Recombinant Coxiella burnetii Ribosome-recycling factor (frr)

What is Ribosome-recycling factor (frr) and what is its function in Coxiella burnetii?

Ribosome-recycling factor (frr) in Coxiella burnetii is responsible for the release of ribosomes from messenger RNA at the termination of protein biosynthesis. It belongs to the RRF family of proteins which are essential components of bacterial translational machinery. The primary function of frr is to dissociate the ribosome from mRNA after completion of protein synthesis, thereby allowing the ribosomes to be recycled for subsequent rounds of translation. This recycling process significantly increases the efficiency of protein translation by making ribosomes available for new synthesis cycles .

In the context of C. burnetii biology, frr functions as part of the core translational machinery. While not directly identified as a virulence factor like the QpH1 plasmid, efficient protein synthesis is crucial for bacterial survival and adaptation within host cells, particularly given C. burnetii's lifestyle as an intracellular pathogen that replicates within acidified phagolysosome-like parasitophorous vacuoles (CCVs) in mononuclear phagocytes .

How can recombinant C. burnetii frr be effectively expressed and purified for research applications?

Recombinant expression of C. burnetii frr can be accomplished through several expression systems, with the baculovirus system being specifically mentioned in the available data . This approach offers advantages for expressing potentially toxic bacterial proteins in eukaryotic cells.

Expression Systems Comparison for C. burnetii frr:

For purification, the following methodological approach is recommended:

• Initial Clarification: After cell lysis, centrifugation to remove cell debris (10,000-20,000 × g for 30 minutes).

• Affinity Chromatography: If the recombinant protein includes an affinity tag (often determined during manufacturing) , use appropriate affinity resin (His-tag, GST, etc.).

• Quality Assessment: SDS-PAGE to confirm purity (target >85% as indicated in commercial preparations) .

• Storage Preparation: Aliquot in appropriate buffer and add glycerol (5-50% final concentration) before storing at -20°C or -80°C for extended storage .

For optimal results in downstream applications, it's recommended to avoid repeated freeze-thaw cycles and to work with aliquots at 4°C for up to one week .

What are the appropriate storage and handling conditions for maintaining recombinant C. burnetii frr activity?

Proper storage and handling of recombinant C. burnetii frr is critical for maintaining its structural integrity and biological activity. Based on standard protocols for similar proteins and specific information from the search results, the following guidelines are recommended:

• Long-term Storage: Store the lyophilized or concentrated protein at -20°C for routine storage, or at -80°C for extended preservation .

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% to prevent freeze-thaw damage

  • Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Working Storage: For active experiments, store working aliquots at 4°C for up to one week to maintain activity .

• Stability Considerations: Avoid repeated freezing and thawing as this can lead to protein denaturation and loss of functional activity. Each freeze-thaw cycle can result in approximately 10-30% loss of activity depending on protein stability.

• Quality Monitoring: Periodically verify protein integrity using methods such as SDS-PAGE (target purity >85%) or activity assays if conducting functional studies.

These storage and handling protocols aim to preserve the native conformation and functional capacity of the recombinant frr protein, ensuring reliable and reproducible experimental results.

How does C. burnetii frr compare to RRF proteins in other bacterial species?

While the search results don't provide a direct comparison between C. burnetii frr and RRF proteins from other bacterial species, we can infer several important points based on the available information and general knowledge about RRF proteins:

Functional Conservation:
The primary function of releasing ribosomes from mRNA at termination and increasing translational efficiency is likely conserved across bacterial species . This functional conservation reflects the essential nature of the translation termination and ribosome recycling process in all bacteria.

Species-Specific Adaptations:
C. burnetii's unique lifestyle as an intracellular pathogen that thrives in acidified phagolysosome-like vacuoles may have driven adaptive changes in its frr sequence or regulation. These adaptations could potentially relate to protein stability under acidic conditions or optimization for the specific translational needs of C. burnetii in its distinctive niche.

Evolutionary Context:
As a member of the gamma proteobacteria (order Legionellales), C. burnetii's frr likely shares higher sequence similarity with closely related bacterial species than with more distantly related bacteria. This phylogenetic relationship could be valuable for researchers investigating the evolution of translation machinery in diverse bacterial lineages.

Comparative studies examining sequence alignments, structural modeling, and functional analyses would be necessary to fully characterize the unique aspects of C. burnetii frr relative to other bacterial RRFs.

What experimental approaches can be used to study frr's role in C. burnetii protein synthesis?

Investigating the role of frr in C. burnetii protein synthesis requires sophisticated experimental approaches that address both its biochemical function and biological significance. Several methodological strategies can be employed:

In vitro Translation Systems:

• Reconstituted Translation Assay: Develop a purified translation system using C. burnetii ribosomes, translation factors, and recombinant frr to directly measure recycling activity.

• Ribosome Binding Assays: Use techniques such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify binding kinetics between frr and C. burnetii ribosomes.

• Cryo-EM Analysis: Determine structural interactions between frr and C. burnetii ribosomes during different stages of translation termination and recycling.

Cellular and Genetic Approaches:

• Conditional Expression Systems: Since frr is likely essential, develop a conditional expression system in C. burnetii to modulate frr levels and observe effects on translation and growth.

• Shuttle Vector Complementation: Similar to the approach used with the QpH1 plasmid , develop shuttle vectors to express wild-type or mutant frr proteins to assess functional complementation.

• Fluorescent Tagging: Use fluorescent protein fusions to track frr localization during different growth phases in C. burnetii.

Comparative Analyses:

• Cross-Species Complementation: Test whether frr proteins from other bacterial species can functionally complement C. burnetii frr, potentially using the plasmid-based approaches described for QpH1 studies .

• Mutational Analysis: Create point mutations in conserved residues to identify regions critical for C. burnetii frr function.

Transcriptomics and Proteomics:

• Ribosome Profiling: Apply ribosome profiling (Ribo-seq) under conditions where frr activity is modulated to assess global impacts on translation.

• Quantitative Proteomics: Use stable isotope labeling approaches to measure effects of frr modulation on protein synthesis rates across the C. burnetii proteome.

These methodological approaches would provide comprehensive insights into how frr contributes to C. burnetii protein synthesis, potentially revealing unique aspects related to its pathogenesis and adaptation to intracellular life.

How can I validate the functional activity of recombinant C. burnetii frr in experimental systems?

Validating the functional activity of recombinant C. burnetii frr requires establishing assays that specifically measure ribosome recycling. Here are methodological approaches to assess frr functionality:

Biochemical Validation Assays:

• Polysome Dissociation Assay:

  • Methodology: Incubate purified polysomes with recombinant frr along with EF-G and GTP

  • Measurement: Monitor the decrease in polysome fraction and increase in monosome fraction using sucrose gradient centrifugation

  • Controls: Include reactions without frr or with heat-inactivated frr

  • Expected outcome: Active frr will promote conversion of polysomes to monosomes

• mRNA Release Assay:

  • Methodology: Use fluorescently labeled mRNA bound to post-termination complexes

  • Measurement: Quantify mRNA release upon addition of frr, EF-G, and GTP

  • Analysis: Calculate kinetic parameters (Km, Vmax) to compare with frr from other species

• Light Scattering Assay:

  • Methodology: Monitor changes in light scattering as ribosomes dissociate

  • Advantages: Real-time measurement, requires smaller amounts of purified components

  • Quantification: Derive initial rates of dissociation as a measure of activity

Cellular Validation Approaches:

• Complementation in Heterologous Systems:

  • Express C. burnetii frr in temperature-sensitive E. coli frr mutants

  • Assess growth restoration at non-permissive temperatures

  • Measure polysome profiles to confirm functional restoration of ribosome recycling

• In vitro Translation Complementation:

  • Deplete endogenous RRF from bacterial extracts using antibodies

  • Add recombinant C. burnetii frr and measure restoration of translation activity

  • Compare activity with positive controls (e.g., E. coli RRF)

Quality Control Metrics:

Establish the following parameters to demonstrate that purified recombinant frr is functionally active:

• Specific activity (μmol ribosomes recycled per minute per mg frr)

• Temperature and pH optima for activity

• Stability under various storage conditions

• Minimum effective concentration in recycling assays

These validation protocols would confirm that the recombinant C. burnetii frr not only maintains its structural integrity (>85% purity by SDS-PAGE ) but also retains the functional capacity to perform its biological role in ribosome recycling.

What is the relationship between frr and the pathogenicity of C. burnetii?

The relationship between frr and the pathogenicity of C. burnetii represents an interesting but underexplored area of research. While direct evidence linking frr to virulence is not explicitly stated in the search results, several conceptual frameworks can guide investigation of this relationship:

Essential Function in Bacterial Survival:
As a core component of the translation machinery, frr likely plays an essential role in C. burnetii viability. Efficient protein synthesis is particularly critical for intracellular pathogens that must adapt to changing host environments. The efficiency of ribosome recycling could impact the bacterium's ability to rapidly respond to stress conditions encountered during infection .

Potential Interaction with Virulence Mechanisms:
The search results reveal that C. burnetii strains carry one of four plasmid types (QpH1, QpRS, QpDV, or QpDG) or a chromosomally integrated sequence that contributes to virulence . The QpH1 plasmid (37,319 bp encoding 40 ORFs) is essential for C. burnetii colonization of murine bone marrow-derived macrophages (BMDMs) . While no direct interaction between frr and plasmid-encoded virulence factors is mentioned, investigating such potential interactions could reveal important functional relationships.

Experimental Approaches to Investigate frr's Role in Pathogenicity:

• Conditional Expression Studies:

  • Develop regulatable frr expression systems in C. burnetii

  • Assess impacts on growth in different host cell types (comparing human THP-1 cells vs. murine BMDMs)

  • Compare with the differential growth patterns observed with QpH1-deficient strains

• Comparative Analysis Between Strains:

  • Compare frr sequence and expression levels between C. burnetii isolates with different virulence profiles

  • Investigate whether frr variants correlate with plasmid type or virulence in animal models

• Host Response Studies:

  • Examine whether frr-modulated C. burnetii strains differ in their ability to manipulate host immune responses

  • Assess CCV biogenesis and maintenance when frr activity is altered

• Integration with Type IV Secretion Studies:

  • The search results note that C. burnetii utilizes a type IV secretion system and effectors for CCV biogenesis and growth in host cells

  • Investigate whether optimal frr function impacts the expression or secretion of these effectors

While frr itself may not be a classic virulence factor, its fundamental role in protein synthesis positions it as a potential indirect contributor to pathogenicity by enabling optimal expression of true virulence factors and supporting bacterial adaptation to the intracellular niche.

How does frr interact with the QpH1 plasmid system in C. burnetii strains?

Theoretical Framework for frr-QpH1 Interactions:

• Translational Regulation of Plasmid-Encoded Genes:
  As the ribosome-recycling factor, frr may influence the efficiency of translation for plasmid-encoded proteins, including the three identified secretion effectors (CBUA0006, CBUA0013, and CBUA0023) that are conserved across all plasmid types and secreted via the type IV secretion system . Efficient ribosome recycling could be particularly important for optimal expression of these effectors.

• Potential Co-regulation:
  The expression of frr and plasmid genes might be co-regulated under specific conditions, such as during phagolysosomal adaptation or host cell infection. This coordinated regulation could ensure that translation capacity (influenced by frr) is aligned with the expression needs of virulence factors.

Experimental Approaches to Investigate Potential Interactions:

• Comparative Expression Analysis:

  • Perform RNA-seq and proteomics to compare expression profiles of frr and plasmid genes under various conditions

  • Use techniques like ribosome profiling to examine translation efficiency of plasmid-encoded genes in the presence of normal versus reduced frr activity

• Functional Complementation Studies:

  • Similar to the QpH1-deficient strain studies described in the search results , develop frr-modulated strains

  • Compare the phenotypes of frr-modulated strains, QpH1-deficient strains, and combined variants in different host cell types

• Regulatory Network Analysis:

  • Investigate whether common transcription factors regulate both frr and key plasmid genes

  • Perform chromatin immunoprecipitation sequencing (ChIP-seq) to identify potential shared regulatory elements

• Host Cell Response Integration:

  • Compare host cell responses to infection with strains having different frr activity levels and QpH1 status

  • Focus particularly on differences in the CCV formation and maintenance

While direct molecular interactions between frr and QpH1 components seem unlikely based on their different cellular functions (translation versus secreted effectors), their functional integration within C. burnetii's pathogenic mechanisms represents an important area for investigation, potentially revealing how core cellular processes are coordinated with virulence factor expression during infection.

How can recombinant frr be used in structural biology studies of C. burnetii ribosomes?

Recombinant C. burnetii frr offers valuable opportunities for structural biology studies aimed at understanding the unique features of C. burnetii ribosomes and translation machinery. Here are methodological approaches for utilizing recombinant frr in structural investigations:

Cryo-Electron Microscopy (Cryo-EM) Studies:

• Ribosome-frr Complex Visualization:

  • Reconstitute post-termination ribosomal complexes with purified C. burnetii ribosomes and recombinant frr

  • Use cryo-EM to capture structures at different stages of the recycling process

  • Compare with structures from model organisms to identify unique features of C. burnetii translation termination

• Time-resolved Cryo-EM:

  • Implement time-resolved approaches to capture transient states during frr-mediated ribosome recycling

  • Visualize conformational changes in both the ribosome and frr during the recycling process

X-ray Crystallography Applications:

• frr Structure Determination:

  • Crystallize the purified recombinant frr (>85% purity as indicated in the specifications)

  • Determine the high-resolution structure of C. burnetii frr

  • Compare with structures from other bacterial species to identify unique structural adaptations

• Co-crystallization Studies:

  • Attempt co-crystallization of frr with ribosomal components or other translation factors

  • Focus on interactions with ribosomal proteins or rRNA fragments that might be unique to C. burnetii

Molecular Dynamics and Computational Approaches:

• Simulation Studies:

  • Use the experimentally determined structures to perform molecular dynamics simulations

  • Model the interaction of frr with the complete C. burnetii ribosome

  • Investigate the energetics and dynamics of binding and conformational changes

• Structure-Based Drug Design:

  • Identify unique structural features of C. burnetii frr that could be targeted for antimicrobial development

  • Perform virtual screening to identify potential inhibitors specific to C. burnetii frr

Hybrid Structural Biology Approaches:

• Integrative Modeling:

  • Combine data from multiple structural techniques (cryo-EM, X-ray, NMR, SAXS) to build comprehensive models

  • Incorporate crosslinking mass spectrometry data to validate interaction interfaces

• In situ Structural Studies:

  • Develop approaches to visualize frr-ribosome interactions within C. burnetii cells

  • Apply cellular cryo-electron tomography to examine ribosome states in their native context

These structural biology approaches would provide unprecedented insights into the molecular mechanisms of translation termination and ribosome recycling in C. burnetii, potentially revealing adaptations related to its unique lifestyle as an intracellular pathogen that thrives in acidified parasitophorous vacuoles.

What are the key methodological challenges when working with C. burnetii frr in research settings?

Working with C. burnetii frr presents several methodological challenges that researchers should anticipate and address:

Biosafety Considerations:
C. burnetii is classified as a select agent (BSL-3) due to its high infectivity and potential for aerosol transmission. While recombinant frr itself doesn't pose such risks, researchers should be aware that:

• Rigorous biosecurity screening and export control compliance are essential when ordering materials related to C. burnetii

• Working with native frr purified from C. burnetii would require appropriate containment facilities

• Proper decontamination protocols should be established for all equipment and materials

Protein Stability and Solubility Challenges:

• Aggregation Potential: Bacterial ribosomal proteins and translation factors often have challenges with solubility when overexpressed

• Storage Sensitivity: Recommended storage at -20°C or -80°C with the addition of 5-50% glycerol indicates potential stability issues

• Working Timeframe Limitations: The recommendation to use working aliquots at 4°C for only up to one week suggests activity loss over time

Functional Assay Development:

• System Reconstitution: Creating a functional C. burnetii translation system requires multiple purified components beyond just frr

• Specificity Verification: Confirming that observed effects are specific to frr function rather than contaminants or buffer effects

• Heterologous Systems: When using E. coli or other model systems to study C. burnetii frr, compatibility issues may arise

Technical Optimization Strategies:

Addressing these methodological challenges requires careful experimental design, rigorous controls, and potentially iterative optimization of protocols to achieve reliable and reproducible results when working with C. burnetii frr.

How can gene knockout or mutation studies be designed to investigate frr function in C. burnetii?

Designing effective genetic manipulation studies to investigate frr function in C. burnetii requires careful consideration of both technical approaches and biological constraints, particularly since frr likely serves an essential function:

Conditional Expression Systems:

• Tetracycline-Regulated Expression:

  • Construct a conditional frr mutant where the native gene is deleted and replaced with a copy under control of a tetracycline-inducible promoter

  • Modulate expression levels by varying tetracycline concentration

  • Monitor growth, morphology, and protein synthesis rates at different expression levels

• Degradation Tag Approach:

  • Fuse the frr protein with an inducible degradation tag (e.g., ssrA-based degron tags)

  • Allow controlled depletion of the protein upon inducer addition

  • Track cellular responses to progressive frr depletion

Domain-Specific Mutagenesis:

• Structure-Guided Mutations:

  • Based on conserved domains in the RRF family, introduce point mutations in predicted functional regions

  • Target residues involved in ribosome binding, conformational changes, or interactions with other factors

  • Assess functional impact using in vitro and cellular assays

• Complementation Analysis:

  • Create a library of frr variants with different mutations

  • Test their ability to complement growth in conditional mutants under non-permissive conditions

  • Identify residues essential for different aspects of frr function

Plasmid-Based Approaches:

The search results describe successful approaches for creating QpH1 plasmid-deficient strains of C. burnetii , which could be adapted for frr studies:

• Incompatibility-Based Replacement:

  • Construct shuttle vectors containing wild-type or mutant frr versions

  • Transform these into C. burnetii containing a conditional frr expression system

  • Switch off the conditional expression and assess the ability of plasmid-encoded variants to support growth

• CRISPR-Cas9 Editing:

  • Develop CRISPR-Cas9 systems optimized for C. burnetii

  • Target the chromosomal frr gene while providing plasmid-encoded variants

  • Screen for successful editing events

Experimental Controls and Validation:

• Phenotypic Analysis Pipeline:

  • Growth kinetics in axenic media (similar to the analysis performed for QpH1-deficient strains)

  • Intracellular growth in different cell types (BGMK cells, THP-1 cells, murine BMDMs)

  • Ribosome profiles to directly assess impact on translation

• Complementation Controls:

  • Include heterologous RRFs from related bacteria

  • Test chimeric proteins combining domains from different species

  • Use these to identify C. burnetii-specific functional regions

These genetic approaches would provide valuable insights into frr function while acknowledging and addressing the challenges associated with manipulating potentially essential genes in an intracellular bacterial pathogen.

What emerging technologies could advance C. burnetii frr research in the next decade?

Several cutting-edge technologies are poised to transform research on C. burnetii frr and protein synthesis mechanisms over the next decade:

Single-Molecule Translation Imaging:

• Real-time Ribosome Recycling Visualization:

  • Single-molecule fluorescence resonance energy transfer (smFRET) to directly observe frr-mediated ribosome recycling events

  • Nanoscale imaging of individual ribosomes during the recycling process

  • Mapping of conformational changes during interaction with frr at unprecedented resolution

• In vivo Translation Dynamics:

  • Development of non-disruptive fluorescent tags for ribosomes and frr

  • Live-cell imaging of translation events in C. burnetii under various conditions

  • Correlation with intracellular pathogen behavior and host cell responses

Advanced Structural Biology Approaches:

• Cryo-Electron Tomography:

  • Visualization of ribosomes and translation factors in their native cellular context

  • 3D reconstructions of C. burnetii translation machinery within intact cells

  • Integration with focused ion beam milling for examining intracellular bacteria

• Time-Resolved Structural Studies:

  • Serial femtosecond crystallography at X-ray free-electron lasers

  • Capturing transient states in the frr-ribosome interaction pathway

  • Millisecond-to-microsecond resolution of conformational changes

Synthetic Biology and Genome Engineering:

• Minimal Translation Systems:

  • Construction of minimal reconstituted translation systems using C. burnetii components

  • Systematic complexity reduction to identify essential interactions

  • Engineering of orthogonal translation systems with modified frr proteins

• Genome-wide Interaction Mapping:

  • High-throughput genetic interaction screens using CRISPRi technologies

  • Identification of genes that display synthetic lethality with frr mutations

  • Construction of comprehensive genetic interaction networks centered on frr

Computational and AI-Enhanced Approaches:

• Machine Learning for Structure Prediction:

  • Implementation of AlphaFold-like approaches specific to bacterial translation systems

  • Prediction of species-specific variations in frr-ribosome interactions

  • Computational design of C. burnetii-specific frr inhibitors

• Multi-scale Molecular Simulations:

  • Integration of quantum mechanics, molecular dynamics, and coarse-grained simulations

  • Modeling the complete ribosome recycling process across relevant timescales

  • Identification of transition states and energy landscapes specific to C. burnetii frr

These emerging technologies will enable unprecedented insights into the structural, functional, and regulatory aspects of frr in C. burnetii, potentially revealing novel therapeutic targets and fundamental principles of bacterial translation that could be exploited for antimicrobial development.

How might understanding frr function contribute to developing new antimicrobial strategies against C. burnetii?

Understanding the structure and function of C. burnetii frr offers promising avenues for novel antimicrobial development, particularly given the challenges of treating Q fever infections:

Rational Drug Design Opportunities:

• Structural Vulnerability Exploitation:

  • High-resolution structures of C. burnetii frr, alone and in complex with ribosomes, could reveal unique pockets for selective targeting

  • Computational screening of compound libraries against these unique structural features

  • Fragment-based approaches to develop high-affinity inhibitors that disrupt frr function

• Species-Specific Targeting:

  • Comparison of frr sequences and structures across bacterial species to identify regions unique to C. burnetii

  • Development of inhibitors that selectively target C. burnetii frr over human translational components

  • Structure-activity relationship studies to optimize selectivity and potency

Mechanism-Based Intervention Strategies:

• Disruption of Ribosome Recycling:

  • Compounds that prevent frr from binding to ribosomes or that block its release

  • Molecules that lock frr into non-productive conformations

  • Agents that trap ribosomes in post-termination complexes, preventing recycling

• Interference with frr-EF-G Interactions:

  • targeting the interface between frr and elongation factor G (EF-G)

  • Development of peptidomimetics that mimic interaction interfaces

  • Allosteric inhibitors that prevent productive complex formation

Therapeutic Potential and Advantages:

• Addressing Intracellular Persistence:

  • C. burnetii's ability to persist in acidified vacuoles contributes to treatment challenges

  • Inhibiting frr could prevent the efficient protein synthesis required for adaptation to this niche

  • Combination with current therapies could reduce treatment duration and relapse rates

• Resistance Considerations:

  • The essential nature of frr function may present a higher barrier to resistance development

  • Multiple binding sites could be targeted simultaneously to further reduce resistance potential

  • Conservation across strains suggests broad efficacy against different C. burnetii isolates

• Delivery Strategies for Intracellular Targeting:

  • Development of nanoparticle formulations to deliver frr inhibitors to phagolysosomes

  • Prodrug approaches that become activated in the acidic environment of CCVs

  • Liposomal or polymer-based systems that follow similar uptake pathways as C. burnetii

The research on QpH1 plasmid as a virulence factor demonstrates that C. burnetii has specific adaptations for colonizing different host cell types, particularly in rodent models. This suggests that targeting frr could potentially disrupt the pathogen's ability to adapt and survive in diverse host environments, offering a new therapeutic strategy that complements existing approaches for treating Q fever.