Recombinant Brucella abortus Lectin-like protein BA14k (BAB2_0505)

What is the BA14K protein in Brucella abortus?

BA14K is a 14-kilodalton immunoreactive protein identified in Brucella abortus, a facultative intracellular zoonotic pathogen that causes undulant fever in humans and abortions in cattle. This protein was first characterized as being immunogenic in animals infected with Brucella species. Subsequent research has revealed that BA14K possesses lectin-like properties, with a particular affinity toward mannose, as well as immunoglobulin binding and hemagglutination capabilities . The gene encoding BA14K has been cloned and characterized, with sequence analysis revealing no significant homology to previously described proteins, indicating its uniqueness to Brucella .

How does BA14K contribute to Brucella virulence?

BA14K plays an essential role in the virulence of B. abortus, primarily through its direct or indirect involvement in smooth lipopolysaccharide (LPS) synthesis. Experimental studies involving gene disruption have demonstrated that when the BA14K gene is disrupted in virulent B. abortus strain 2308, the resulting mutant exhibits:

• A rough-like phenotype

• An altered smooth LPS immunoblot profile

• Significantly reduced ability to replicate in mouse spleens

What immunogenic properties does BA14K exhibit?

BA14K has been identified as an immunoreactive protein capable of eliciting responses in both humoral and cell-mediated immunity. Specifically:

• It is reactive with antibodies from both naturally and experimentally infected hosts

• It stimulates T lymphocytes from experimentally infected BALB/c mice

• It demonstrates cross-reactivity across all currently recognized Brucella species, as confirmed by Southern blot analysis

These properties make BA14K a protein of significant interest for understanding the antigenic specificity of protective immunity to brucellosis and for potential vaccine development.

What experimental designs are most effective for studying BA14K function?

Rigorous experimental design is critical for investigating BA14K function. Based on published research methodologies, the following approaches have proven effective:

Gene Disruption Studies:

• Construction of gene disruption vectors containing a selectable marker (e.g., chloramphenicol resistance)

• Electroporation of the vector into target Brucella strains

• Selection of recombinant colonies resistant to the marker but sensitive to the vector-specific antibiotic

• Confirmation of gene disruption via Southern blotting

• Complementation studies to verify phenotype causation

Comparative Strain Analysis:
Testing BA14K function across different strains (virulent vs. attenuated) allows researchers to correlate protein function with specific bacterial phenotypes. The experimental design should include:

This design allows for isolation of BA14K-specific effects from strain-specific characteristics .

How can researchers effectively disrupt the BA14K gene for functional studies?

Based on published methodologies, the following protocol has been successfully employed for BA14K gene disruption:

• Vector Construction:

  • Amplify the BA14K-encoding gene with its upstream region using PCR

  • Clone the amplified fragment into a suitable vector (e.g., pCR2.1)

  • Insert an antibiotic resistance cassette (e.g., chloramphenicol) into the coding sequence

  • Confirm construct integrity via nucleotide sequence analysis

• Transformation:

  • Electroporate the disruption construct into the target Brucella strain

  • Select transformants on media containing appropriate antibiotics

  • Screen for double crossover events (resistant to the cassette marker but sensitive to vector marker)

• Confirmation:

  • Prepare genomic DNA from potential mutants

  • Digest with appropriate restriction enzymes (e.g., HindIII)

  • Perform Southern blotting using digoxigenin-labeled DNA probes specific for both the BA14K gene and the disruption cassette

  • Verify disruption by Western blot analysis to confirm absence of BA14K protein

• Complementation:

  • Clone the intact BA14K gene with its native promoter into a broad-host-range plasmid (e.g., pBBR4MCS)

  • Transform the mutant strain with this construct

  • Select transformants containing the complementation plasmid

  • Verify protein expression and phenotype restoration

What methodologies can assess the lectin-like properties of BA14K?

The lectin-like properties of BA14K can be assessed through several complementary approaches:

• Hemagglutination Assays:

  • Prepare serial dilutions of purified recombinant BA14K

  • Add appropriate erythrocyte suspensions

  • Incubate and observe for hemagglutination

  • Include positive and negative controls

• Hemagglutination Inhibition:

  • Pre-incubate BA14K with various carbohydrates (mannose, glucose, galactose, etc.)

  • Add erythrocyte suspension

  • Assess inhibition of hemagglutination

  • Calculate inhibition concentrations

  Research has shown that mannose specifically inhibits BA14K-mediated hemagglutination, indicating its affinity for this sugar .

• Immunoglobulin Binding Assays:

  • Immobilize various immunoglobulins on solid support

  • Incubate with labeled recombinant BA14K

  • Wash and detect bound protein

  • Assess binding in the presence of potential inhibitors

• Glycan Array Analysis:
  For comprehensive characterization of carbohydrate specificity:

  • Test purified BA14K against arrays containing various glycan structures

  • Detect binding using fluorescently labeled antibodies against BA14K

  • Analyze binding patterns to determine glycan preference profiles

How does BA14K gene disruption affect virulence in different experimental models?

The effects of BA14K gene disruption on Brucella virulence have been systematically studied in mouse models, with the following results:

Mouse Spleen Colonization Data:

These data demonstrate that:

• The wild-type strain (2308) establishes progressive infection with high bacterial loads

• The BA14K mutant (2308Δ14) shows significantly reduced replication but maintains persistence

• Complementation restores the virulent phenotype, confirming BA14K's role in virulence

In contrast, disruption of BA14K in the already attenuated rough strain RB51 did not further alter its clearance pattern in mice, suggesting that BA14K's virulence contribution is linked to smooth LPS synthesis, which is already compromised in RB51 .

How can recombinant BA14K be effectively expressed and purified for research applications?

Based on published methodologies, the following protocol can be used for recombinant BA14K expression and purification:

• Cloning Strategy:

  • Amplify the BA14K gene using PCR with primers containing appropriate restriction sites

  • Clone into an expression vector (e.g., pET system) with a suitable tag (His-tag recommended)

  • Transform into an expression host (E. coli BL21 or similar)

• Expression Optimization:

  • Test multiple induction conditions (temperature, IPTG concentration, induction time)

  • Optimal conditions: 0.5 mM IPTG, 25°C, 4-6 hours

  • Monitor expression by SDS-PAGE

• Purification Protocol:

  • Harvest cells and lyse by sonication in appropriate buffer (e.g., phosphate buffer with 8M urea for inclusion bodies)

  • Clarify lysate by centrifugation

  • Purify using nickel affinity chromatography for His-tagged protein

  • Perform dialysis to remove denaturants if needed

  • Verify purity by SDS-PAGE and Western blotting

• Activity Verification:

  • Assess hemagglutination activity of purified protein

  • Confirm mannose-binding properties

  • Test immunoreactivity with sera from infected animals

Alternative expression systems such as Brucella RB51 can also be used, with the gene cloned under control of the groE promoter in a broad-host-range plasmid (e.g., pBBGroERBS14) .

What considerations are important when incorporating BA14K into experimental vaccine designs?

When designing experimental vaccines incorporating BA14K, researchers should consider:

How can researchers distinguish between natural infection and vaccination when using BA14K-based diagnostics?

This is a critical consideration for brucellosis control programs. Researchers can implement the following strategies:

• Epitope Modification:

  • Identify immunodominant B-cell epitopes in BA14K

  • Introduce specific mutations that preserve T-cell epitopes but alter B-cell recognition

  • This allows differentiation between vaccine-induced and infection-induced antibodies

• Companion Diagnostic Tests:

  • Develop ELISA tests using both wild-type BA14K and modified BA14K

  • Compare antibody reactivity patterns

  • Establish algorithms to differentiate infection from vaccination

• Multi-Antigen Panels:

  • Combine BA14K with other Brucella antigens in diagnostic panels

  • Natural infection typically elicits responses to multiple antigens

  • Vaccines can be designed to exclude certain immunogenic components

• Recombinant DNA Vaccine Approach:

  • DNA vaccines encoding selected epitopes from BA14K (25-70 amino acids in length)

  • These epitopes are chosen to be present across multiple Brucella species (99% identical at DNA level)

  • Such vaccines eliminate many safety concerns of live attenuated vaccines and can be designed for DIVA (Differentiating Infected from Vaccinated Animals) capability

What are the optimal conditions for overexpressing BA14K in bacterial systems?

Based on published methodologies, researchers have successfully overexpressed BA14K using the following approaches:

• Expression in E. coli:

  • Vector: pET expression system with T7 promoter

  • Host strain: BL21(DE3) or similar

  • Induction: 0.5 mM IPTG

  • Temperature: 25-30°C (lower temperatures reduce inclusion body formation)

  • Duration: 4-6 hours post-induction

  • Growth media: LB with appropriate antibiotics

• Expression in Brucella RB51:

  • Vector: pBBGroERBS14 or pBB14up (broad-host-range plasmids)

  • Transformation: Electroporation (2.5 kV, 25 μF, 400 Ω)

  • Selection: TSA plates with 30 μg/ml chloramphenicol

  • Confirmation: Western blot analysis of heat-killed bacteria

Key considerations for optimizing expression include:

• Codon optimization for the host organism

• Inclusion of appropriate tags for purification

• Management of protein toxicity if encountered

• Optimization of culture conditions to maximize yield

What experimental approaches can be used to study BA14K's role in LPS synthesis?

The connection between BA14K and LPS synthesis can be investigated through:

• LPS Profile Analysis:

  • Extract LPS from wild-type, mutant, and complemented strains

  • Perform SDS-PAGE and silver staining

  • Conduct immunoblot analysis using anti-smooth LPS antibodies

  • Compare banding patterns to identify specific alterations

• Glycosyltransferase Activity Assays:

  • Prepare membrane fractions from various strains

  • Measure incorporation of radiolabeled sugar precursors into LPS

  • Compare enzymatic activities between strains with and without BA14K

• Protein-Protein Interaction Studies:

  • Perform pull-down assays with tagged BA14K

  • Identify binding partners involved in LPS biosynthesis

  • Confirm interactions using techniques such as bacterial two-hybrid systems

• Complementation Analysis:

  • Construct a series of BA14K variants with specific mutations

  • Test their ability to restore smooth LPS in the BA14K mutant

  • Map functional domains important for LPS synthesis

• Subcellular Localization:

  • Determine where BA14K localizes within the bacterial cell

  • Assess co-localization with known LPS biosynthesis machinery

  • This can provide insights into its mechanism of action

How can researchers effectively analyze immune responses to BA14K in different host species?

To comprehensively analyze immune responses to BA14K across host species:

• Antibody Response Analysis:

  • Develop ELISA systems using purified recombinant BA14K

  • Test sera from various naturally and experimentally infected hosts

  • Characterize antibody isotypes and subclasses

  • Perform Western blot analysis to confirm specificity

• T-Cell Response Assessment:

  • Isolate peripheral blood mononuclear cells (PBMCs) from infected hosts

  • Stimulate with purified BA14K

  • Measure proliferation responses using techniques such as 3H-thymidine incorporation

  • Analyze cytokine production profiles by ELISA or intracellular cytokine staining

• Cross-Species Comparison Protocol:

  Host Species	Sample Collection	Antibody Detection	T-Cell Assays	Analysis Methods
  Mice	Serum, splenocytes	ELISA, Western blot	Proliferation, cytokine ELISA	Flow cytometry, IFN-γ ELISPOT
  Cattle	Serum, PBMCs	ELISA, Western blot	Proliferation, cytokine ELISA	Flow cytometry, IFN-γ ELISPOT
  Humans	Serum, PBMCs	ELISA, Western blot	Proliferation, cytokine ELISA	Flow cytometry, IFN-γ ELISPOT
  Other ruminants	Serum, PBMCs	ELISA, Western blot	Proliferation	Comparative analysis

• Epitope Mapping:

  • Generate overlapping peptides spanning the BA14K sequence

  • Test reactivity with T cells and antibodies from different host species

  • Identify conserved and species-specific epitopes

This comprehensive approach allows researchers to characterize the immunological significance of BA14K across different host species relevant to brucellosis .

What are promising areas for future BA14K research that could advance brucellosis control?

Several promising research directions could significantly advance our understanding and utilization of BA14K:

• Structure-Function Analysis:

  • Determine the crystal structure of BA14K

  • Map the carbohydrate binding domain and immunodominant epitopes

  • Design structure-based modifications to enhance vaccine potential

• Host-Pathogen Interaction Mechanisms:

  • Investigate how BA14K's lectin properties influence host cell recognition

  • Determine whether BA14K plays a role in bacterial attachment or invasion

  • Explore potential interactions with host mannose-containing receptors

• Improved Vaccine Designs:

  • Development of BA14K-based subunit vaccines with appropriate adjuvants

  • Design of recombinant attenuated strains expressing modified BA14K

  • Creation of vectored vaccines expressing BA14K epitopes

• Diagnostic Applications:

  • Development of BA14K-based serological tests

  • Use of BA14K in multiplex diagnostic platforms

  • Creation of point-of-care tests for field diagnosis in endemic regions

• Comparative Analysis Across Brucella Species:

  • Characterize BA14K homologs in other Brucella species

  • Identify species-specific variations that correlate with host preference

  • Determine whether BA14K contributes to host specificity differences

These research directions would contribute significantly to our fundamental understanding of Brucella pathogenesis and provide practical applications for disease control and prevention .

How might the lectin-like properties of BA14K be exploited for novel therapeutic approaches?

The unique lectin-like properties of BA14K offer several potential therapeutic applications:

• Targeted Drug Delivery:

  • Develop BA14K-conjugated nanoparticles for delivering antimicrobials

  • Target these to mannose-rich environments where Brucella may reside

  • Enhance intracellular delivery of antibiotics to infected cells

• Anti-Adhesion Therapy:

  • If BA14K mediates bacterial attachment, soluble mannose analogs could block this interaction

  • Design high-affinity mannose derivatives that competitively inhibit BA14K

  • This could prevent initial colonization or spread within the host

• Immunomodulatory Applications:

  • BA14K could be modified to target specific immune cell populations

  • This might enhance protective responses against Brucella

  • Could potentially be used as an adjuvant for other vaccines

• Diagnostic Imaging:

  • Label BA14K for use in imaging Brucella infections

  • Target mannose-rich environments in infected tissues

  • Develop novel approaches for tracking infection progression

Each of these approaches would require detailed understanding of BA14K's structure, binding specificity, and role in pathogenesis to be effectively implemented.