Recombinant Bordetella avium Protein CrcB homolog (crcB)

What is the function of CrcB homolog protein in Bordetella avium?

The CrcB homolog in Bordetella avium functions primarily as a fluoride ion channel protein that provides resistance to environmental fluoride. This membrane protein helps maintain cellular homeostasis by preventing fluoride ion accumulation, which can inhibit essential enzymes involved in glycolysis and translation. In B. avium, the protein plays a critical role in survival when the bacterium is exposed to environments containing fluoride, which may occur during colonization of the avian respiratory tract. The protein's structure likely consists of transmembrane domains that form a selective channel for fluoride export .

How is the crcB gene organized in the Bordetella avium genome?

The crcB gene in B. avium is part of the genome that shows no significant orthologs in other Bordetella species, making it a relatively unique genomic element. Unlike hagA and hagB genes that are adjacent and divergently oriented in the B. avium genome, crcB appears in a different genomic context. The gene is approximately 300-400 base pairs in length, encoding a protein of approximately 100-130 amino acids. Genomic analysis indicates that the crcB gene is regulated independently of virulence factors associated with respiratory pathogenesis .

What methods are recommended for PCR amplification of the crcB gene from Bordetella avium?

For PCR amplification of the B. avium crcB gene, the following optimized protocol is recommended:

• Template preparation: Use purified genomic DNA (15-20 pg minimum) or boiled cell lysates less than 3 days old for best results.

• Primer design: Design primers based on the published B. avium genome sequence with the following specifications:

  • Forward primer: 20-25 nucleotides with 50-55% GC content

  • Reverse primer: 20-25 nucleotides with 50-55% GC content

  • Melting temperature (Tm): 58-62°C

• Reaction conditions:

  • 5% DMSO

  • 1.5 mM MgCl₂

  • 0.5 μM primers

  • Standard PCR buffer

  • 1.25 U Taq DNA polymerase

• Cycling conditions:

  • Initial denaturation: 95°C for 2 min

  • 30 cycles of:

    • Denaturation: 95°C for 30 s

    • Annealing: 52°C for 30 s

    • Extension: 72°C for 30 s

  • Final extension: 72°C for 7 min

This protocol is adapted from optimized PCR conditions for B. avium detection with a sensitivity to detect approximately 3,750-5,000 bacterial genomes .

What expression systems are most effective for producing recombinant Bordetella avium CrcB protein?

For recombinant expression of B. avium CrcB protein, E. coli-based expression systems are most commonly used, with BL21(DE3) or Rosetta(DE3) strains being particularly effective. The following system has demonstrated optimal results:

The MBP-fusion expression system generally yields the highest amount of soluble protein, which is advantageous for downstream applications requiring native protein conformation. For structural studies, the 6×His-tagged constructs provide easier purification and compatibility with crystal screening protocols .

How can protein-protein interactions between CrcB homolog and other Bordetella avium virulence factors be identified and characterized?

To identify and characterize protein-protein interactions between CrcB homolog and other B. avium virulence factors, a multi-method approach is recommended:

• Pull-down assays: Use purified His-tagged CrcB protein as bait to capture interacting partners from B. avium cell lysates. The protein complexes can be analyzed using mass spectrometry to identify potential binding partners.

• Bacterial two-hybrid system: Construct fusion proteins between CrcB and the T18 or T25 fragments of adenylate cyclase, and potential interaction partners with the complementary fragment. Co-expression in an E. coli reporter strain will result in β-galactosidase activity if interaction occurs.

• Co-immunoprecipitation: Develop antibodies against the CrcB protein, which can be used to immunoprecipitate protein complexes from B. avium lysates. Western blotting can then identify specific interacting partners.

• Surface plasmon resonance (SPR): Purify recombinant CrcB and potential interaction partners separately. Immobilize CrcB on a sensor chip and measure binding kinetics of purified candidate partners flowing over the chip surface.

For analyzing interactions with hemagglutination factors like HagA and HagB, incorporate competitive binding assays to determine if CrcB affects the ability of these proteins to bind to host cells. This approach has been successful with other B. avium outer membrane proteins and could reveal functional relationships between virulence factors .

What are the best methods for determining the fluoride ion channel activity of recombinant CrcB homolog protein?

To accurately assess the fluoride ion channel activity of recombinant CrcB homolog protein, several complementary approaches can be employed:

• Fluoride-sensitive electrode measurements: Reconstitute purified CrcB protein in liposomes and measure fluoride ion flux across the membrane using a fluoride-selective electrode. The following protocol has proven effective:

  • Reconstitute protein at 1:100 protein:lipid ratio in phosphatidylcholine liposomes

  • Load liposomes with 100 mM KCl buffer (pH 7.4)

  • Measure fluoride efflux after addition of NaF to external buffer

• Fluorescent probes: Use PBFI (potassium-binding benzofuran isophthalate) with potassium as the counterion for fluoride transport. Monitor fluorescence changes as fluoride ions are transported across the membrane.

• Patch-clamp electrophysiology: Express CrcB in Xenopus oocytes or mammalian cell lines (e.g., HEK293) and perform patch-clamp recording to directly measure channel conductance and ion selectivity.

• Bacterial fluoride sensitivity assay: Complement a ΔcrcB E. coli strain with the B. avium crcB gene and assess growth in media containing increasing concentrations of NaF. Compare growth curves to determine the level of functional complementation.

The bacterial complementation assay provides a rapid screening method, while the biophysical approaches offer more detailed analysis of channel properties. Together, these methods can generate a comprehensive understanding of CrcB ion channel activity and selectivity .

How can structural studies of the CrcB homolog in Bordetella avium be optimized?

Structural studies of the B. avium CrcB homolog require specific optimization strategies due to the challenges associated with membrane protein crystallization:

• Protein purification optimization:

  • Use mild detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) for extraction

  • Employ size exclusion chromatography as the final purification step to ensure homogeneity

  • Assess protein stability using thermal shift assays to identify optimal buffer conditions

• Crystallization approaches:

  • Lipidic cubic phase (LCP) crystallization has proven more successful than detergent-based methods for membrane proteins similar to CrcB

  • Screen various LCP host lipids (monoolein, monopalmitolein) at different temperatures (4-22°C)

  • Add cholesterol hemisuccinate (CHS) to stabilize the protein during crystallization

• Cryo-EM sample preparation:

  • Reconstitute purified CrcB in nanodiscs using MSP1D1 scaffold protein

  • Optimize protein:lipid:scaffold ratios (typically 1:50:2)

  • Screen various grid types and freezing conditions to minimize preferred orientation

• NMR studies:

  • For solution NMR, express ¹³C/¹⁵N-labeled protein in detergent micelles

  • For solid-state NMR, reconstitute labeled protein in lipid bilayers

  • Focus on specific domains for initial structural characterization

Successful structural studies of CrcB homologs from other bacterial species have utilized LCP crystallization with 35% PEG 400, 100 mM sodium citrate pH 5.5, and 100 mM sodium chloride. Similar conditions may serve as a starting point for B. avium CrcB crystallization trials .

What role does the CrcB homolog play in Bordetella avium pathogenesis and host adaptation?

The role of CrcB homolog in B. avium pathogenesis and host adaptation can be investigated through various experimental approaches:

• Construction of crcB knockout mutants:

  • Create unmarked, in-frame deletion mutants using allelic exchange

  • Compare growth of wild-type and ΔcrcB strains in various conditions, including different fluoride concentrations

  • Assess colonization efficiency in turkey poult tracheal models

• Transcriptional analysis:

  • Perform RNA-seq or qRT-PCR to measure crcB expression during different growth phases

  • Compare expression levels in standard media versus conditions mimicking the avian respiratory tract

  • Identify potential transcriptional regulators by analyzing promoter binding sites

• In vivo colonization studies:

  • Inoculate turkey poults with wild-type and ΔcrcB mutant strains

  • Enumerate bacteria recovered from tracheal tissues at various time points

  • Evaluate clinical signs and histopathological changes in the respiratory tract

• Fluoride concentration measurement in host tissues:

  • Determine fluoride levels in avian respiratory tissues using ion-selective electrodes

  • Compare fluoride concentrations in infected versus uninfected tissues

  • Correlate environmental fluoride levels with crcB expression in vivo

Previous studies with hagA and hagB mutants demonstrated that in vivo tracheal colonization assays provide valuable insights into the role of specific genes in B. avium pathogenesis. Similar approaches with crcB mutants would help establish whether fluoride resistance contributes to survival in the avian host. Preliminary data suggest that crcB expression is upregulated during the initial colonization phase, indicating a potential role in adaptation to the host environment .

What are common challenges in purifying recombinant CrcB homolog protein and how can they be addressed?

Purification of recombinant CrcB homolog protein presents several challenges due to its hydrophobic nature as a membrane protein. Common issues and solutions include:

To optimize yield and purity, a recommended workflow includes:

• Initial extraction with 1% DDM from membrane fraction

• IMAC purification with imidazole gradient (20-300 mM)

• Buffer exchange to reduce detergent concentration to 0.05% DDM

• Size exclusion chromatography as final polishing step

This approach typically yields 1-2 mg of purified protein per liter of bacterial culture with >90% purity suitable for functional and structural studies .

How can researchers troubleshoot inconsistent results in CrcB functional assays?

When facing inconsistent results in CrcB functional assays, consider the following troubleshooting strategies:

• Protein quality issues:

  • Verify protein integrity by SDS-PAGE and Western blot

  • Assess protein homogeneity by size exclusion chromatography

  • Confirm proper folding using circular dichroism spectroscopy

  • Use fresh protein preparations (<1 week old) for all assays

• Liposome-based assay variability:

  • Standardize liposome preparation (size, composition)

  • Verify protein incorporation using fluorescent labeling

  • Control for non-specific leakage using calcein entrapment

  • Calibrate fluoride electrodes before each experiment

• Cell-based assay inconsistencies:

  • Maintain consistent cell passage numbers

  • Standardize transfection efficiency using reporter genes

  • Verify expression levels by Western blot prior to functional testing

  • Control for endogenous channels/transporters with specific inhibitors

• Bacterial growth assay problems:

  • Use fresh media preparations

  • Standardize starting inoculum (OD₆₀₀ = 0.05)

  • Control for effects of antibiotics used for selection

  • Include positive controls (known fluoride exporters) and negative controls

For electrophysiology experiments, inconsistent results often stem from variable protein incorporation into membranes. Pre-screening protein batches using a simpler fluorescence-based flux assay before proceeding to patch-clamp studies can identify problematic preparations. Additionally, recording multiple independent samples (n ≥ 5) helps establish reliable trends despite individual measurement variability .

What strategies can overcome difficulties in generating specific antibodies against Bordetella avium CrcB protein?

Generating specific antibodies against B. avium CrcB protein can be challenging due to its membrane-associated nature and potentially low immunogenicity. The following strategies can overcome these difficulties:

• Antigen design optimization:

  • Focus on hydrophilic regions predicted to be exposed (typically 15-20 amino acids)

  • Generate multiple peptide antigens from different regions of the protein

  • Avoid transmembrane domains which are poorly immunogenic

  • Consider using predicted extracellular loops as immunogens

• Recombinant fragment approach:

  • Express soluble domains of CrcB as GST or MBP fusions

  • Use these fragments for immunization rather than full-length protein

  • Purify under native conditions to preserve epitope conformation

• Fusion protein strategy:

  • Create genetic fusions with highly immunogenic carrier proteins

  • Express the fusion protein in E. coli

  • Purify under denaturing conditions if necessary

• Adjuvant selection:

  • For peptide antigens, conjugate to KLH or BSA carrier proteins

  • Use stronger adjuvants such as Freund's complete/incomplete or TiterMax

  • Consider multiple immunization routes (subcutaneous, intradermal, intraperitoneal)

• Antibody screening optimization:

  • Develop ELISA assays using both peptide and recombinant protein

  • Validate antibodies by Western blot against recombinant protein and B. avium lysates

  • Confirm specificity using lysates from crcB knockout strains

When using similar approaches for generating antibodies against B. avium outer membrane proteins such as HagB, researchers have successfully produced antisera capable of blocking functional activities of the target proteins. This suggests similar strategies may succeed for CrcB, particularly when focusing on predicted surface-exposed regions of the protein .

How can CrcB homolog be utilized in the development of diagnostic tools for Bordetella avium infection?

The CrcB homolog protein from B. avium can be leveraged for developing diagnostic tools through several innovative approaches:

• PCR-based detection systems:

  • Design primers specific to the crcB gene sequence unique to B. avium

  • Develop multiplex PCR assays that simultaneously detect crcB and other B. avium-specific genes

  • Implement quantitative PCR (qPCR) protocols for determining bacterial load

• Serological assays:

  • Produce recombinant CrcB protein as an antigen for ELISA development

  • Create lateral flow immunoassays using anti-CrcB antibodies

  • Develop microagglutination tests similar to those used for other B. avium antigens

• Biosensor development:

  • Immobilize purified CrcB protein on sensor chips for surface plasmon resonance detection

  • Develop aptamer-based detection systems targeting CrcB

  • Create immunosensors using anti-CrcB antibodies coupled with electrochemical detection

• Integrated diagnostic platforms:

  • Combine PCR detection of crcB with serological testing for comprehensive diagnosis

  • Develop point-of-care testing devices for field use in poultry operations

  • Create multipathogen arrays that include CrcB-based detection alongside other respiratory pathogens

The specificity of the crcB gene in B. avium makes it a valuable target for diagnostic development. PCR assays targeting species-specific genes in B. avium have demonstrated 100% sensitivity and 98.8% specificity, suggesting similar performance could be achieved with crcB-based diagnostics. For optimal results, assays should be designed to detect approximately 20 pg of bacterial DNA, corresponding to 3,750-5,000 bacterial genomes .

What is the potential for using CrcB homolog as a target for novel antimicrobial development against Bordetella avium?

The CrcB homolog in B. avium presents several attributes that make it a promising target for novel antimicrobial development:

• Target validation strategies:

  • Confirm essentiality through conditional knockout studies

  • Demonstrate growth inhibition in high-fluoride conditions when CrcB function is impaired

  • Establish the relationship between CrcB inhibition and bacterial survival in host environments

• Small molecule inhibitor development:

  • Perform in silico docking studies to identify potential binding sites

  • Screen chemical libraries for compounds that inhibit fluoride channel activity

  • Design fluoride analogs that may block the channel without being transported

• Peptide inhibitor approach:

  • Design synthetic peptides that mimic CrcB interaction domains

  • Develop cyclic peptides that block the channel pore

  • Create cell-penetrating peptides that interfere with CrcB assembly

• Structure-based drug design:

  • Once the structure is determined, identify druggable pockets

  • Design compounds with specificity for B. avium CrcB over host proteins

  • Optimize lead compounds for pharmacokinetic properties suitable for respiratory delivery

A table of potential inhibition strategies and their predicted efficacies:

How does the genetic diversity of crcB genes across Bordetella avium isolates impact protein function and potential research applications?

Understanding the genetic diversity of crcB genes across B. avium isolates is crucial for research applications and functional analysis:

• Sequence analysis approach:

  • Perform whole-genome sequencing of multiple B. avium isolates from diverse geographic locations

  • Conduct comparative genomic analysis focusing on the crcB locus

  • Identify single nucleotide polymorphisms (SNPs) and insertion/deletion variants

  • Assess selection pressure through Ka/Ks ratio analysis

• Diversity impact on protein function:

  • Express variants with different mutations in heterologous systems

  • Compare fluoride export efficiency among variants

  • Evaluate protein stability and membrane integration of different variants

  • Assess the impact of polymorphisms on protein-protein interactions

• Implications for diagnostic development:

  • Design primers/probes targeting conserved regions for reliable detection

  • Develop multiple primer sets to ensure detection of all variants

  • Establish a database of known polymorphisms to interpret diagnostic results

• Research and therapeutic considerations:

  • Select representative variants for structural studies

  • Test inhibitor efficacy across diverse protein variants

  • Evaluate immunogenicity of different protein variants for vaccine development

What are the most promising approaches for studying the interaction between CrcB homolog and the host immune system during Bordetella avium infection?

To investigate the interaction between CrcB homolog and the host immune system during B. avium infection, researchers should consider these promising approaches:

• In vitro immune response studies:

  • Stimulate avian immune cells (macrophages, dendritic cells) with purified CrcB protein

  • Measure cytokine production using ELISA or qRT-PCR

  • Assess activation of pattern recognition receptors (TLRs, NLRs)

  • Evaluate antigen presentation and T-cell activation in response to CrcB

• Animal model investigations:

  • Compare immune responses to wild-type and crcB-deficient B. avium strains

  • Monitor antibody development against CrcB during infection progression

  • Perform adoptive transfer experiments to identify protective immune components

  • Use immunohistochemistry to track CrcB expression in infected tissues

• Omics-based approaches:

  • Conduct transcriptomics of host tissues infected with wild-type versus ΔcrcB mutants

  • Perform proteomics on immune cells responding to CrcB stimulation

  • Use systems biology to identify key immune pathways involved in recognition

  • Deploy single-cell RNA sequencing to identify responsive immune cell populations

• Immunoinformatics analysis:

  • Predict T-cell and B-cell epitopes within the CrcB sequence

  • Identify potential molecular mimicry between CrcB and host proteins

  • Model HLA/MHC binding of CrcB-derived peptides

  • Design experiments to validate predicted immunogenic regions

Previous studies with B. avium virulence factors such as HagB have demonstrated that these proteins can elicit specific antibody responses that block bacterial functions. Similar approaches with CrcB would help establish whether this protein is immunogenic during natural infection and whether anti-CrcB antibodies contribute to bacterial clearance. Additionally, analysis of immune responses in the turkey model would provide valuable insights into host-pathogen interactions specific to the natural host .