Recombinant Campylobacter concisus Translation initiation factor IF-2 (infB), partial

What is the role of Translation Initiation Factor IF-2 in bacterial protein synthesis?

Translation Initiation Factor IF-2 mediates the binding of formylmethionyl-tRNA (fMet-tRNA) to the 30S ribosomal subunit during translation initiation. The protein recognizes the formyl group on the initiator tRNA, positioning it at the P-site of the ribosome in a GTP-dependent manner. Following GTP hydrolysis after 50S subunit joining, IF-2 is released, allowing translation elongation to proceed. In pathogenic bacteria like C. concisus, IF-2 may play additional roles in adaptation to stressful environments encountered during host infection.

How does Campylobacter concisus differ from other Campylobacter species?

Campylobacter concisus has emerged as an oral and intestinal pathogen associated with inflammatory bowel disease (IBD) and Crohn's disease, while C. jejuni and C. coli are established primarily as gastrointestinal pathogens. C. concisus possesses a relatively smaller genome (1.8-2.1 Mb), suggesting evolutionary adaptation through versatile respiratory pathways and multifunctional enzymes . The species demonstrates significant strain-to-strain variability, with strains isolated from chronic intestinal diseases showing higher invasive potential than those from acute gastroenteritis . Additionally, C. concisus can grow under both microaerobic and anaerobic conditions, utilizing various N- or S-oxides as terminal electron acceptors during anaerobic respiration .

What is known about genetic diversity among Campylobacter concisus strains?

Campylobacter concisus exhibits considerable genetic heterogeneity across strains isolated from different clinical sources and disease states. Genomic analyses reveal that strains from chronic intestinal diseases possess significantly higher invasive potential than those from acute diseases or healthy controls . This variation in virulence appears linked to specific plasmids . Certain genes, such as bisA, are present in intestinal strains (13826 and 51562) but absent in oral strains (33237), contributing to different metabolic capabilities and virulence factors . This genetic diversity explains the heterogeneity observed in clinical outcomes associated with C. concisus infection and highlights the importance of strain-specific characterization in research.

What expression systems are typically used for recombinant production of bacterial translation factors?

For recombinant production of bacterial translation factors like IF-2, E. coli-based expression systems are predominantly employed. Common vectors include pET series plasmids with T7 promoters for high-level inducible expression. When expressing potentially toxic proteins, tightly regulated systems such as pBAD (arabinose-inducible) may be preferable. E. coli BL21(DE3) and its derivatives are frequently used due to their reduced protease activity. For challenging proteins, specialized strains providing rare codons (Rosetta) or cold-adapted chaperones (Arctic Express) can enhance soluble expression. Purification typically involves affinity chromatography using His6 tags, often with protease cleavage sites for tag removal post-purification. When expressing C. concisus proteins specifically, codon optimization may be necessary due to the significant differences in codon usage between Campylobacter and E. coli.

How might strain variation in C. concisus affect the structure and function of translation initiation factors?

The genetic diversity observed between C. concisus strains isolated from different clinical sources likely extends to variations in translation machinery proteins including IF-2. These variations may manifest as amino acid substitutions affecting IF-2's binding affinity for GTP, fMet-tRNA, or ribosomal components. Strains with enhanced invasive potential from chronic intestinal diseases might possess IF-2 variants optimized for protein synthesis under inflammatory conditions, similar to those observed in Crohn's disease patients .

To investigate these variations, researchers should implement:

• Comparative sequence analysis of infB genes across strains

• Recombinant expression and purification of IF-2 variants

• Structural characterization using X-ray crystallography or cryo-EM

• Functional assays measuring GTPase activity and translation initiation rates

• Assessment of protein stability under varying pH and oxidative stress conditions

Methodological approaches should include site-directed mutagenesis to test the functional significance of strain-specific amino acid differences and ribosome binding assays to quantify interaction dynamics between variant IF-2 proteins and ribosomes.

What experimental approaches can be used to study the interaction between IF-2 and the host immune system during C. concisus infection?

Given that C. concisus infection activates immune pathways including interleukin-12 production, proteasome activation, and NF-κB signaling , investigating IF-2's potential role in these processes requires sophisticated experimental approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation using recombinant IF-2 and host cell lysates

  • Surface plasmon resonance to quantify binding affinities

  • Proximity labeling (BioID/APEX) to identify in vivo interaction partners

• Immunological assays:

  • Exposure of intestinal epithelial cells to purified IF-2 with measurement of cytokine production (IL-12, IFN-γ)

  • Flow cytometry analysis of immune cell activation markers

  • Reporter cell lines for NF-κB activation assessment

• Comparative studies:

  • Wild-type C. concisus versus strains with modified infB expression

  • Analysis of host response to invasive versus non-invasive strains

• Localization studies:

  • Immunofluorescence microscopy with anti-IF-2 antibodies during infection

  • Fractionation of infected cells to track IF-2 distribution

These approaches would determine whether IF-2 directly interacts with host immune components or if its role in pathogenesis is primarily through maintaining bacterial protein synthesis during infection.

How does the redox environment affect the stability and activity of C. concisus IF-2, and what implications does this have for pathogenesis?

C. concisus must adapt to varying oxygen tensions throughout the gastrointestinal tract, growing under both microaerobic and anaerobic conditions . This adaptation likely extends to its translation machinery, including IF-2. Research suggests C. concisus possesses sophisticated mechanisms for managing oxidative stress, exemplified by the BisA protein's dual role in respiration and protein methionine sulfoxide repair .

To investigate redox effects on IF-2:

Understanding how IF-2 maintains functionality during oxidative stress could explain C. concisus persistence during intestinal inflammation, where reactive oxygen species are abundant, and potentially reveal targets for therapeutic intervention.

What computational approaches can predict strain-specific variations in IF-2 structure and their functional implications?

Computational approaches offer powerful tools for predicting how strain-specific variations in C. concisus IF-2 might impact function:

• Sequence analysis:

  • Multiple sequence alignment of infB genes from diverse C. concisus strains

  • Phylogenetic analysis correlating sequence clusters with clinical sources

  • Identification of positively selected residues under evolutionary pressure

• Structural prediction:

  • Homology modeling based on resolved bacterial IF-2 structures

  • Ab initio modeling for divergent regions

  • Molecular dynamics simulations (100ns-1μs) to assess conformational differences

• Functional prediction:

  • GTP binding site analysis

  • Ribosome and tRNA interaction surface mapping

  • Identification of strain-specific surface properties

• Integration with experimental data:

  • Correlation of computational predictions with invasive potential

  • Validation through site-directed mutagenesis and biochemical assays

  • Iterative refinement of models based on experimental results

This computational-experimental pipeline could identify strain-specific adaptations in IF-2 that contribute to the varying pathogenic potential observed among C. concisus strains from different disease states .

What proteomic approaches are most effective for studying post-translational modifications of C. concisus IF-2?

Studying post-translational modifications (PTMs) of C. concisus IF-2 requires an integrated proteomic approach:

• Sample preparation strategies:

  • Multiple proteases (trypsin, chymotrypsin, Glu-C) to maximize sequence coverage

  • Enrichment methods for specific PTMs (TiO₂ for phosphorylation, antibody-based for acetylation)

  • Differential alkylation to preserve in vivo redox states

• Mass spectrometry approaches:

  • High-resolution LC-MS/MS with ETD fragmentation to preserve labile modifications

  • Top-down proteomics for intact protein analysis to detect modification combinations

  • Parallel reaction monitoring (PRM) for targeted quantification of modified peptides

• Specific considerations for C. concisus:

  • Focus on redox-related modifications given the organism's adaptation to varying oxygen conditions

  • Comparison of modifications between strains with different invasive potential

  • Investigation of PTM changes during oxidative stress response

• Validation methods:

  • Site-directed mutagenesis of modified residues

  • Functional assays comparing wild-type and modification-site mutants

  • Structural analysis of modification impact on protein conformation

This comprehensive approach would reveal how PTMs regulate IF-2 function in response to the changing environments encountered during C. concisus infection and colonization.

How can researchers differentiate between the direct effects of IF-2 mutations and broader genomic variations when studying C. concisus strain differences?

Differentiating direct IF-2 effects from broader genomic influences requires a systematic approach:

By isolating the IF-2 variable while controlling other genomic factors, researchers can establish causal relationships between specific IF-2 variants and phenotypic differences observed among C. concisus strains.

What statistical approaches are most appropriate for analyzing differences in IF-2 activity across multiple C. concisus strains?

When analyzing IF-2 activity across multiple C. concisus strains, robust statistical frameworks are essential:

• Experimental design considerations:

  • Balanced designs with sufficient biological replicates (minimum n=5 per strain)

  • Inclusion of technical replicates to quantify measurement error

  • Blocking factors to control for batch effects

• Statistical models:

  • Mixed-effects linear models accounting for fixed (strain type, growth conditions) and random effects (biological variation)

  • ANCOVA when controlling for covariates like growth rate or protein expression level

  • Non-parametric approaches when normality assumptions are violated

• Multiple testing corrections:

  • Benjamini-Hochberg procedure for controlling false discovery rate

  • Tukey's HSD for post-hoc comparisons between multiple strains

• Advanced analytical techniques:

  • Principal component analysis for multivariate activity data

  • Hierarchical clustering to identify strain groupings based on activity profiles

  • Bayesian methods for incorporating prior knowledge about IF-2 function

• Validation strategies:

  • Cross-validation of predictive models

  • Bootstrap resampling to establish robust confidence intervals

  • Independent verification experiments with distinct methodologies

Appropriate statistical analysis is particularly important given the known heterogeneity among C. concisus strains , which can introduce substantial biological variability.

How can isotope labeling techniques be applied to study the turnover and dynamics of IF-2 during C. concisus infection?

Isotope labeling techniques provide powerful tools for studying IF-2 dynamics during infection:

• Protein synthesis and turnover:

  • SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) with heavy isotope-labeled amino acids (¹³C₆-lysine, ¹³C₆-arginine)

  • Pulse-chase experiments to determine IF-2 half-life during different infection phases

  • Quantification of synthesis/degradation rates during stress response

• Structural dynamics:

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to map conformational changes

  • Limited proteolysis coupled with MS to identify flexible regions

  • Cross-linking Mass Spectrometry (XL-MS) to capture interaction interfaces

• Protein-protein interactions:

  • Isotope-labeled crosslinking reagents to capture transient interactions

  • Proximity-dependent labeling with isotope-coded tags

  • Co-immunoprecipitation with isotope-labeled antibodies

• In vivo applications:

  • ¹⁵N-labeled bacterial cultures for infection experiments

  • Tissue sampling at various timepoints for MS analysis

  • Comparison between strains with different invasive potential

These techniques should be applied within physiologically relevant models that recapitulate the intestinal environment, including appropriate oxygen tension and pH conditions that C. concisus would naturally encounter .

What are the most common challenges in expressing and purifying recombinant C. concisus IF-2, and how can they be overcome?

Each of these challenges requires a tailored approach, and often multiple strategies must be combined for successful recombinant production of functional C. concisus IF-2.

What cell-based assays can measure the impact of IF-2 variants on C. concisus virulence and host cell interactions?

Cell-based assays to evaluate IF-2 variants' impact on C. concisus virulence should include:

• Adhesion and invasion assays:

  • Gentamicin protection assay to quantify bacterial internalization

  • Differential immunofluorescence staining to distinguish adherent from invasive bacteria

  • Comparison between C. concisus strains from chronic and acute intestinal diseases

• Host cell response measurements:

  • ELISA quantification of cytokine production (IL-12, IFN-γ)

  • Western blot analysis of NF-κB activation and immunoproteasome induction

  • Flow cytometry assessment of host cell apoptosis/pyroptosis

• Barrier function assessment:

  • Transepithelial electrical resistance (TEER) measurements

  • FITC-dextran permeability assays

  • Immunostaining for tight junction proteins

• Translation impact analysis:

  • Puromycin incorporation to measure global protein synthesis

  • Polysome profiling to assess translation efficiency

  • Metabolic labeling with ³⁵S-methionine

• Advanced models:

  • Co-culture systems with immune and epithelial cells

  • Intestinal organoids from patient biopsies

  • Microfluidic gut-on-chip platforms

These assays should be conducted using isogenic strains differing only in IF-2 sequence to directly attribute phenotypic differences to IF-2 variants.

What are the best approaches for creating conditional knockdowns or modifications of the infB gene in C. concisus?

Creating conditional modifications of the essential infB gene requires specialized approaches:

• Inducible expression systems:

  • Engineer a merodiploid strain with native infB and an inducible copy

  • Use tetracycline-responsive or iron-regulated promoters

  • CRISPR-Cas9 targeting of native copy after establishing inducible expression

• Post-translational control:

  • Fusion of degron tags (auxin-inducible or temperature-sensitive) to IF-2

  • Small-molecule induced protein destabilization

  • Split protein complementation systems

• Targeted mutagenesis:

  • Recombineering with single-stranded DNA oligonucleotides

  • Lambda Red recombination system adapted for C. concisus

  • Allelic exchange vectors with counterselectable markers

• Considerations for C. concisus:

  • Exploit natural transformation ability of some strains

  • Adapt methylation patterns to overcome restriction barriers

  • Develop electroporation protocols optimized for C. concisus

• Validation strategies:

  • qRT-PCR to confirm transcriptional changes

  • Western blotting to verify protein levels

  • Growth curves to assess physiological impact

  • Ribosome profiling to measure translation effects

Each approach must be carefully validated, as even partial reduction of IF-2 function may cause pleiotropic effects that complicate interpretation of results.

How can researchers effectively model the interaction between C. concisus IF-2 and the human intestinal environment?

Modeling C. concisus IF-2 interactions with the intestinal environment requires systems that recapitulate key physiological features:

• Advanced culture systems:

  • Intestinal organoids derived from human stem cells

  • Microfluidic gut-on-chip platforms with peristaltic movement

  • Transwell co-cultures with differentiated intestinal epithelium

• Environmental parameter control:

  • Oxygen gradient systems (aerobic to anaerobic) reflecting intestinal conditions

  • pH variation (5.5-7.5) mimicking different intestinal regions

  • Inclusion of physiological concentrations of bile acids, mucins, and short-chain fatty acids

• Host-pathogen interaction components:

  • Addition of relevant immune cells (macrophages, dendritic cells)

  • Incorporation of cytokines found in inflammatory conditions (IL-12, IFN-γ)

  • Introduction of commensal bacterial populations

• Analytical approaches:

  • Real-time monitoring of bacterial gene expression using reporter constructs

  • Proteomic analysis of IF-2 modifications under varying conditions

  • Microscopy to track bacterial localization and host cell interactions

• Comparative studies:

  • Parallel testing of C. concisus strains from chronic versus acute intestinal diseases

  • Analysis of wild-type versus IF-2 variant strains

  • Evaluation under healthy versus inflammatory conditions

These complex models provide more physiologically relevant contexts for studying how IF-2 contributes to C. concisus adaptation and virulence in the human intestinal environment.