The recombinant virB2 is expressed in E. coli and purified via affinity chromatography due to its His tag. Key production parameters include:
Parameter | Specification |
---|---|
Expression System | E. coli |
Tag | N-terminal His tag |
Molecular Weight | ~8.5 kDa (32–107aa) |
Purity | >90% (SDS-PAGE verified) |
Storage Buffer | Tris/PBS-based buffer with 6% trehalose, pH 8.0 |
Handling instructions emphasize avoiding repeated freeze-thaw cycles and storing aliquots at -20°C or -80°C .
virB2 participates in a network of interactions critical for T4SS functionality:
Homodimerization:
Heterodimerization with virB5:
Indirect Interactions via virB3:
Yeast Two-Hybrid (YTHS) Assays: Identified virB2-virB2 and virB2-virB5 interactions in B. henselae .
Binding Assays: Confirmed virB3-virB5 interactions, reinforcing the structural conduit between ATPases (virB4/virB11) and the pilus .
Recombinant virB2 is utilized in:
Pathogenesis Studies:
Diagnostic Development:
Structural Analysis:
A comparison of virB2 with other T4SS proteins highlights its unique role:
Protein | Function | Key Interactions | Localization |
---|---|---|---|
virB2 | Pilus subunit, substrate transfer | virB2 (homodimer), virB5 | Outer membrane/pilus |
virB5 | Outer membrane protein, pilus assembly | virB2, virB3, virB7 | Outer membrane |
virB3 | Outer membrane protein (novel role) | virB5 | Outer membrane |
virB11 | ATPase, energy source for T4SS | virB4, virB8, virB10, virB9 | Inner membrane |
Stability Issues:
Mechanistic Gaps:
Host-Specific Interactions:
KEGG: bhe:BH13260
STRING: 283166.BH13260
VirB2 serves as the major pilus subunit of the Type IV Secretion System (T4SS) in Bartonella henselae. The T4SS is a critical virulence factor that enables B. henselae to deliver effector proteins (Beps) into host cells, facilitating the manipulation of host cell functions. VirB2 forms the structural backbone of the T-pilus, which is essential for establishing contact between the bacterium and host cells during the infection process .
The VirB/VirD4 T4SS plays a crucial role in translocation of Bartonella effector proteins into host cells, where they mediate various functions including prevention of apoptosis and triggering angiogenic reprogramming of endothelial cells, which are key to the pathogenesis of B. henselae infections .
VirB2 forms the pilus structure of the T4SS with distinct structural properties:
The T-pilus assembled from VirB2 exhibits 5-fold rotational symmetry, similar to pED208 and F-pilus structures, but different from some other conjugative pili like pKpQIL .
Unlike previous mass spectrometry studies of purified T-pili that suggested cyclic VirB2 subunits, cryo-EM structural analysis reveals that VirB2 may not form cyclic subunits in the assembled pilus. The modeled termini of VirB2 were found to be approximately 30 Å apart, too distant for a cyclic linkage .
Cyclic VirB2 may represent either an intermediate form that is later linearized or a different functional form, as VirB2 appears to have multiple roles during mating pair formation .
Researchers employ several complementary approaches to identify and characterize the virB gene cluster:
PCR-Based Methods: Amplification of specific regions of the virB genes using polymerase chain reaction, followed by either sequencing or restriction fragment length polymorphism (RFLP) analysis .
Gene-Specific Targets: Several genes are commonly used as targets for PCR-based identification, including:
Enrichment Culture with Molecular Detection: Due to the fastidious nature of Bartonella, conventional culturing techniques are often supplemented with enrichment culture methods (like BAPGM - Bartonella Alpha Proteobacteria Growth Medium) followed by molecular detection targeting genes such as ITS, gltA, and ribC .
Culture Conditions: Bartonella species require specific growth conditions:
While the search results don't explicitly detail all VirB2 interactions, the T4SS in Bartonella henselae involves multiple protein-protein interactions that collectively form a functional secretion apparatus. The VirB proteins function together to create a pathway from the bacterial cytoplasm to the host cell.
Key interactions in the T4SS system include:
VirB9 occupies a central position, interacting with VirB7, VirB8, VirB10, and VirB11, in addition to forming homo-interactions .
VirB7 and VirB9 show bidirectional interaction, with the strongest interaction between VirB9 prey and VirB7 bait .
A novel interaction between VirB3 and VirB5 was identified using both yeast two-hybrid system and confirmed through binding assays with His-VirB3 and MBP-VirB5 .
These interactions collectively suggest a structural conduit between the cytoplasm-facing ATPases (VirB11/VirB4) and the pilus subunit, which includes VirB2 .
The virB gene cluster in B. henselae demonstrates fascinating evolutionary patterns characterized by enhanced recombination:
Based on the research approaches described in the search results, the following experimental methods have proven effective for studying protein-protein interactions in the T4SS, including those involving VirB2:
Yeast Two-Hybrid System (YTHS): This has been successfully employed to identify pair protein interactions between structural components of the B. henselae secretion apparatus. The advantage of this approach is that it allows the use of full-length protein subunits to determine interactions between T4SS proteins .
In vitro Binding Assays: Specific protein interactions can be confirmed through binding assays using purified recombinant proteins. For example, the interaction between VirB3 and VirB5 was confirmed through coelution experiments with His-tagged VirB3 and MBP-tagged VirB5 .
Cryo-Electron Microscopy (Cryo-EM): This technique has been valuable for determining the structural arrangement of T4SS components, including how VirB2 assembles to form the T-pilus and interacts with other system components. Cryo-EM has revealed important structural insights such as the 5-fold rotational symmetry of the T-pilus and the spatial arrangement of VirB2 subunits .
Genetic Approaches: Analysis of mutant alleles in T4SS components has identified uncoupling phenotypes that maintain virulence but lack detectable pili (Vir+, Pil- phenotype). These studies help elucidate the functional relationships between different VirB proteins .
While the search results don't directly address purification challenges for recombinant VirB2, several methodological considerations can be inferred based on the nature of this protein and general challenges in T4SS research:
Protein Structure Complexity: VirB2 undergoes processing and potentially forms cyclic structures in certain contexts. This structural complexity presents challenges for expression and purification of functional protein .
Native Conformation Maintenance: Ensuring that recombinant VirB2 maintains its native conformation during purification is crucial for structural studies, especially given the evidence that VirB2 may exist in different forms (cyclic versus linear) depending on its functional state .
Cultivation Challenges: B. henselae is fastidious, requiring specific growth conditions including hemin-dependent media, blood-enriched agar, 35-37°C temperature, and 5% CO₂ atmosphere with high humidity. These requirements complicate the cultivation of the organism for native protein extraction .
Identification Methods: Researchers have employed various techniques for identification and characterization of B. henselae, including:
The high recombination frequency observed in the virB gene cluster has significant implications for VirB2 function and T4SS assembly:
The relationship between VirB2 and the translocation of Bartonella effector proteins (Beps) involves several key aspects:
Structural Foundation: VirB2 forms the major component of the T-pilus, which is essential for establishing contact with host cells. This physical connection is a prerequisite for the translocation of effector proteins .
Translocation Signal Recognition: Bartonella effector proteins are recognized by the VirB/VirD4 T4SS via a bipartite translocation signal composed of:
BID Domain Structure: BID domains adopt a conserved structural fold consisting of an anti-parallel four-helix bundle topped with a hook, despite having variable sequences. This conserved fold and elongated shape appear crucial for recognition by the secretion system .
Surface Charge Distribution: BID domains display a conserved pattern of surface charge distribution with two positively charged areas separated by one negatively charged patch. This charge distribution may play a role in the interaction with the T4SS machinery .
Functional Duality: While the conserved fold of BID domains is essential for secretion via the VirB/VirD4 T4SS, the highly variable sequences enable secondary functions that are important for different stages of the infection cycle, including bacterial uptake through induction of stress fiber formation and inhibition of apoptosis .
Based on the information provided in the search results, the following culture conditions would be suitable for working with Bartonella henselae and potentially for expressing recombinant VirB2:
Growth Medium: Tryptic soy agar supplemented with 5% sheep blood is recommended by ATCC for Bartonella cultivation. This medium supports the growth of colonies that are shiny, smooth, circular with entire edges, low convex, non-hemolytic, auto-adherent, and not embedded in the agar .
Environmental Conditions:
Hemin Requirement: Growth in axenic medium is hemin-dependent, which is a critical factor to consider when designing expression systems .
Enrichment Techniques: For enhanced growth and detection, specialized enrichment culture techniques like BAPGM (Bartonella Alpha Proteobacteria Growth Medium) can be employed .
For recombinant expression specifically, heterologous systems in E. coli would likely require optimization of codon usage and expression conditions to account for the unique properties of VirB2, particularly if it undergoes post-translational modifications like those suggested by the cyclic versus linear forms observed in different contexts .
To effectively analyze the structure-function relationship of VirB2, researchers should employ a multi-faceted approach:
Structural Analysis Techniques:
Cryo-EM: This has proven valuable for elucidating the structural arrangement of VirB2 in the assembled T-pilus, revealing features such as 5-fold rotational symmetry and the spatial arrangement of subunits .
Mass Spectrometry: This can be used to determine post-translational modifications and confirm structural features such as the cyclic versus linear nature of VirB2 in different contexts .
Functional Analysis Approaches:
Mutational Studies: Analysis of mutant alleles can identify uncoupling phenotypes that maintain virulence but affect pilus formation (Vir+, Pil- phenotype), helping to separate different functional aspects of VirB2 .
Protein Interaction Studies: The yeast two-hybrid system has been successfully used to identify protein-protein interactions in the T4SS and could be applied to study VirB2 interactions specifically .
In vitro Binding Assays: These can confirm specific protein interactions, as demonstrated for other VirB components .
Evolutionary Analysis:
Comparative Genomics: Analysis of recombination patterns in the virB gene cluster across different Bartonella strains and species can provide insights into evolutionary pressures on VirB2 and functional constraints .
Population Structure Studies: Examining the population structure of different Bartonella species can reveal horizontal gene transfers that might affect VirB2 function .
Integration of Data:
Correlating structural features with functional outcomes
Mapping conservation patterns onto structural models to identify functionally important regions
Combining in silico predictions with experimental validation
This integrated approach would provide comprehensive insights into how the structure of VirB2 relates to its functions in T-pilus formation and effector protein translocation.
The interpretation of conflicting data regarding VirB2 structure requires careful consideration of methodological differences and biological context:
Cyclic versus Linear Structure Discrepancy: Previous mass spectrometry studies of purified T-pili suggested that VirB2 forms cyclic subunits, whereas cryo-EM structural analysis indicates that VirB2 may not form cyclic subunits in the assembled pilus (with modeled termini approximately 30 Å apart) . Researchers should consider:
Methodological differences: Different sample preparation methods for mass spectrometry versus cryo-EM might influence the observed structure.
Biological states: Cyclic VirB2 might represent an intermediate form that is later linearized during pilus assembly.
Functional forms: VirB2 may exist in different structural forms depending on its functional state, as it appears to have multiple roles during mating pair formation .
Resolution of Conflicting Data:
Employ complementary structural techniques to validate findings
Consider the biological context of each preparation (purified protein versus assembled complex)
Design experiments that can directly test competing models
Examine VirB2 at different stages of T4SS assembly
Structural Variation Across Species:
Compare VirB2 structure across different bacterial species to identify conserved versus variable features
Correlate structural variations with functional differences
Based on the methodologies described in the search results, the following statistical approaches are appropriate for analyzing recombination patterns in the virB gene cluster:
Recombination to Mutation Ratio (r/m): This metric provides a quantitative measure of the relative frequency of recombination compared to mutation. For B. henselae, this was estimated using ClonalFrame with multiple independent runs, yielding consistent r/m values of approximately 1.13-1.14 .
GENECONV Analysis: This tool has been successfully used to identify genes most strongly affected by recombination events. When applied to orthologous gene sets in B. henselae, it highlighted the virB and trw gene clusters as the most recombination-prone sequences in the genome .
Nucleotide Sequence Divergence Analysis: Measuring divergence levels between different strains (e.g., less than 1% divergence observed among B. henselae strains IC11, UGA10, and Houston-1) provides a baseline against which to identify regions with enhanced divergence due to recombination .
Segment Identification: Statistical methods can be used to identify genomic segments with significantly enhanced divergence levels. Four to eight such segments were identified per genome in B. henselae, with the virB and trw gene clusters showing the most pronounced divergence .
Phylogenetic Analysis: Constructing phylogenetic trees for specific genes across multiple strains and species can reveal different population structures and identify horizontal gene transfer events. This approach was successfully applied to study a gene putatively involved in iron metabolism across 80 strains of Bartonella quintana, B. henselae, and B. grahamii .
These statistical approaches, when combined, provide a comprehensive framework for analyzing recombination patterns in the virB gene cluster and understanding their evolutionary significance.
Several emerging techniques and approaches hold promise for advancing our understanding of VirB2 function:
Advanced Structural Biology Methods:
Higher resolution cryo-EM techniques to better resolve fine structural details of the T-pilus
Integrative structural biology approaches combining multiple techniques (X-ray crystallography, NMR, cryo-EM, mass spectrometry)
Time-resolved structural studies to capture different states of VirB2 during T4SS assembly and function
Advanced Genetic Approaches:
CRISPR-Cas9 based genome editing to create precise mutations in virB2
Site-specific incorporation of non-canonical amino acids for studying protein dynamics and interactions
Conditional expression systems to control VirB2 expression with temporal precision
Single-Molecule Techniques:
Single-molecule FRET to study conformational changes in VirB2
Super-resolution microscopy to visualize T4SS assembly and dynamics in living cells
Optical tweezers or atomic force microscopy to study mechanical properties of the T-pilus
Systems Biology Approaches:
Multi-omics integration (genomics, transcriptomics, proteomics) to understand VirB2 in the context of the entire T4SS
Network analysis of protein-protein interactions to identify novel functional relationships
Machine learning approaches to predict functional consequences of sequence variations
Host-Pathogen Interface Studies:
Advanced in vitro models of host-pathogen interactions
Real-time imaging of T4SS-mediated effector translocation
Host cell proteomics to identify novel targets of translocated effectors
These emerging approaches, particularly when used in combination, have the potential to significantly advance our understanding of VirB2 structure, function, and role in Bartonella pathogenesis.
Understanding VirB2 in Bartonella henselae has significant implications for broader research on type IV secretion systems:
Evolutionary Insights:
The high recombination frequency observed in the virB gene cluster of B. henselae suggests that similar evolutionary mechanisms might operate in other bacterial species with T4SSs .
Comparative studies of VirB2 across different species could reveal conserved functional constraints versus adaptable regions that respond to host-specific pressures.
Structural Conservation and Variation:
The observation that the T-pilus in Agrobacterium tumefaciens has 5-fold rotational symmetry, similar to pED208 and F-pilus but different from pKpQIL, indicates both conservation and divergence in T4SS architecture across species .
Understanding the structural basis for these similarities and differences could inform broader principles of T4SS assembly.
Mechanism of Substrate Recognition:
Insights into how the VirB/VirD4 T4SS recognizes effector proteins via the BID domain and C-terminal tail could inform our understanding of substrate recognition in other T4SSs .
The conserved structural fold of BID domains despite sequence variability suggests functional constraints that might apply to other T4SS substrates.
Therapeutic Implications:
Understanding the structure and function of VirB2 could inform the development of inhibitors targeting T4SS in various pathogens.
The essential role of T4SS in the virulence of multiple human pathogens (Brucella spp., Campylobacter jejuni, Coxiella burnetii, Legionella pneumophila, Rickettsia prowazekii, Helicobacter pylori, and Bordetella pertussis) makes it an attractive target for broad-spectrum therapeutic approaches .
Methodological Advances:
Techniques developed for studying VirB2 in B. henselae, such as the successful use of the yeast two-hybrid system with full-length protein subunits, could be applied to study T4SS components in other bacterial species .
Integrated approaches combining structural, functional, and evolutionary analyses could serve as a template for comprehensive studies of other T4SS components.