Recombinant Campylobacter lari Protein CrcB homolog (crcB)

What is Campylobacter lari and what is known about its crcB protein?

Campylobacter lari is a species within the Campylobacter genus of Gram-negative bacteria that typically appear comma or s-shaped and are motile. While less studied than C. jejuni and C. coli, C. lari is clinically significant as a known cause of recurrent diarrhea in children . The crcB protein in C. lari is a homolog of the crcB (camphor resistance) gene family found across various bacterial species.

The crcB protein (amino acids 1-122) is involved in cellular functions related to fluoride ion channels and resistance mechanisms. Current research suggests it functions in maintaining cellular homeostasis under environmental stress conditions, though the specific molecular mechanisms in C. lari require further characterization through comparative genomic and functional analyses .

How does C. lari crcB differ from other Campylobacter species' crcB proteins?

The crcB protein in C. lari demonstrates structural and functional similarities to orthologs in other Campylobacter species, but with notable differences that reflect their evolutionary divergence. Transcriptomic studies reveal that C. lari has distinct expression patterns compared to C. coli and C. jejuni under stress conditions, suggesting species-specific regulatory mechanisms .

Analysis of conserved domains indicates that while the core functional elements remain similar across species, C. lari's crcB exhibits unique amino acid substitutions that may influence protein-protein interactions and substrate specificity. These differences are likely related to C. lari's ecological niche and may contribute to its specific pathogenicity profile .

What expression systems are suitable for producing recombinant C. lari crcB protein?

Recombinant C. lari crcB protein can be effectively expressed using several heterologous systems. The most common expression platforms include:

• E. coli expression systems: Offer high yields and straightforward protocols, typically using BL21(DE3) or similar strains with pET-based vectors

• Yeast expression systems: Useful when post-translational modifications are required

• Baculovirus expression systems: Provide eukaryotic processing capabilities for complex proteins

• Mammalian cell expression systems: Offer the most authentic post-translational modifications

The choice of expression system should be determined by the research objectives, with E. coli being preferred for structural studies due to higher yields, while mammalian systems may be more appropriate for functional analyses requiring native protein conformation.

What are the optimal conditions for studying heat stress response of crcB in C. lari compared to other Campylobacter species?

To effectively study heat stress response involving crcB in C. lari, researchers should implement a controlled experimental design similar to those used in comparative studies of Campylobacter species. Based on previous research with C. lari RM2100, optimal conditions include:

• Baseline cultivation: Grow C. lari on Mueller-Hinton agar containing 5% sheep blood (MHB) for 48 hours or in Brucella broth for 24 hours at 37°C under microaerobic conditions (6% O₂, 7% CO₂, 7% H₂, 80% N₂)

• Heat stress induction: Subject cultures to 46°C for periods ranging from 15 to 60 minutes, with sampling at 15, 30, and 60-minute intervals to capture both immediate and sustained responses

• Comparative analysis: Include reference strains of C. lari alongside field isolates to account for strain variability, with parallel experiments using C. coli and/or C. jejuni strains for inter-species comparison

• Transcriptomic assessment: Apply RNA-seq or RT-qPCR to quantify expression changes of crcB alongside known heat-responsive genes (dnaK, groES, groEL, clpB)

This methodology enables identification of species-specific patterns in crcB regulation during thermal stress, which differs significantly between C. lari and other Campylobacter species as demonstrated by previous transcriptomic analyses .

How can researchers effectively design experiments to characterize the functional role of crcB in C. lari pathogenicity?

To characterize the functional role of crcB in C. lari pathogenicity, researchers should implement a multi-faceted experimental approach:

• Gene knockout/knockdown studies:

  • Create crcB deletion mutants using homologous recombination or CRISPR-Cas9

  • Develop conditional expression systems for dose-dependent phenotypic analysis

  • Evaluate mutants in both in vitro and in vivo pathogenicity models

• Protein-protein interaction analysis:

  • Apply yeast two-hybrid or co-immunoprecipitation to identify binding partners

  • Validate interactions with orthogonal methods (FRET, SPR, or biolayer interferometry)

  • Map interaction domains through truncation and site-directed mutagenesis

• Transcriptomic and proteomic comparisons:

  • Compare wild-type and crcB-deficient strains under various stress conditions

  • Analyze differential expression patterns using RNA-seq and comparative proteomics

  • Correlate findings with phenotypic outcomes in infection models

• Infection models:

  • Employ cellular infection assays with relevant human cell lines

  • Utilize animal models that recapitulate human diarrheal disease

  • Quantify bacterial colonization, persistence, and host immune responses

This comprehensive experimental framework enables researchers to dissect the specific contributions of crcB to C. lari pathogenicity and compare these functions with other Campylobacter species where crcB may play different roles in virulence and stress response .

What technical challenges exist in crystallizing C. lari crcB protein for structural studies, and how can they be overcome?

Crystallizing C. lari crcB protein presents several technical challenges that researchers must address for successful structural determination:

• Protein solubility and stability issues:

  • Challenge: The small size (122 amino acids) and potentially hydrophobic regions of crcB can lead to aggregation

  • Solution: Implement solubility screening with various buffers and additives; consider fusion partners (MBP, SUMO, or thioredoxin) to enhance solubility while preserving native structure

• Protein purity and homogeneity concerns:

  • Challenge: Heterogeneous protein populations impede crystal formation

  • Solution: Apply rigorous multi-step purification including affinity chromatography followed by size exclusion and ion exchange; confirm homogeneity by dynamic light scattering

• Crystal nucleation and growth difficulties:

  • Challenge: Small proteins often form crystals that diffract poorly

  • Solution: Implement high-throughput screening of crystallization conditions (pH, salt concentrations, precipitants); explore seeding techniques and crystallization in lipidic environments for membrane-associated regions

• Phase determination complications:

  • Challenge: Lack of homologous structures for molecular replacement

  • Solution: Incorporate selenomethionine for SAD/MAD phasing or prepare heavy atom derivatives; alternatively, explore microcrystal electron diffraction (MicroED) for crystals that are too small for traditional X-ray crystallography

• Functional conformation capture:

  • Challenge: Ensuring the crystal structure represents a biologically relevant conformation

  • Solution: Co-crystallize with ligands, substrate analogs, or binding partners; validate structural findings with complementary biophysical techniques (HDX-MS, SAXS)

By systematically addressing these challenges, researchers can overcome the technical barriers to obtaining high-resolution structures of C. lari crcB, enabling structure-function relationship studies essential for understanding its role in bacterial physiology and pathogenesis .

How do transcriptomic profiles of crcB expression differ between C. lari and other Campylobacter species under various stress conditions?

Transcriptomic analysis reveals significant differences in crcB expression patterns between Campylobacter species when exposed to environmental stressors:

These differential expression patterns suggest that while core stress response mechanisms involving molecular chaperones (dnaK, groES, groEL, clpB) are conserved across Campylobacter species, regulatory networks controlling crcB differ substantially . The pronounced differences in expression profiles between C. lari and other species reflect their phylogenetic distance and corresponding adaptations to their ecological niches.

Notably, the generally lower and more transient expression of stress-responsive genes in C. lari compared to C. coli suggests different survival strategies employed by these species when facing environmental challenges. These findings emphasize that stress response mechanisms described for one Campylobacter species cannot be directly applied to others, necessitating species-specific experimental approaches .

What biomedical research applications benefit from studying recombinant C. lari crcB protein?

Recombinant C. lari crcB protein offers valuable applications across several biomedical research domains:

• Vaccine development:

  • The recombinant protein serves as a potential antigenic target for subunit vaccine development against C. lari infections

  • Comparative immunogenicity studies with crcB proteins from different Campylobacter species enable identification of conserved epitopes for broad-spectrum vaccine candidates

• Diagnostic test development:

  • Species-specific antibodies against unique epitopes in C. lari crcB facilitate rapid and precise detection systems

  • Recombinant protein-based assays enable differentiation between Campylobacter species in clinical and environmental samples

• Antimicrobial resistance research:

  • Structure-function studies of crcB contribute to understanding bacterial resistance mechanisms

  • The protein can serve as a target for novel antimicrobial development focusing on species-specific inhibitors

• Host-pathogen interaction studies:

  • Purified recombinant protein enables investigation of interactions with host cell receptors and immune components

  • These studies reveal mechanisms of pathogenesis specific to C. lari compared to other Campylobacter species

• Structural biology and drug discovery:

  • High-resolution structural data from purified recombinant crcB facilitates rational drug design

  • Structure-based virtual screening identifies potential small-molecule inhibitors targeting species-specific features

These diverse applications demonstrate the significant value of recombinant C. lari crcB protein as a research tool for addressing the burden of Campylobacter infections, particularly in vulnerable populations such as children where C. lari has been identified as a cause of recurrent diarrhea .

How can researchers address contradictions in published data regarding crcB function in different Campylobacter species?

Researchers encountering conflicting data regarding crcB function across Campylobacter species should implement a systematic approach to resolve these contradictions:

• Standardization of experimental conditions:

  • Establish uniform growth conditions, stress parameters, and analytical techniques

  • Conduct parallel experiments with multiple Campylobacter species in the same laboratory

  • Document microaerobic conditions precisely (6% O₂, 7% CO₂, 7% H₂, 80% N₂) as variations significantly impact results

• Strain selection and characterization:

  • Use well-characterized reference strains (e.g., C. lari RM2100, C. coli RM2228) alongside clinical isolates

  • Perform whole-genome sequencing to identify strain-specific genetic variations

  • Create isogenic mutants differing only in crcB to eliminate confounding variables

• Multi-omics integration:

  • Apply transcriptomics, proteomics, and metabolomics to the same experimental setup

  • Cross-validate findings across different analytical platforms

  • Identify discrepancies between mRNA expression and protein abundance that might explain functional contradictions

• Time-course analysis:

  • Implement fine-grained temporal sampling (15, 30, 60 minutes) during stress responses

  • Account for species-specific timing differences in gene expression (e.g., C. lari's more transient response compared to C. coli's sustained expression)

  • Correlate molecular changes with phenotypic outcomes at each timepoint

• Phylogenetic context:

  • Consider evolutionary relationships when interpreting functional differences

  • Recognize that C. lari's greater phylogenetic distance from C. jejuni and C. coli explains many functional discrepancies

  • Analyze synteny and genomic context of crcB across species to identify regulatory differences

This methodical approach acknowledges that approximately 20% of genes show differential expression between C. lari and C. coli under stress conditions, with significant differences in the proportion of up-regulated versus down-regulated genes (C. coli: 67.1% up-regulated; C. lari: 43.6% up-regulated) . By addressing these species-specific differences systematically, researchers can resolve apparent contradictions and develop a more nuanced understanding of crcB function across the Campylobacter genus.

What protein purification strategies yield the highest purity and activity for recombinant C. lari crcB protein?

For optimal purification of recombinant C. lari crcB protein (aa 1-122), researchers should implement a strategic multi-step approach:

• Expression system optimization:

  • E. coli BL21(DE3) with codon optimization for rare codons in C. lari

  • Induction at lower temperatures (16-18°C) to enhance proper folding

  • Addition of solubility tags (His6, GST, or MBP) with precision protease cleavage sites

• Primary capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged constructs

  • Glutathione affinity chromatography for GST-fusion proteins

  • Precipitation with ammonium sulfate (40-60% saturation) as an alternative initial step

• Intermediate purification:

  • Ion exchange chromatography (typically anion exchange at pH 8.0)

  • Affinity tag removal using site-specific proteases (TEV, PreScission, or Factor Xa)

  • Second IMAC step to remove uncleaved protein and the cleaved tag

• Polishing steps:

  • Size exclusion chromatography using Superdex 75 or equivalent to achieve >95% purity

  • Removal of aggregates and separation of monomeric from oligomeric species

  • Buffer optimization to maintain stability (typically 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol)

• Quality control assessment:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Dynamic light scattering to verify homogeneity

  • Activity assays specific to crcB function (fluoride ion binding or transport assays)

  • Mass spectrometry to confirm protein integrity and modifications

This optimized workflow typically yields 2-5 mg of pure protein per liter of bacterial culture with >95% purity as determined by densitometric analysis of SDS-PAGE gels. The purified protein maintains stability for 1-2 weeks at 4°C and can be stored for extended periods at -80°C in buffer containing 10% glycerol without significant loss of activity .

How should researchers design experiments to investigate the role of crcB in C. lari's heat stress response?

To investigate crcB's role in C. lari's heat stress response, researchers should implement this comprehensive experimental design:

• Strain preparation and characterization:

  • Generate crcB deletion mutants (ΔcrcB) and complemented strains in C. lari RM2100

  • Create point mutations in conserved residues to identify critical functional domains

  • Verify mutants by whole genome sequencing to ensure no secondary mutations

• Stress exposure protocol:

  • Establish baseline growth in Brucella broth for 24h at 37°C under microaerobic conditions

  • Expose cultures to 46°C with sampling at precise intervals (0, 15, 30, 60 minutes)

  • Monitor viability using standardized plate counting on Mueller-Hinton agar with 5% sheep blood

• Molecular response analysis:

  • Perform RNA-seq to capture genome-wide transcriptional changes

  • Conduct RT-qPCR validation focusing on crcB and known heat shock genes (dnaK, groES, groEL, clpB)

  • Analyze protein expression changes with quantitative proteomics

• Comparative experimental controls:

  • Parallel analysis of wild-type, ΔcrcB mutant, and complemented strains

  • Side-by-side comparison with C. coli and/or C. jejuni under identical conditions

  • Inclusion of other stress conditions (oxidative, acid, osmotic) to determine stress-specificity

• Functional characterization:

  • Measure membrane integrity under heat stress using fluorescent dyes

  • Assess protein aggregation and misfolding in wild-type vs. ΔcrcB strains

  • Evaluate metabolic activity through ATP production and respiratory capacity

This experimental design enables comprehensive characterization of crcB's contribution to heat stress response in C. lari, building upon previous findings that C. lari exhibits distinct stress response patterns compared to C. coli, with approximately 19.4% of its genes showing differential expression during heat stress .

What are the most effective approaches for studying protein-protein interactions involving C. lari crcB?

To effectively study protein-protein interactions (PPIs) involving C. lari crcB, researchers should employ multiple complementary approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Tag crcB with affinity tags (FLAG, HA, or Strep-tag II) that minimally impact function

  • Express in native C. lari or heterologous systems under relevant stress conditions

  • Perform gentle lysis and affinity capture followed by LC-MS/MS identification

  • Implement SILAC or TMT labeling for quantitative comparison across conditions

• Yeast two-hybrid (Y2H) screening:

  • Use crcB as bait against C. lari genomic DNA library or predicted interactome

  • Employ membrane Y2H variants for membrane-associated regions of crcB

  • Validate positive interactions through reverse Y2H and co-immunoprecipitation

  • Map interaction domains using truncation constructs

• Proximity-based labeling techniques:

  • Fuse crcB to BioID, TurboID, or APEX2 enzymes

  • Express in C. lari under native regulation during heat or other stress

  • Identify proximal proteins through streptavidin purification and MS analysis

  • Distinguish stable from transient interactions through time-course experiments

• Biophysical validation methods:

  • Surface plasmon resonance (SPR) or biolayer interferometry for kinetic parameters

  • Microscale thermophoresis for interactions in complex solutions

  • Isothermal titration calorimetry for thermodynamic characterization

  • Förster resonance energy transfer (FRET) for in vivo interaction validation

• Computational prediction and analysis:

  • Apply machine learning algorithms trained on known bacterial interactomes

  • Identify conserved interaction motifs through comparative genomics

  • Perform molecular docking studies based on structural models

  • Integrate interaction data with transcriptomics to identify co-regulated partners

This multi-faceted approach enables the identification of interaction partners under various conditions, particularly during heat stress where expression patterns of numerous genes are significantly altered. Given that approximately 35 genes show similar expression patterns between C. lari and C. coli during heat stress, these may represent conserved interaction partners within core stress response pathways .

How can structural biology techniques be applied to understand the functional mechanism of C. lari crcB?

Structural biology offers powerful approaches to elucidate the functional mechanisms of C. lari crcB:

• X-ray crystallography:

  • Crystallize purified recombinant crcB in various conformational states

  • Determine high-resolution structures (≤2.5Å) to reveal atomic details

  • Co-crystallize with putative ligands or interacting partners

  • Analyze crystal contacts for potential oligomerization interfaces

• Cryo-electron microscopy (cryo-EM):

  • Apply single-particle analysis for structures of crcB complexes

  • Utilize cryo-electron tomography to visualize crcB in membrane contexts

  • Implement subtomogram averaging for in situ structural determination

  • Capture conformational ensembles representing functional states

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Conduct solution NMR for dynamic regions and conformational changes

  • Map binding interfaces through chemical shift perturbation experiments

  • Measure ¹⁵N relaxation parameters to characterize protein dynamics

  • Determine solution structures of domains or the complete protein (given its manageable size of 122 amino acids)

• Integrative structural biology approaches:

  • Combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with computational modeling

  • Implement small-angle X-ray scattering (SAXS) for solution state conformations

  • Apply crosslinking mass spectrometry (XL-MS) to identify domain interactions

  • Utilize molecular dynamics simulations to explore conformational landscapes

• Structure-guided functional studies:

  • Design site-directed mutagenesis based on structural insights

  • Create chimeric proteins with other Campylobacter species' crcB

  • Develop structure-based fluorescent biosensors for real-time activity monitoring

  • Perform virtual screening for small-molecule modulators

This comprehensive structural biology approach provides molecular-level understanding of how C. lari crcB functions, particularly in response to environmental stressors such as heat. The structural insights can explain why C. lari demonstrates different expression patterns and stress responses compared to other Campylobacter species, despite sharing core chaperone machinery .

What genomic and evolutionary analyses can reveal about the divergence of crcB function across Campylobacter species?

Genomic and evolutionary analyses provide critical insights into crcB functional divergence across Campylobacter species:

• Comparative genomics:

  • Analyze synteny and genomic context of crcB across species

  • Identify conservation patterns of upstream regulatory elements

  • Quantify selection pressures through Ka/Ks ratios across orthologous sequences

  • Map species-specific insertions, deletions, and substitutions onto structural models

• Phylogenetic profiling:

  • Construct phylogenetic trees based on whole genomes and crcB sequences

  • Correlate crcB sequence divergence with species habitat and host range

  • Identify lineage-specific adaptations through ancestral sequence reconstruction

  • Apply coevolution analysis to identify functionally linked genes

• Population genomics:

  • Analyze intraspecies variation in crcB across C. lari isolates

  • Identify potential horizontal gene transfer events affecting crcB

  • Detect selective sweeps indicating recent adaptive events

  • Compare core vs. accessory genome elements interacting with crcB

• Experimental evolution:

  • Subject C. lari to long-term serial passage under selective pressures

  • Sequence evolved populations to identify adaptive mutations in crcB

  • Compare parallel adaptations across Campylobacter species

  • Validate fitness effects of observed genetic changes

• Pan-genome analysis:

  • Determine if crcB belongs to core or accessory genome components

  • Identify species-specific gene neighborhoods that may influence function

  • Analyze correlation between crcB variants and pathogenicity islands

  • Construct species-specific protein-protein interaction networks

These approaches reveal that the significant differences in stress response between C. lari and other Campylobacter species (with only 35 similarly expressed genes during heat stress) likely reflect their evolutionary divergence and adaptation to different ecological niches . This divergence is consistent with the higher number of orthologous genes between C. jejuni and C. coli compared to C. lari, explaining the greater similarity in heat stress response between the former two species.

How can systems biology approaches integrate transcriptomic, proteomic, and metabolomic data to understand crcB's role in C. lari biology?

Systems biology approaches enable holistic understanding of crcB's role in C. lari through multi-omics integration:

• Multi-omics data generation and normalization:

  • Perform RNA-seq, proteomics, and metabolomics on identical samples

  • Apply standardized stress conditions (46°C heat shock) with precise timing

  • Compare wild-type, crcB knockout, and complemented strains

  • Include multiple timepoints (0, 15, 30, 60 minutes) to capture dynamic responses

• Network reconstruction methods:

  • Develop gene regulatory networks through time-series expression data

  • Construct protein-protein interaction networks centered on crcB

  • Map metabolic pathway alterations resulting from crcB perturbation

  • Integrate networks across omics layers using machine learning approaches

• Computational modeling techniques:

  • Develop ordinary differential equation models of crcB-related pathways

  • Apply flux balance analysis to quantify metabolic consequences

  • Implement Bayesian network inference to identify causal relationships

  • Utilize constraint-based modeling to predict system behaviors

• Perturbation studies with integrated analysis:

  • Systematically perturb pathways connected to crcB

  • Analyze system-wide responses across transcriptome, proteome, and metabolome

  • Identify buffering mechanisms and compensatory responses

  • Validate model predictions through targeted experiments

• Comparative systems analysis:

  • Perform parallel analyses in C. lari, C. coli, and C. jejuni

  • Identify conserved versus species-specific network modules

  • Quantify differences in network topology and information flow

  • Correlate network differences with phenotypic variations

This systems biology framework reveals that crcB functions within complex stress response networks that differ significantly between Campylobacter species. While C. lari and C. coli both differentially express approximately 20% of their genes during heat stress, the regulatory architecture differs substantially, with C. coli predominantly up-regulating genes (67.1%) while C. lari shows more balanced regulation (43.6% up-regulated) . These network-level differences explain the species-specific adaptation strategies and highlight why stress response mechanisms described for one Campylobacter species cannot be directly applied to others.