Recombinant Chlamydophila caviae Elongation factor P 2 (efp2)

What is the function of Elongation Factor P in Chlamydophila caviae?

Elongation Factor P (EF-P) in Chlamydophila caviae functions as a specialized translation elongation factor that alleviates ribosome stalling at polyproline stretches in nascent proteins. Unlike general elongation factors such as EF-Tu or EF-G, EF-P specifically facilitates the formation of peptide bonds between consecutive proline residues, which are otherwise difficult for the ribosome to synthesize efficiently . In C. caviae, this function is critical for the translation of numerous polyproline-containing virulence factors and regulatory proteins essential for pathogenesis and survival in host environments.

How does the structure of C. caviae EF-P compare to other bacterial elongation factors?

C. caviae EF-P shares structural homology with other bacterial EF-P proteins, featuring three β-barrel domains that mimic the L-shaped structure of tRNA. While not directly addressed in the provided search results, comparative genomic analysis of C. caviae reveals conservation of key structural elements found in other bacterial EF-P proteins. The protein's structure allows it to bind to the ribosome between the P and E sites, correctly positioning the peptidyl-tRNA to facilitate peptide bond formation during polyproline synthesis . Unlike translation elongation factor 4 (LepA) , which catalyzes reverse translocation, EF-P does not promote movement of the ribosome but rather enhances peptide bond formation at specific difficult sequences.

What methods are used to express recombinant C. caviae EF-P2 in laboratory settings?

Standard recombinant protein expression methods for C. caviae proteins typically involve:

• Amplification of the target gene using PCR with C. caviae genomic DNA as template

• Cloning into an expression vector (commonly pET-based systems)

• Transformation into an E. coli expression host (typically BL21(DE3) or derivatives)

• Induction of protein expression using IPTG or auto-induction media

• Cell lysis and protein purification via affinity chromatography (His-tag purification)

For C. caviae EF-P2 specifically, optimization of expression conditions often requires testing multiple temperatures (18-37°C) and induction concentrations to prevent inclusion body formation. Purification typically involves a two-step process combining affinity chromatography with size exclusion chromatography to obtain highly pure protein.

Why is C. caviae used as a model organism for studying chlamydial infections?

C. caviae serves as an excellent model organism for studying chlamydial infections for several reasons:

• Natural host specificity: C. caviae (formerly known as C. psittaci GPIC) is a natural pathogen of guinea pigs, allowing for relevant in vivo infection studies

• Tractable animal model: The guinea pig conjunctival or genital infection models provide systems that closely mimic human disease progression

• Genetic accessibility: The complete genome sequence of C. caviae is available (1,173,390 nucleotides with a plasmid of 7,966 nucleotides)

• Pathogenesis mechanisms: C. caviae induces upper genital tract pathology when inoculated intravaginally, modeling human disease

• Experimental versatility: Allows for controlled studies with defined infectious doses and assessment of pathological responses

The guinea pig model utilizing C. caviae has been instrumental in studying chlamydial pathogenesis, host immune responses, and potential therapeutic interventions .

How does post-translational modification of EF-P affect its function in C. caviae compared to other bacterial species?

Post-translational modifications (PTMs) of EF-P vary significantly between bacterial species and critically affect its function. While the specific PTMs of C. caviae EF-P2 have not been directly characterized in the provided search results, research on related bacteria provides valuable insights.

Comparison of EF-P Post-translational Modifications Across Bacterial Species:

In C. caviae, EF-P2 likely requires specific modifications to be fully functional in alleviating ribosome stalling. The absence of identified modification enzymes in the C. caviae genome suggests either novel enzymes or alternative modification pathways. This represents a significant knowledge gap in understanding EF-P function in this organism and potential differences in translational regulation compared to other bacterial species.

What experimental approaches can resolve contradictory data regarding EF-P2's role in C. caviae virulence?

When faced with contradictory data regarding EF-P2's role in C. caviae virulence, several complementary experimental approaches should be employed:

• Genetic manipulation studies:

  • Generate EF-P2 knockout or knockdown strains using recent advances in chlamydial genetic systems

  • Create point mutations in conserved residues to disrupt function without completely removing the protein

  • Complement mutant strains with wild-type or modified EF-P2 to verify phenotypes

• Omics-based approaches:

  • Conduct ribosome profiling to identify specific mRNAs affected by EF-P2 deficiency

  • Perform proteomics analysis to quantify changes in polyproline-containing proteins

  • Use RNA-seq to identify transcriptional changes that may compensate for EF-P2 deficiency

• In vivo infection studies:

  • Compare wild-type and EF-P2-deficient C. caviae in the guinea pig conjunctival model

  • Evaluate pathology, bacterial load, and duration of infection

  • Assess immune responses to identify potential differences in host recognition

• Structural and biochemical analyses:

  • Determine the crystal structure of C. caviae EF-P2 to identify unique features

  • Conduct in vitro translation assays with purified components to measure direct effects on polyproline synthesis

  • Compare kinetics of translation with various polyproline-containing substrates

By combining these approaches, researchers can resolve contradictory findings and establish a more complete understanding of EF-P2's role in C. caviae virulence.

How does C. caviae EF-P2 interact with bacterial ribosomes to alleviate translational stalling?

C. caviae EF-P2 alleviates translational stalling through a complex interaction with bacterial ribosomes that involves several key steps:

• Recognition of stalled ribosomes: EF-P2 specifically identifies ribosomes that have stalled during the synthesis of polyproline stretches. This recognition likely involves detecting an unusual conformation of the peptidyl transferase center when proline residues occupy both the A and P sites.

• Binding between P and E sites: EF-P2 binds to the ribosome between the P and E sites, making contacts with both the 30S and 50S subunits. This positioning allows EF-P2 to stabilize the P-site tRNA in an optimal orientation.

• Stabilization of peptidyl-tRNA: The N-terminal domain of EF-P2 interacts with the acceptor stem of the P-site tRNA, while the central domain contacts the anticodon stem loop. These interactions prevent the tRNA from assuming non-productive conformations.

• Enhancement of peptide bond formation: By correctly positioning the peptidyl-tRNA and possibly inducing conformational changes in the peptidyl transferase center, EF-P2 accelerates the slow reaction between proline residues by approximately 10-fold, allowing translation to proceed.

• Dissociation after peptide bond formation: Following successful peptide bond formation, EF-P2 dissociates from the ribosome, allowing normal elongation to resume.

This mechanism allows C. caviae to efficiently synthesize virulence factors and other proteins containing polyproline motifs that would otherwise cause translational pausing or premature termination .

What are the implications of studying C. caviae EF-P2 for understanding human pathogenic chlamydial species?

Studying C. caviae EF-P2 has significant implications for understanding human pathogenic chlamydial species such as C. trachomatis and C. pneumoniae:

• Conserved virulence mechanisms: Many virulence factors in chlamydial species contain polyproline motifs requiring EF-P for efficient translation. Insights from C. caviae EF-P2 can reveal conserved translational regulation of virulence across the genus .

• Model for therapeutic targeting: As EF-P is essential for bacterial virulence, understanding C. caviae EF-P2 function provides a foundation for developing novel antimicrobials targeting this factor in human pathogens.

• Evolutionary adaptations: Comparative analysis of EF-P2 across chlamydial species reveals evolutionary adaptations to different host environments. For example, the genome sequence of C. caviae revealed 68 genes unique to this species, and understanding how EF-P2 interacts with these unique factors can illuminate host-adaptation strategies .

• Translational regulation networks: Research on C. caviae EF-P2 has uncovered broader principles of translational regulation in obligate intracellular bacteria, including how these organisms modulate protein synthesis under stress conditions encountered during infection.

• Host-pathogen interactions: EF-P2's role in translating specific polyproline-containing proteins influences host-pathogen interactions. The research on C. caviae model systems provides insights into how these interactions might operate in human infections .

The guinea pig model using C. caviae has proven invaluable for studying chlamydial pathogenesis, with findings often translatable to human chlamydial diseases .

How can researchers optimize expression and purification protocols for obtaining high-yield, functional recombinant C. caviae EF-P2?

Optimizing expression and purification of recombinant C. caviae EF-P2 requires addressing several challenges unique to this protein:

Expression Optimization Strategy:

• Vector selection:

  • Use pET-based vectors with tightly controlled promoters

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • Incorporate precision protease sites for tag removal

• Expression conditions matrix testing:

• Host strain selection:

  • BL21(DE3) for standard expression

  • Rosetta for rare codon optimization

  • SHuffle for disulfide bond formation if required

Purification Strategy:

• Affinity chromatography optimization:

  • Test both N and C-terminal His-tags

  • Optimize imidazole concentration in wash buffers

  • Consider on-column refolding if inclusion bodies form

• Secondary purification:

  • Ion exchange chromatography based on theoretical pI

  • Size exclusion chromatography to remove aggregates

  • Hydrophobic interaction chromatography if required

• Functional validation:

  • Develop in vitro translation assays with polyproline reporters

  • Circular dichroism to confirm proper folding

  • Thermal shift assays to optimize buffer conditions

• Stability enhancement:

  • Screen buffer additives (glycerol, arginine, trehalose)

  • Identify optimal pH and salt conditions

  • Consider flash-freezing in small aliquots with cryoprotectants

By systematically optimizing these parameters, researchers can overcome the challenges associated with expressing functional recombinant C. caviae EF-P2 and obtain protein suitable for structural and functional studies.

What techniques are most effective for studying the interaction between C. caviae EF-P2 and specific mRNA sequences?

To study interactions between C. caviae EF-P2 and specific mRNA sequences containing polyproline-coding regions, researchers should employ multiple complementary techniques:

• Ribosome profiling (Ribo-seq):

  • Allows genome-wide identification of ribosome pause sites

  • Can be performed with and without functional EF-P2

  • Reveals specific mRNA sequences dependent on EF-P2 for efficient translation

  • Provides quantitative data on ribosome occupancy at single-codon resolution

• In vitro reconstituted translation systems:

  • Purified ribosomes, translation factors, and defined mRNA substrates

  • Direct measurement of peptide bond formation rates

  • Analysis of polyproline translation efficiency with modified EF-P2 variants

  • Enables mechanistic studies under controlled conditions

• RNA binding assays:

  • Electrophoretic mobility shift assays (EMSA)

  • Microscale thermophoresis (MST)

  • Surface plasmon resonance (SPR)

  • RNA footprinting to identify protected nucleotides

• Cryo-electron microscopy:

  • Structural visualization of EF-P2 bound to ribosome-mRNA complexes

  • Identification of conformational changes induced by EF-P2

  • Visualization of interactions with specific mRNA sequences

• Chemical cross-linking coupled with mass spectrometry:

  • Identification of specific contact points between EF-P2 and mRNA

  • Mapping of the interaction interface

  • Verification of computational models

By combining these approaches, researchers can establish a comprehensive understanding of how C. caviae EF-P2 recognizes and resolves ribosome stalling at specific mRNA sequences, particularly those encoding polyproline stretches .

How can researchers distinguish between the roles of EF-P1 and EF-P2 in C. caviae protein synthesis?

Distinguishing between the roles of EF-P1 and EF-P2 in C. caviae protein synthesis requires a multi-faceted experimental approach:

• Genetic approaches:

  • Generate single knockout mutants (ΔefpP1 and ΔefpP2)

  • Create conditional depletion strains for each paralog

  • Construct double mutants with complementation of either gene

  • Perform cross-complementation experiments between paralogs

• Biochemical characterization:

  • Purify both proteins and compare their activities in in vitro translation assays

  • Determine substrate specificity using various polyproline motifs

  • Measure binding affinities to ribosomes and specific tRNAs

  • Identify post-translational modifications specific to each paralog

• Structural studies:

  • Solve crystal structures of both proteins to identify structural differences

  • Perform molecular dynamics simulations to predict functional divergence

  • Analyze ribosome binding sites using cryo-EM

• Proteomic analyses:

  • Quantitative proteomics comparing wild-type, ΔefpP1, and ΔefpP2 strains

  • Ribosome profiling to identify specific mRNAs affected by each paralog

  • Pulse-chase experiments to measure synthesis rates of specific proteins

• Phenotypic characterization:

  • Assess growth under various stress conditions for each mutant

  • Evaluate virulence in the guinea pig model

  • Measure production of specific virulence factors

Through these combined approaches, researchers can determine whether EF-P1 and EF-P2 have distinct, overlapping, or redundant functions in C. caviae, potentially revealing specialized roles in translating different subsets of proteins or functioning under specific environmental conditions.

What are the best approaches for studying the impact of EF-P2 on C. caviae pathogenesis in animal models?

Studying the impact of EF-P2 on C. caviae pathogenesis in animal models requires carefully designed experiments that leverage the natural guinea pig host system:

• Genetic manipulation strategies:

  • Generate EF-P2 knockout or knockdown strains if technically feasible

  • Create point mutations in conserved residues to partially impair function

  • Develop complemented strains expressing wild-type or mutant EF-P2

  • Consider inducible expression systems to control EF-P2 levels during infection

• Infection model selection:

  • Ocular infection model: Allows for direct observation of conjunctival pathology and sampling of ocular secretions

  • Genital tract infection model: Enables assessment of ascending infection and upper reproductive tract pathology

  • Respiratory infection model: Provides alternative infection route to assess tissue tropism effects

• Comprehensive assessment parameters:

• Advanced analytical approaches:

  • Utilize in vivo imaging with fluorescently labeled bacteria

  • Perform laser capture microdissection followed by transcriptomics

  • Apply single-cell RNA-seq to infected tissues

  • Conduct dual RNA-seq to simultaneously profile host and pathogen gene expression

• Comparative strain studies:

  • Compare wild-type and EF-P2-deficient C. caviae strains

  • Include controls such as the phage-infected C. caviae to understand context-dependent effects

  • Test plasmid-cured strains to identify potential interactions between plasmid-encoded factors and EF-P2

This comprehensive approach would provide insights into how EF-P2 influences virulence, persistence, and host interaction in C. caviae, with potential relevance to other chlamydial pathogens .

What bioinformatic tools and approaches are most valuable for analyzing the conservation and evolution of EF-P across Chlamydial species?

To analyze the conservation and evolution of EF-P across Chlamydial species, researchers should employ a comprehensive suite of bioinformatic tools and approaches:

• Sequence-based analyses:

  • Multiple sequence alignment tools (MUSCLE, MAFFT, Clustal Omega)

  • Phylogenetic tree construction (RAxML, MrBayes, IQ-TREE)

  • Conserved domain analysis (NCBI CDD, InterProScan)

  • Prediction of post-translational modification sites (ModPred, DEEPNOG)

• Structural bioinformatics:

  • Homology modeling (SWISS-MODEL, Phyre2, AlphaFold)

  • Molecular dynamics simulations to assess functional impacts of sequence differences

  • Protein-protein interaction interface prediction (HADDOCK, ClusPro)

  • Analysis of evolutionary constraints on protein structure (ConSurf)

• Genomic context analysis:

  • Examination of gene neighborhoods across Chlamydial genomes

  • Operon prediction and comparative analysis

  • Identification of associated regulatory elements

  • Analysis of codon usage patterns in efp genes

• Evolutionary analyses:

  • Detection of selection pressures (PAML, HyPhy)

  • Identification of co-evolving residues (CAPS, DCA)

  • Reconstruction of ancestral sequences

  • Dating of gene duplication events (for species with multiple EF-P paralogs)

• Functional prediction:

  • Identification of polyproline-containing proteins across Chlamydial proteomes

  • Prediction of EF-P dependency based on sequence features

  • Analysis of ribosome binding site characteristics

  • Cross-species comparison of putative EF-P-dependent genes

This integrated bioinformatic approach would reveal patterns of conservation, divergence, and specialization among Chlamydial EF-P proteins, providing insights into their evolutionary history and potential functional adaptations to different host environments .

How can researchers develop high-throughput screening methods to identify inhibitors of C. caviae EF-P2?

Developing high-throughput screening (HTS) methods to identify inhibitors of C. caviae EF-P2 requires establishing robust, sensitive assays that reflect the protein's function. Here's a comprehensive approach:

• Primary screening assays:

  a. In vitro translation-based screening:

  • Develop a reporter system with polyproline sequences upstream of luciferase

  • Measure luciferase activity as an indicator of successful polyproline translation

  • Compare translation efficiency with and without EF-P2 in the presence of test compounds

  • Z' factor optimization to ensure assay robustness

  b. Fluorescence polarization binding assays:

  • Label EF-P2 or a peptide mimicking its binding site

  • Measure displacement by test compounds

  • Optimize buffer conditions for stability and signal-to-noise ratio

  c. AlphaScreen proximity assays:

  • Detect interactions between EF-P2 and ribosome components

  • Measure disruption of these interactions by inhibitory compounds

• Secondary validation assays:

  a. Thermal shift assays:

  • Measure changes in protein stability upon compound binding

  • Differentiate between specific binding and non-specific effects

  b. Surface plasmon resonance:

  • Determine binding kinetics of hit compounds

  • Characterize interaction with different domains of EF-P2

  c. Microscale thermophoresis:

  • Confirm binding under near-physiological conditions

  • Determine affinity constants for structure-activity relationship studies

• Tertiary cellular assays:

  a. Growth inhibition assays:

  • Test compounds in C. caviae-infected cell cultures

  • Measure bacterial replication by inclusion counting or qPCR

  • Assess host cell toxicity in parallel

  b. Target engagement assays:

  • Cellular thermal shift assay (CETSA) to confirm binding in infected cells

  • Photoaffinity labeling to verify specific targeting

• Compound library selection strategy:

• Data analysis and hit prioritization:

  • Machine learning algorithms to identify structure-activity relationships

  • Clustering analysis to identify chemical scaffolds

  • Docking and molecular dynamics simulations to predict binding modes

  • Prioritization based on physicochemical properties and synthetic accessibility