, four helper T lymphocyte (HTL) epitopes, four linear B cell epitopes, and three conformational B cell epitopes from VirB10 that show promise for vaccine development .

• Construct Engineering:

  • Incorporate selected epitopes with appropriate linkers

  • Add adjuvants such as TLR agonists to enhance immunogenicity

  • Include appropriate trafficking signals for optimal antigen presentation

  • Design the construct to ensure stability and proper folding

• Structural Analysis and Optimization:

  • Predict secondary structure using tools like SOPMA

  • Model tertiary structure using methods such as RoseTTAFold

  • Evaluate physicochemical properties including antigenicity, hydrophilicity, and stability

  • Perform in silico immune simulations to predict vaccine efficacy

• Combined Protein Approach:

  • Consider combining VirB10 epitopes with complementary proteins from the T4SS

  • VirB8 has shown good immunogenicity and is frequently paired with VirB10 in vaccine designs

  • The natural interactions between VirB9-1, VirB9-2, and VirB10 make them good candidates for a linked protein vaccine approach

• Molecular Dynamics Simulations:

  • Conduct molecular dynamics simulations of the vaccine construct with target receptors (e.g., TLR4)

  • Assess stability and binding interactions over simulation time

  • Optimize the construct based on simulation results

This comprehensive approach leverages VirB10's demonstrated immunogenicity in infection models and its conservation across Brucella species to develop potentially effective vaccine candidates against brucellosis.

What are the challenges in using VirB10 as a serological marker compared to traditional LPS-based diagnostics?

While VirB10 shows promise as a serological marker for Brucella infection, researchers face several challenges when comparing it to traditional LPS-based diagnostics:

• Sensitivity Considerations:

  • VirB10-based assays typically show good but slightly lower sensitivity compared to LPS-based tests

  • Recent studies report sensitivity values exceeding 0.91 for VirB10-based ELISAs, which is promising but not consistently superior to LPS tests

  • Researchers must optimize assay conditions (coating concentration, incubation times, detection systems) to maximize sensitivity

• Expression Level Variations:

  • VirB10 expression may vary during different stages of infection

  • This variation could affect the timing of antibody responses and subsequently impact diagnostic sensitivity

  • Development of assays may require temporal studies to determine optimal detection windows

• Protein Stability and Production Issues:

  • Maintaining the native conformation of recombinant VirB10 during production and purification can be challenging

  • Expression under denaturing conditions may be necessary, requiring careful refolding protocols

  • Long-term stability of purified protein for diagnostic kit development needs thorough evaluation

• Reference Standard Establishment:

  • Well-characterized serum panels for validation are essential but sometimes difficult to obtain

  • Establishing appropriate cutoff values requires balancing sensitivity and specificity

  • Correlation with disease progression and treatment response needs further investigation

• Differential Host Responses:

  • Individual variations in immune responses to VirB10 may affect diagnostic reliability

  • Studies indicate that some host MHC class II types may not respond strongly to VirB10

  • Research in cattle with different MHC class II DRB3 restriction fragment length polymorphism types showed variable T-cell responses to T4SS proteins

How do animal models contribute to understanding VirB10 immunogenicity and diagnostic potential?

Animal models play a crucial role in evaluating VirB10's immunogenicity and diagnostic potential through systematic experimental approaches. Researchers utilize these models to gather essential data on antibody responses, T-cell activation, and protection levels:

• Mouse Models for Initial Evaluations:

  • Balb/C mice are commonly used for experimental Brucella infections

  • Studies have demonstrated that VirB10 induces detectable antibody responses following experimental infection with B. melitensis 16M and clinical isolates

  • Mouse models allow for controlled infection and systematic sampling over time to track antibody development patterns

  • These models enable comparative analyses between responses to VirB10 and other antigens such as LPS

• Differential Immune Response Assessment:

  • Animal models permit evaluation of both humoral and cell-mediated immune responses

  • Researchers can assess T-cell proliferation in response to VirB10 stimulation

  • Cytokine profiles can be measured to determine Th1/Th2 balance in the immune response

  • The production of specific immunoglobulin isotypes (IgG1, IgG2a) provides insights into the type of immune response induced

• Cross-Reactivity Studies:

  • Animal models allow for controlled exposure to related pathogens

  • Studies involving Y. enterocolitica O:9 infection in mice help evaluate potential cross-reactivity issues

  • These experiments are crucial for determining the specificity of VirB10-based diagnostics

• Large Animal Models for Translational Research:

  • Cattle models provide insights into host-specific immune responses more relevant to the natural host

  • Studies in cattle have revealed MHC-restricted T-cell responses to VirB proteins

  • Variations in immune responses based on MHC class II DRB3 restriction fragment length polymorphism types have been observed

  • These findings help identify potential limitations in diagnostic application across genetically diverse populations

• Protein Interaction Validation:

  • Animal studies confirm that natural interactions between VirB proteins, as observed in vitro, are immunologically relevant in vivo

  • The linked recognition of VirB9-1, VirB9-2, and VirB10 in cattle validates their potential as combined diagnostic markers or vaccine components

By utilizing these animal models, researchers can systematically evaluate VirB10's potential as a diagnostic marker before advancing to clinical studies in humans, thereby optimizing assay parameters and identifying potential limitations in diverse populations.

What are the key methodological considerations when investigating VirB10's role in T4SS assembly dynamics?

Investigating VirB10's role in T4SS assembly dynamics requires sophisticated methodological approaches to capture the protein's complex structural transitions and interactions. Researchers should consider the following experimental strategies:

• Scanning Cysteine Accessibility Method (SCAM):

  • This technique can determine the membrane topology and structural changes of VirB10

  • By introducing cysteine residues at specific positions and testing their accessibility to membrane-impermeable sulfhydryl reagents, researchers can map which domains are exposed to different cellular compartments

  • This approach has been used to investigate whether VirB10 functions through a "shuttling" mechanism or remains anchored at the inner membrane

• Site-Directed Mutagenesis and Functional Assays:

  • Systematic mutations targeting specific domains (transmembrane domain, PRR, β-barrel) reveal functional contributions

  • "Uncoupling" mutations can be identified that selectively block T pilus biogenesis but not substrate transfer

  • Functional assays such as substrate transfer efficiency and T pilus production provide readouts for VirB10 activity

• Formaldehyde Cross-Linking and Co-Immunoprecipitation:

  • These techniques identify close contacts between VirB10 and other T4SS components or DNA substrates

  • Formaldehyde cross-linking can capture transient interactions during the dynamic assembly process

  • This approach has been used to monitor the passage of DNA substrates through the translocation channel and their interaction with channel subunits

• Energy Coupling Assays:

  • Since VirB10 functions dynamically by coupling inner membrane energy to secretion channel assembly, assays measuring ATP consumption can be correlated with structural changes

  • ATP-dependent conformational changes can be monitored using protease sensitivity assays, fluorescence resonance energy transfer (FRET), or structural probes

• Protein-Protein Interaction Verification:

  • Far-Western blotting and immunoprecipitation assays confirm specific protein interactions

  • These methods have revealed specific interactions of VirB10 with VirB9-1 and VirB9-2

  • Understanding these interactions is crucial for mapping the assembly pathway of the T4SS complex

By employing these methodological approaches, researchers can gain insights into VirB10's dynamic role in T4SS assembly and function, potentially identifying critical steps that could be targeted for intervention in Brucella infections.

How can researchers optimize combined VirB protein formulations for enhanced diagnostic or vaccine applications?

Optimizing combined VirB protein formulations requires a systematic approach to leverage the complementary properties and interactions of different T4SS components. Based on recent research, the following methodological framework is recommended:

• Protein Selection Based on Interaction Networks:

  • Select proteins with confirmed natural interactions, such as the VirB9-1, VirB9-2, and VirB10 complex

  • Natural protein associations can provide linked recognition for T-cell-B-cell interactions

  • Immunoprecipitation and far-Western blotting can identify specific protein-protein interactions that might be advantageous in combined formulations

• Complementary Epitope Analysis:

  • Conduct epitope mapping of multiple VirB proteins to identify complementary epitopes

  • Select combinations that provide broader coverage of both B-cell and T-cell epitopes

  • Address MHC restriction limitations by including proteins that can stimulate T-cell responses in individuals who might not respond to a single protein

  • For example, studies in cattle showed that some animals with specific MHC class II DRB3 types lacked T-cell responses to VirB9-1 or VirB10, suggesting that combinations might overcome individual variation in immune responses

• Optimization of Protein Ratios and Formulation:

  • Test different molar ratios of combined proteins to identify optimal formulations

  • Evaluate various adjuvants and delivery systems for enhanced immunogenicity

  • Consider structural compatibility and stability in combined preparations

  • Assess potential for co-purification of interacting proteins to maintain native complexes

• Diagnostic Performance Evaluation Matrix:

  • Create a systematic testing matrix comparing single proteins versus combinations

  • Evaluate key diagnostic parameters including sensitivity, specificity, and cross-reactivity

  • Recent studies suggest that different VirB proteins (VirB3, VirB4, VirB9, BMEII0036, VirB8, VirB11, VirB10) show varying performance with some exhibiting better specificity (VirB10) and others better sensitivity (VirB4, VirB9)

  Protein Combination	Sensitivity	Specificity	Cross-reactivity	MHC Restriction Coverage
  VirB10 alone	Good	Excellent	Minimal	Moderate
  VirB9+VirB10	Better	Good	Low	Improved
  VirB8+VirB9+VirB10	Excellent	Good	Low	Comprehensive

• Linked Recognition Validation:

  • Confirm that T-cell responses to one component can provide help for B-cell responses to interacting partners

  • Test whether antibody production against VirB10 occurs in individuals lacking direct T-cell responses to VirB10 but responding to partner proteins

  • The concept of linked recognition, demonstrated in studies with VirB9-1, VirB9-2, and VirB10, provides a strong rationale for combined protein approaches

By systematically applying these methodologies, researchers can develop optimized VirB protein combinations that overcome the limitations of single-protein approaches and provide more robust diagnostic tools or vaccine candidates for brucellosis.