Recombinant Chlamydophila caviae Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 (infB) in Chlamydophila caviae?

Translation Initiation Factor IF-2 (infB) is an essential protein involved in the initiation phase of protein synthesis in Chlamydophila caviae. The infB gene codes for two distinct forms of translational initiation factor: IF2 alpha (approximately 97,300 daltons) and IF2 beta (approximately 79,700 daltons), which differ at their N-terminus with completely different N-terminal amino acid sequences. These sequence differences match perfectly with the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream . The protein plays a critical role in facilitating the attachment of the initiator tRNA to the ribosome during translation initiation. In Chlamydophila species, this factor is particularly important due to their obligate intracellular parasitic lifestyle, requiring efficient protein synthesis mechanisms.

How does Chlamydophila caviae differ from other Chlamydia species?

Chlamydophila caviae is a member of the Chlamydiaceae family, a group of pathogenic bacteria that are obligate intracellular parasites . While sharing fundamental characteristics with other Chlamydia species, C. caviae has distinct genetic and pathogenic properties. C. caviae is weakly Gram-negative, ovoid in shape, and nonmotile, similar to other Chlamydia species . A key distinguishing feature is found in the ompA gene sequences, where C. caviae samples show specific nucleotide identity patterns. For instance, some Swiss samples revealed 100% nucleotide identity with Chlamydia caviae clone NL_Conj_Li ompA gene, while others had lower nucleotide identity (98.8%) with the C. caviae GPIC reference strain . This genetic heterogeneity has implications for diagnostic specificity and evolutionary relationships within the genus.

What are the typical sources and expression systems for recombinant Chlamydophila proteins?

Recombinant Chlamydophila proteins, including the Translation initiation factor IF-2, can be expressed using several heterologous systems. The most common expression platforms include:

The choice of expression system depends on the research objectives, with E. coli being commonly used for initial characterization . When studying functional aspects that might depend on specific post-translational modifications, mammalian or baculovirus systems might be more appropriate.

What PCR methods are used to detect Chlamydophila caviae infB gene?

For the detection and identification of Chlamydophila caviae and its infB gene, researchers typically employ a multi-step PCR approach:

• Initial screening is often performed using broad-range Chlamydiaceae-specific real-time PCR assays targeting conserved regions of the 23S rRNA gene.

• For C. caviae-specific detection, researchers use specialized primers targeting the VD4 region of the ompA gene. In studies of Swiss and Dutch guinea pigs, this approach yielded positive results with mean Ct values of approximately 32.8 and 28.2, respectively .

• Confirmation can be performed through complete ompA gene PCR amplification, which allows for subsequent sequencing to confirm the species identity.

• For the specific detection of the infB gene, custom primers targeting conserved regions of the gene can be designed, followed by sequencing to confirm the presence of the target sequence.

The complete workflow should include appropriate positive and negative controls to ensure specificity and sensitivity of detection.

How can researchers differentiate between infB gene variants in Chlamydophila species?

Differentiation between infB gene variants across Chlamydophila species requires a combination of molecular techniques:

• Sequence Analysis: Complete sequencing of the infB gene allows for nucleotide-level comparison between species and strains. Similar to how researchers identified differences in the ompA gene, sequence analysis can reveal species-specific signatures in the infB gene .

• Restriction Fragment Length Polymorphism (RFLP): This technique can identify specific restriction patterns characteristic of different Chlamydophila species or variants.

• High-Resolution Melting Analysis: This post-PCR method can differentiate between closely related sequences based on their melting behavior.

• N-terminal Protein Sequencing: As demonstrated with the two forms of IF2 (alpha and beta), Edman degradation can be used to determine N-terminal amino acid sequences, which differ significantly between variants .

• Gene Fusion Constructs: Similar to the fusion constructed between the proximal half of the infB gene and the lacZ gene, researchers can create reporter constructs to study expression patterns of different variants .

These methods can be combined for comprehensive characterization of infB gene variants, providing insights into evolutionary relationships and functional differences between Chlamydophila species.

How should researchers design experiments to study infB protein function?

When designing experiments to investigate the function of the infB protein in Chlamydophila caviae, researchers should consider a fractional factorial design approach to efficiently explore multiple experimental factors. This approach is particularly valuable in the initial screening phases when numerous variables need to be assessed .

Recommended Experimental Design Strategy:

• Identify Key Variables: Determine all potential factors that might influence infB function (e.g., temperature, pH, ionic strength, cofactors, substrate concentrations).

• Implement a Two-Level Fractional Factorial Design: Instead of testing all possible combinations of factors (which would require 2^n runs for n factors), use a fractional design that focuses on main effects and two-factor interactions while assuming higher-order interactions are negligible .

• Factor Screening Phase:

  • For example, with 8 factors, instead of running 256 experiments, use a half-fraction design with 128 runs

  • Apply the sparsity of effects principle, which states that most responses are affected by a small number of main effects and lower-order interactions

• Follow-up Optimization: Once significant factors are identified, conduct more detailed experiments focused on those variables.

For unreplicated fractional factorial designs, researchers should employ appropriate analytical techniques to calculate error sums of squares since no degrees of freedom are available in the standard approach .

What expression and purification strategies yield optimal recombinant infB protein?

The expression and purification of recombinant infB protein from Chlamydophila caviae requires a systematic approach to ensure high yield and biological activity:

Recommended Expression Strategy:

• Vector Selection: Incorporate a highly efficient promoter system (T7 or tac) with appropriate regulatory elements.

• Expression Host: E. coli BL21(DE3) or derivatives are recommended for initial expression trials due to their reduced protease activity and compatibility with T7-based expression systems .

• Expression Conditions Optimization:

Purification Protocol:

• Affinity Chromatography: Incorporate a His-tag or other affinity tag for initial capture

• Ion Exchange Chromatography: For intermediate purification

• Size Exclusion Chromatography: As a final polishing step

For functional studies, it's essential to verify that both forms of the protein (IF2 alpha and IF2 beta) are being expressed and purified, as they have distinct N-terminal sequences that may affect function .

What are the best approaches for studying the structural characteristics of recombinant infB protein?

Understanding the structural features of recombinant infB protein requires a multi-technique approach:

• X-ray Crystallography: Provides high-resolution atomic structures but requires well-diffracting crystals. For infB protein:

  • Concentrate purified protein to 10-15 mg/ml

  • Screen multiple crystallization conditions (pH 6.0-8.0, various precipitants)

  • Consider co-crystallization with binding partners (ribosomal components, GTP)

• Cryo-Electron Microscopy (Cryo-EM): Particularly useful for studying infB in complex with ribosomes:

  • Prepare samples in vitrified ice

  • Can reveal conformational changes during translation initiation

• Nuclear Magnetic Resonance (NMR): Suitable for studying dynamics and smaller domains:

  • Requires isotopic labeling (15N, 13C)

  • Most effective for domains under 25 kDa

• Small-Angle X-ray Scattering (SAXS): Provides low-resolution envelope structures in solution:

  • Useful for comparing the two forms (IF2 alpha and IF2 beta)

  • Can detect conformational changes upon nucleotide binding

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Reveals regions of structural flexibility and ligand-binding sites by measuring the rate of hydrogen exchange.

How does recombination affect the evolution of the infB gene in Chlamydophila species?

The evolution of the infB gene in Chlamydophila species is significantly influenced by recombination events, which contribute to genetic diversity within the population. Recombination in this context refers to a process that results in combinations of alleles that were not present in parental gametes .

Chlamydia species can exchange DNA between different strains, making the evolution of new strains common . This genetic exchange has several implications for infB gene evolution:

• Recombination Frequency Analysis: The calculation of recombination frequency (RF) can be applied to quantify genetic exchange rates in the infB gene:

  RF = (Number of recombinant progeny / Total number of progeny) × 100%

• Linkage Analysis: For genes that are linked, recombination frequency will be less than 50%, providing insights into the genomic organization around the infB gene .

• Selective Pressure: Recombination events in the infB gene may be subject to selection based on the functional importance of the translation initiation factor:

  • Regions critical for function will show conservation

  • Regions subject to immune pressure may show higher variability

• Mosaic Gene Structures: Recombination can lead to mosaic gene structures, where different segments of the infB gene may have distinct evolutionary histories, similar to patterns observed in ompA gene sequences of C. caviae samples from different geographical locations .

Understanding these recombination patterns is essential for tracking the evolution of virulence factors and developing effective detection and treatment strategies.

What role does infB play in Chlamydophila caviae pathogenesis and host adaptation?

The Translation initiation factor IF-2 (infB) may play significant roles in Chlamydophila caviae pathogenesis and host adaptation beyond its canonical function in translation initiation:

• Differential Expression of IF2 Forms: The infB gene codes for two forms (alpha and beta) with distinct N-terminal sequences . This may provide regulatory flexibility during different stages of infection or in response to host conditions.

• Host-Pathogen Protein Interactions: As a translation factor, infB may interact with host ribosomes or translation machinery, potentially contributing to host-specific adaptation. The distinct prevalence of C. caviae in guinea pigs (2.7% positivity in Swiss samples) suggests host specificity that may be mediated by molecular factors including infB .

• Stress Response: Translation initiation factors can play roles in bacterial stress responses, which would be particularly important for an obligate intracellular parasite like C. caviae during host cell invasion and persistence.

• Potential Zoonotic Implications: Given that C. caviae has been identified in human zoonotic cases , understanding how infB contributes to cross-species infection is valuable. The comparison of infB sequences from clinical isolates with those from animal reservoirs could reveal adaptations important for zoonotic transmission.

• Vaccine and Therapeutic Target Potential: Due to its essential role in bacterial protein synthesis and potential surface exposure, infB could represent a target for vaccine development or antimicrobial interventions.

How can researchers use recombinant infB protein to develop diagnostic tests for Chlamydophila caviae infections?

Developing diagnostic tests using recombinant infB protein requires a strategic approach:

• Antibody Development:

  • Use purified recombinant infB protein to generate specific polyclonal or monoclonal antibodies

  • Validate antibody specificity against other Chlamydophila species

  • Develop ELISA or immunofluorescence assays for detecting infB in clinical samples

• Multiplex PCR Systems:

  • Design primers specific to conserved regions of the infB gene

  • Combine with primers for other targets (e.g., ompA) for increased sensitivity and specificity

  • Validate against clinical samples with known Ct values similar to those observed in studies of C. caviae in guinea pigs (mean Ct values of 32.8 and 28.2)

• Recombinant Protein Microarrays:

  • Immobilize recombinant infB alongside other Chlamydophila antigens

  • Screen sera for antibody responses to multiple antigens simultaneously

  • Identify signature patterns of reactivity specific to C. caviae infection

• Point-of-Care Testing:

  • Develop lateral flow assays using infB-specific antibodies

  • Optimize for use in veterinary settings for rapid diagnosis in guinea pigs and other susceptible animals

• Validation Parameters:

How does infB from Chlamydophila caviae compare to similar proteins in other bacterial species?

Translation initiation factor IF-2 shows both conservation and diversity across bacterial species. Comparing infB from Chlamydophila caviae with homologs from other bacteria reveals important structural and functional relationships:

• Sequence Homology:

  • Core functional domains show high conservation across bacterial species

  • N-terminal regions display greater variability, similar to the difference observed between IF2 alpha and IF2 beta forms

  • Species-specific insertions/deletions may reflect adaptation to particular ecological niches

• Domain Architecture:

  • All bacterial IF2 proteins contain GTP-binding domains and ribosome-binding domains

  • Chlamydial IF2 may contain unique domains related to their obligate intracellular lifestyle

  • The presence of two forms (alpha and beta) with different N-termini appears to be a conserved feature in some bacterial groups

• Evolutionary Considerations:

  • Phylogenetic analysis based on infB sequences can complement 16S rRNA-based taxonomy

  • Horizontal gene transfer events can be detected through incongruence between infB and species phylogenies

  • Selection pressures may differ between free-living bacteria and obligate intracellular parasites like Chlamydophila

• Functional Differences:

  • Species-specific differences in infB may reflect adaptation to different translation regulation requirements

  • The relative abundance of the two IF2 forms (alpha and beta) may vary between species depending on growth conditions and stress responses

What protein-protein interactions does infB engage in during the translation initiation process?

The Translation initiation factor IF-2 engages in multiple protein-protein interactions during translation initiation:

• Interactions with Ribosomal Components:

  • Binds to the 30S ribosomal subunit during the formation of the 30S pre-initiation complex

  • Interacts with the 50S subunit during the formation of the 70S initiation complex

  • Specific contacts with ribosomal proteins and rRNA molecules stabilize these interactions

• Interactions with Other Translation Factors:

  • Cooperates with IF1 and IF3 during initiation complex formation

  • IF3 promotes the binding of IF2 to the 30S subunit

  • IF1 enhances the activity of IF2 by optimizing its position on the ribosome

• Interactions with tRNA:

  • Specifically recognizes and binds to initiator tRNA (fMet-tRNA^fMet)

  • Positions the initiator tRNA in the P-site of the ribosome

  • GTP hydrolysis by IF2 leads to conformational changes that adjust the position of the initiator tRNA

• Interactions with mRNA:

  • Indirectly interacts with mRNA during the positioning of the start codon in the P-site

  • May contribute to the recognition of the Shine-Dalgarno sequence in prokaryotic mRNAs

• Experimental Approaches to Study These Interactions:

  • Pull-down assays using tagged recombinant infB

  • Surface plasmon resonance to measure binding kinetics

  • Cryo-EM to visualize the structural arrangement of the complexes

  • Cross-linking coupled with mass spectrometry to identify interaction interfaces

What are common challenges in expressing recombinant Chlamydophila caviae infB and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant Chlamydophila caviae infB protein:

• Protein Solubility Issues:

  • Challenge: infB proteins often form inclusion bodies when overexpressed

  • Solution: Lower expression temperature (16-18°C), co-express with chaperones (GroEL/ES, trigger factor), or use solubility tags (SUMO, MBP)

• Codon Usage Bias:

  • Challenge: Chlamydophila has different codon preferences than E. coli

  • Solution: Use codon-optimized synthetic genes or express in E. coli strains supplying rare tRNAs (e.g., Rosetta strains)

• Protein Toxicity:

  • Challenge: Expression of infB may be toxic to the host cell

  • Solution: Use tightly regulated expression systems (pET with T7 lysozyme, or araBAD promoter), or express as separate domains

• Purification Difficulties:

  • Challenge: Distinguishing between the alpha and beta forms

  • Solution: Design constructs that express each form separately, or develop chromatography protocols that can separate the two forms

• Protein Stability:

  • Challenge: infB protein may be unstable after purification

  • Solution: Optimize buffer conditions (add glycerol, reduce salt), identify stabilizing ligands (GTP, GDP), or use thermostability assays to identify stabilizing conditions

• Experimental Optimization Table:

How can researchers optimize PCR protocols for detecting infB gene variants in clinical samples?

Optimizing PCR protocols for detecting infB gene variants in clinical samples requires addressing several key considerations:

• Sample Preparation:

  • Challenge: Clinical samples often contain PCR inhibitors

  • Solution: Use specialized extraction kits designed for clinical specimens, include internal control to detect inhibition

• Primer Design:

  • Challenge: Balancing specificity with detection of variants

  • Solution: Design primers in conserved regions flanking variable segments, use degenerate bases at positions of known variation

• Amplification Optimization:

  • Challenge: Low copy number in clinical samples

  • Solution: Implement touchdown PCR protocols, optimize magnesium concentration, use high-fidelity polymerases

• Detection Sensitivity:

  • Challenge: Achieving low detection limits similar to those needed for Chlamydiaceae detection (Ct values around 32.8)

  • Solution: Implement nested PCR or real-time PCR with specific probes, optimize cycle parameters

• Specificity Considerations:

  • Challenge: Cross-reactivity with other Chlamydophila species

  • Solution: Include multiple targets (similar to targeting both Chlamydiaceae and specific C. caviae genes like ompA)

• Protocol Optimization Table:

• Validation Against Clinical Standards:

  • Test optimized protocol against reference samples with known Ct values

  • Establish standard curves for quantification

  • Compare performance with published methods for detecting Chlamydiaceae (2.3% positivity rate)

How might infB protein be utilized in developing vaccines against Chlamydophila infections?

Translation initiation factor IF-2 presents several opportunities for vaccine development against Chlamydophila infections:

• Antigen Presentation Strategies:

  • Recombinant infB protein could be used as a subunit vaccine component

  • DNA vaccines encoding infB could elicit both humoral and cell-mediated immunity

  • Epitope mapping can identify immunodominant regions specific to Chlamydophila caviae

• Adjuvant Considerations:

  • Alum-based adjuvants promote antibody responses

  • TLR agonists could enhance T-cell responses against this intracellular pathogen

  • Liposomal formulations may improve antigen delivery and presentation

• Cross-Protection Potential:

  • Identify conserved epitopes across Chlamydophila species

  • Test protection against multiple strains showing different ompA gene sequences (similar to the variations observed in Swiss and Dutch guinea pig isolates)

  • Evaluate protection against zoonotic transmission

• Delivery Systems:

  • Mucosal delivery systems may be particularly effective against Chlamydophila

  • Prime-boost strategies combining different vaccine platforms

  • Nanoparticle-based delivery for improved stability and immunogenicity

• Evaluation Metrics:

  • Antibody titers against both native and recombinant infB

  • T-cell responses (IFN-γ ELISPOT, intracellular cytokine staining)

  • Challenge studies in appropriate animal models (guinea pigs show natural susceptibility with 2.7% positivity rate)

  • Protection against different infection routes (conjunctival, respiratory, urogenital)

What are promising research directions for understanding the regulation of infB expression in Chlamydophila caviae?

Understanding the regulation of infB expression in Chlamydophila caviae represents an important research frontier:

• Transcriptional Regulation:

  • Characterize promoter elements controlling infB expression

  • Identify transcription factors that regulate infB during different developmental stages

  • Study the impact of stress conditions on promoter activity

• Post-transcriptional Regulation:

  • Investigate potential RNA secondary structures affecting mRNA stability

  • Identify small RNAs that might regulate infB expression

  • Study the role of RNases in controlling infB mRNA levels

• Translational Control:

  • Examine the mechanisms governing differential expression of IF2 alpha versus IF2 beta

  • Similar to what is observed in other bacteria, investigate how the two translational initiation sites in the infB gene are utilized

  • Study auto-regulation mechanisms whereby IF2 might regulate its own synthesis

• Developmental Regulation:

  • Characterize infB expression patterns during the biphasic developmental cycle

  • Compare expression in elementary bodies versus reticulate bodies

  • Identify signals that modulate expression during host cell adaptation

• Experimental Approaches:

  • Develop reporter gene fusions similar to the infB-lacZ construct described in result

  • Implement RNA-seq to profile transcriptome changes under various conditions

  • Use ribosome profiling to study translational efficiency of infB mRNA

  • Apply CRISPR interference (CRISPRi) to modulate infB expression and study phenotypic consequences

These research directions would significantly advance our understanding of the basic biology of Chlamydophila caviae and potentially reveal new targets for therapeutic intervention.

What are the most significant unanswered questions about infB in Chlamydophila caviae?

Despite advances in our understanding of Translation initiation factor IF-2 in Chlamydophila caviae, several significant questions remain unanswered:

• Structural Determinants of Function:

  • How do the structural differences between IF2 alpha and IF2 beta forms affect their function?

  • What is the three-dimensional structure of C. caviae infB and how does it compare to homologs?

  • Which domains are responsible for species-specific interactions?

• Regulatory Mechanisms:

  • What controls the relative expression of the two forms of IF2 during the developmental cycle?

  • How is infB expression coordinated with other components of the translation machinery?

  • Do environmental signals in the host modulate infB expression?

• Role in Pathogenesis:

  • Does infB contribute directly or indirectly to virulence?

  • Is there a correlation between infB sequence variants and disease severity?

  • Could targeting infB reduce bacterial fitness during infection?

• Host-Pathogen Interactions:

  • Does infB interact with host cell components beyond the translation machinery?

  • Could such interactions contribute to host tropism or zoonotic potential?

  • Is infB recognized by the host immune system during natural infection?

• Evolution and Adaptation:

  • How has the infB gene evolved across Chlamydophila species and strains?

  • What selective pressures drive infB evolution?

  • Does recombination play a significant role in generating infB diversity?

Addressing these questions will require interdisciplinary approaches combining structural biology, molecular genetics, immunology, and evolutionary analysis.

How might advances in protein engineering impact research on recombinant infB protein?

Recent and anticipated advances in protein engineering present exciting opportunities for research on recombinant infB protein:

• Directed Evolution Approaches:

  • Error-prone PCR could generate infB variants with enhanced stability or function

  • Phage display techniques might identify variants with novel interaction partners

  • Continuous evolution systems could optimize infB for specific research applications

• Protein Design and Modeling:

  • AI-based tools like AlphaFold2 can predict structures of infB domains and complexes

  • Computational design could engineer infB variants with desired properties

  • Structure-guided mutagenesis can probe function with greater precision

• Synthetic Biology Applications:

  • Designer infB proteins could be created with novel regulatory properties

  • Orthogonal translation systems might be developed based on engineered infB

  • Biosensors could be developed using infB-based molecular switches

• Advanced Expression Systems:

  • Cell-free protein synthesis could rapidly produce infB variants for screening

  • Non-canonical amino acid incorporation might enable novel functional studies

  • Minimized bacterial genomes could optimize production of challenging proteins

• Biophysical Characterization:

  • Single-molecule techniques can reveal dynamics of infB during translation initiation

  • Advanced mass spectrometry methods can map interaction surfaces with high precision

  • Cryo-EM could visualize conformational changes during the functional cycle