Recombinant Campylobacter jejuni Protein CrcB homolog (crcB)

How is recombinant CrcB typically expressed and purified for research applications?

The recombinant CrcB protein is typically expressed in E. coli expression systems with an N-terminal histidine tag to facilitate purification. The protein is expressed as a full-length construct (amino acids 1-122) and purified using immobilized metal affinity chromatography (IMAC) .

For optimal expression:

• Transform the expression vector containing the crcB gene into a suitable E. coli strain

• Culture the transformed bacteria in appropriate media with induction conditions

• Harvest cells and lyse using mechanical disruption or detergent-based methods

• Purify using Ni-NTA or similar IMAC resin

• Elute with imidazole and perform buffer exchange

• Store as lyophilized powder for maximum stability

What are the optimal storage and reconstitution protocols for recombinant CrcB?

Repeated freeze-thaw cycles significantly reduce protein activity and should be avoided. Centrifugation of the vial before opening is recommended to ensure all material is at the bottom of the tube .

How does CrcB structure relate to its ion transport function?

The CrcB protein forms a homodimeric channel in the membrane with each monomer containing multiple transmembrane helices. The transmembrane topology analysis of the amino acid sequence reveals hydrophobic regions consistent with membrane integration. The protein's structure creates a selective pore that allows F⁻ ions to pass while excluding other ions.

Researchers investigating structure-function relationships should consider:

• Analyzing the conserved regions across CrcB homologs

• Performing site-directed mutagenesis of key residues

• Conducting electrophysiological measurements to assess ion selectivity

• Using molecular dynamics simulations to model ion transport mechanisms

What experimental approaches can determine CrcB's role in C. jejuni pathogenicity?

While direct evidence linking CrcB to C. jejuni pathogenicity is limited, established experimental approaches include:

• Gene knockout studies comparing wild-type and ΔcrcB mutants in:

  • Colonization models

  • Fluoride resistance assays

  • Stress response experiments

  • Transcriptomic analyses

• Protein-protein interaction studies to identify binding partners:

  • Pull-down assays with tagged CrcB

  • Two-hybrid screening

  • Cross-linking experiments followed by mass spectrometry

• Expression analysis during different stages of infection:

  • qRT-PCR

  • Western blotting with specific antibodies

  • Reporter gene assays

How might CrcB relate to C. jejuni's potential role in colorectal carcinogenesis?

Recent research has demonstrated that C. jejuni promotes colorectal tumorigenesis primarily through its cytolethal distending toxin (CDT), which causes DNA double-strand breaks . While no direct link between CrcB and carcinogenesis has been established, researchers might investigate:

• Whether CrcB expression influences CDT production or delivery

• If CrcB contributes to bacterial persistence in the intestinal environment

• Whether CrcB affects host cell interactions that could promote carcinogenesis

Studies have shown that C. jejuni colonization significantly increases tumor development in susceptible mouse models, with CDT-deficient mutants showing attenuated tumorigenesis . CrcB's role in bacterial survival could indirectly contribute to this process by enhancing bacterial fitness in the host environment.

What assays can be used to measure CrcB fluoride transport activity?

Several methodologies can quantify CrcB-mediated fluoride transport:

• Fluoride-selective electrode measurements:

  • Reconstitute purified CrcB in liposomes

  • Monitor F⁻ concentration changes in internal and external compartments

  • Calculate transport rates under various conditions

• Fluorescence-based assays:

  • Utilize fluoride-sensitive fluorescent probes (e.g., PBFI)

  • Monitor fluorescence changes in real-time

  • Determine transport kinetics (Km, Vmax)

• Cellular survival assays:

  • Express CrcB in fluoride-sensitive bacterial strains

  • Measure growth inhibition at different fluoride concentrations

  • Compare wild-type vs. mutant CrcB variants

• Electrophysiological measurements:

  • Incorporate CrcB into planar lipid bilayers

  • Measure current under voltage-clamp conditions

  • Determine ion selectivity and conductance properties

How can recombinant CrcB be incorporated into cell-based research models?

For researchers interested in studying CrcB in cellular contexts:

• Transfection approaches:

  • Clone crcB into mammalian expression vectors

  • Optimize transfection for the target cell line (e.g., CRC cell lines)

  • Verify expression by Western blot or immunofluorescence

• Bacterial infection models:

  • Create fluorescently-tagged CrcB in C. jejuni

  • Infect intestinal epithelial cells or organoids

  • Track protein localization during infection

• For colorectal cancer studies:

  • Utilize established CRC cell lines (e.g., CoLo 205, established in 1957)

  • Develop co-culture systems with C. jejuni expressing wild-type or mutant CrcB

  • Assess effects on cellular transformation markers

What potential exists for CrcB as a therapeutic target?

Given CrcB's role in bacterial fluoride resistance, it represents a potential target for antimicrobial development:

• High-throughput screening approaches:

  • Develop fluorescence-based assays suitable for screening compound libraries

  • Identify small molecules that inhibit CrcB transport function

  • Validate hits using secondary assays and structure-activity relationship studies

• Peptide-based inhibitors:

  • Design peptides that mimic CrcB interaction partners

  • Test for inhibition of transport activity

  • Optimize for stability and cellular uptake

• Combination therapy potential:

  • Investigate synergistic effects with existing antibiotics

  • Determine if CrcB inhibition sensitizes C. jejuni to fluoride-containing compounds

How might CrcB research contribute to understanding C. jejuni's role in colorectal cancer?

C. jejuni has been shown to promote colorectal cancer through its CDT toxin, which induces DNA damage . Future research directions include:

• Investigating whether CrcB influences C. jejuni persistence in the intestinal environment, which could indirectly affect carcinogenesis

• Determining if CrcB expression correlates with C. jejuni virulence in patient-derived samples

• Examining potential interactions between CrcB and host cellular processes relevant to cancer development

Studies have demonstrated that C. jejuni colonization in mouse models leads to significantly more and larger tumors compared to uninfected controls, with this effect being dependent on CDT production . Understanding how CrcB contributes to bacterial fitness in this context could provide new insights into the mechanisms of bacteria-associated carcinogenesis.

What are the emerging techniques for studying CrcB structure and function?

Contemporary research methodologies offering new insights include:

• Cryo-electron microscopy:

  • Determine high-resolution structure of CrcB in native lipid environments

  • Visualize conformational changes during transport cycle

• Single-molecule fluorescence resonance energy transfer (smFRET):

  • Monitor real-time conformational dynamics

  • Correlate structural changes with transport events

• In silico molecular dynamics:

  • Model fluoride ion passage through the channel

  • Identify critical residues for substrate specificity

• CRISPR-Cas9 genome editing:

  • Create precise modifications in crcB gene in C. jejuni

  • Assess phenotypic consequences in infection models