Recombinant Bartonella henselae Type IV secretion system protein virB2 (virB2)

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Description

Production and Purification

The recombinant virB2 is expressed in E. coli and purified via affinity chromatography due to its His tag. Key production parameters include:

ParameterSpecification
Expression SystemE. coli
TagN-terminal His tag
Molecular Weight~8.5 kDa (32–107aa)
Purity>90% (SDS-PAGE verified)
Storage BufferTris/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 .

Research Findings and Protein Interactions

virB2 participates in a network of interactions critical for T4SS functionality:

Key Interactions

  1. Homodimerization:

    • virB2 forms homodimers, a conserved feature across T4SS systems, stabilizing the pilus structure .

  2. Heterodimerization with virB5:

    • virB2 binds virB5 (outer membrane protein), a key interaction for pilus assembly .

  3. Indirect Interactions via virB3:

    • virB3 (outer membrane protein) interacts with virB5, indirectly linking virB2 to the T4SS core complex .

Experimental Validation

  • 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 .

Applications in Research

Recombinant virB2 is utilized in:

  1. Pathogenesis Studies:

    • Investigating T4SS-mediated translocation of effector proteins (e.g., Beps A–G) into host endothelial cells .

  2. Diagnostic Development:

    • Detecting anti-B. henselae antibodies using virB2 fragments (e.g., 17-kDa virB5) .

  3. Structural Analysis:

    • Mapping pilus assembly and substrate transfer mechanisms via mutagenesis or cryo-EM .

Comparative Analysis with T4SS Components

A comparison of virB2 with other T4SS proteins highlights its unique role:

ProteinFunctionKey InteractionsLocalization
virB2Pilus subunit, substrate transfervirB2 (homodimer), virB5Outer membrane/pilus
virB5Outer membrane protein, pilus assemblyvirB2, virB3, virB7Outer membrane
virB3Outer membrane protein (novel role)virB5Outer membrane
virB11ATPase, energy source for T4SSvirB4, virB8, virB10, virB9Inner membrane

Challenges and Future Directions

  1. Stability Issues:

    • Recombinant virB2’s stability in solution requires optimization (e.g., glycerol addition) .

  2. Mechanistic Gaps:

    • The precise mechanism of virB2-mediated substrate transfer remains unclear, necessitating further structural studies .

  3. Host-Specific Interactions:

    • Investigating virB2’s role in endothelial cell invasion (e.g., invasome formation) requires in vivo models .

Product Specs

Form
Lyophilized powder
Note: While we prioritize shipping the format currently in stock, we can accommodate specific format requests. Please indicate your preference in order notes, and we will fulfill your requirements.
Lead Time
Delivery time may vary depending on the purchase method and location. Please contact your local distributor for specific delivery estimates.
Note: All proteins are shipped with standard blue ice packs. If dry ice shipping is required, please notify us in advance as additional fees will apply.
Notes
Repeated freezing and thawing is not recommended. For optimal results, store working aliquots at 4°C for up to one week.
Reconstitution
We recommend briefly centrifuging the vial prior to opening to ensure the contents are collected at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard final glycerol concentration is 50%. Customers may use this as a reference.
Shelf Life
Shelf life is influenced by various factors including storage conditions, buffer composition, temperature, and the protein's inherent stability.
Generally, liquid forms have a 6-month shelf life at -20°C/-80°C. Lyophilized forms have a 12-month shelf life at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is necessary for multiple use. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type will be determined during the manufacturing process.
Tag type is determined during production. If you have a specific tag type requirement, please inform us, and we will prioritize its development.
Synonyms
virB2; BH13260; Type IV secretion system protein virB2
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
32-107
Protein Length
Full Length of Mature Protein
Species
Bartonella henselae (strain ATCC 49882 / DSM 28221 / Houston 1) (Rochalimaea henselae)
Target Names
virB2
Target Protein Sequence
ATGSASGLGNVDNVLQSIVTMMTGTTAKLIATICVAAVGIGWMYGFIDLRKAAYCLIGIG IVFGASALVSKLTSAS
Uniprot No.

Target Background

Function
The type IV secretion system VirB/VirD4 is a major virulence factor in the subversion of human endothelial cell (HEC) function. VirB-mediated changes in HEC include significant cytoskeletal rearrangements, proinflammatory activation via nuclear factor NF-kappa-B, inhibition of early and late apoptotic events leading to increased cell survival. At high infection doses, a cytostatic or cytotoxic effect is observed, interfering with a potent VirB-independent mitogenic activity. These HEC alterations require the T4S coupling protein VirD4 and at least one of the effector proteins BepA-G. VirB2 appears to be the primary pilus component.
Database Links

KEGG: bhe:BH13260

STRING: 283166.BH13260

Protein Families
VirB2 family
Subcellular Location
Cell outer membrane; Multi-pass membrane protein.

Q&A

What is the role of VirB2 in the Type IV secretion system of Bartonella henselae?

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 .

What are the structural characteristics of VirB2 in T4SS assembly?

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 .

How do researchers identify and characterize the virB gene cluster in B. henselae?

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:

    • Citrate synthase (gltA) gene

    • Riboflavin synthase (ribC) gene

    • groEL gene

    • RNA polymerase beta subunit gene

    • Cell division protein (ftsZ) gene

  • 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:

    • Temperature: 35-37°C

    • Atmosphere: 5% CO₂ with high humidity

    • Medium: Hemin-dependent, blood-enriched agar such as tryptic soy agar with 5% sheep blood

What protein interactions does VirB2 participate in within the T4SS complex?

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:

  • VirB4 forms homodimers and heterodimers with VirB10 .

  • 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 .

What are the evolutionary patterns observed in the virB gene cluster of B. henselae?

The virB gene cluster in B. henselae demonstrates fascinating evolutionary patterns characterized by enhanced recombination:

What experimental approaches are most effective for studying VirB2 protein-protein interactions?

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 .

What methodological challenges exist in purifying recombinant VirB2 for structural studies?

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:

    • Culture on specialized media

    • Various staining methods including electron microscopy

    • Biochemical analysis by conventional methods and API

    • PCR amplification of marker genes with subsequent RFLP analysis

How does recombination in the virB gene cluster affect VirB2 function and T4SS assembly?

The high recombination frequency observed in the virB gene cluster has significant implications for VirB2 function and T4SS assembly:

What is the relationship between VirB2 and the translocation of Bartonella effector proteins?

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:

    • A ~100-amino-acid BID (Bartonella Intracellular Delivery) domain

    • A short positively charged C-terminal tail

  • 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 .

What culture conditions are optimal for expressing recombinant VirB2 protein?

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:

    • Temperature: 35-37°C

    • Atmosphere: 5% CO₂

    • Humidity: High humidity levels are required

    • Incubation time: Extended due to the slow-growing nature of Bartonella

  • 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 .

How can researchers effectively analyze the structure-function relationship of VirB2?

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.

How do researchers interpret conflicting data about VirB2 structure?

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

What statistical approaches are appropriate for analyzing recombination patterns in the virB gene cluster?

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.

What are the emerging techniques that could advance our understanding of VirB2 function?

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.

How might understanding VirB2 inform broader research on type IV secretion systems?

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.

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