Recombinant Chlamydophila abortus Ribosome-recycling factor (frr)

What is the functional role of Ribosome-recycling factor in Chlamydophila abortus?

Ribosome-recycling factor (RRF) in Chlamydophila abortus functions to dissociate ribosomes from mRNA after the termination of translation. It works by splitting the ribosome into its small and large subunits, acting in conjunction with elongation factor G . This process is critical for bacterial protein synthesis as it allows ribosomes to be "recycled" for subsequent rounds of translation. Without effective ribosome recycling, post-termination 70S complexes accumulate in 3′-UTRs, and elongating ribosomes become blocked by non-recycled ribosomes at stop codons, severely impacting protein synthesis .

How does C. abortus RRF compare structurally to RRF in other bacterial species?

While specific structural data for C. abortus RRF is limited, ribosome recycling factors generally exhibit a tRNA-like structure despite functioning differently than tRNA at the ribosome. C. abortus RRF would likely maintain the conserved functional domains found in other bacterial RRFs while potentially displaying species-specific variations that could affect binding specificity or efficiency.

The RRF protein acts as a structural mimic of tRNA but binds to the ribosome differently than the A-site, P-site, or E-site tRNA . Comparative structural analysis with E. coli RRF, which has been extensively studied, would be valuable for understanding C. abortus-specific features that might relate to its unique lifecycle as an obligate intracellular pathogen.

What methods are most effective for expressing recombinant C. abortus RRF?

For effective expression of recombinant C. abortus RRF, researchers should consider:

Recommended expression protocol:

• Clone the frr gene from C. abortus genomic DNA using PCR with primers designed from the annotated genome sequence (C. abortus genome contains 961 coding sequences )

• Insert the frr gene into a vector with a strong promoter (like T7) and a 6xHis-tag for purification

• Transform into E. coli expression hosts (BL21(DE3) is recommended)

• Induce expression with IPTG at lower temperatures (18-25°C) to enhance protein solubility

• Purify using Ni-NTA affinity chromatography followed by size exclusion chromatography

This approach addresses the challenges of expressing proteins from an organism with a markedly different codon usage pattern than typical expression hosts.

How can ribosome profiling be applied to study RRF function in C. abortus?

Ribosome profiling offers powerful insights into RRF function in C. abortus by capturing genome-wide translational dynamics. Based on methodologies used for studying RRF in E. coli , researchers should:

• Establish conditional knockdown system: Develop a method to rapidly deplete RRF levels in C. abortus (challenging due to its obligate intracellular nature)

• Collect sequential samples: Harvest samples at multiple time points after RRF depletion

• Prepare ribosome footprints: Isolate ribosomes and create libraries of ribosome-protected mRNA fragments

• Deep sequencing: Sequence these fragments to determine ribosome positions throughout the transcriptome

• Data analysis: Analyze how ribosome density changes when recycling is inhibited, focusing on:

  • Accumulation patterns in 3′-UTRs

  • Ribosome stacking upstream of stop codons

  • Changes in translational efficiency across genes

Expected patterns based on E. coli studies:

This approach would reveal C. abortus-specific features of ribosome recycling, potentially identifying unique aspects related to its pathogenicity or developmental cycle .

What are the key considerations for studying RRF-ribosome interactions in C. abortus?

To effectively study RRF-ribosome interactions in C. abortus, researchers should consider:

• Ribosome isolation: Develop protocols for isolating intact C. abortus ribosomes from infected cells, accounting for contamination with host ribosomes

• Cryo-EM analysis: Use cryo-electron microscopy to visualize RRF-ribosome complexes at different states of recycling

• Binding kinetics: Employ surface plasmon resonance or microscale thermophoresis to determine binding affinities between purified C. abortus RRF and ribosomes

• Crosslinking studies: Identify specific points of interaction between RRF and ribosomal components using chemical crosslinking followed by mass spectrometry

• Mutational analysis: Create targeted mutations in the C. abortus frr gene to identify essential residues for ribosome interaction

Key challenges to address:

• C. abortus is an obligate intracellular pathogen requiring specialized growth conditions

• The biphasic developmental cycle (elementary body/reticulate body) may affect ribosomal states

• Potential interactions with host factors that might influence recycling efficiency

How does the developmental cycle of C. abortus influence RRF expression and function?

C. abortus exhibits a biphasic developmental cycle, alternating between elementary bodies (EBs, infectious but metabolically inactive) and reticulate bodies (RBs, non-infectious but metabolically active) . This unique lifecycle likely influences RRF expression and function:

Proposed developmental regulation of RRF:

To experimentally determine this pattern, researchers should:

• Use RT-qPCR to measure frr transcript levels throughout the developmental cycle

• Employ ribosome profiling at different timepoints to assess translational activity

• Use immunofluorescence microscopy with anti-RRF antibodies to visualize protein localization

• Correlate RRF expression with other markers of developmental transitions

How might RRF depletion affect C. abortus pathogenesis and persistence?

RRF depletion would likely have profound effects on C. abortus pathogenesis based on findings from other bacterial systems :

Predicted impacts on pathogenesis:

The C. abortus infection cycle involves latency and reactivation during pregnancy, mediated by changes in the host immune environment . Interferon-γ (IFN-γ) plays a critical role in controlling infection by inducing indoleamine-2,3-dioxygenase (IDO), which degrades tryptophan needed for bacterial growth . Unlike C. trachomatis, C. abortus lacks the tryptophan biosynthetic operon (trp) , suggesting it may rely on efficient protein synthesis machinery (including RRF) to maximize resource utilization when tryptophan is limited.

Experimental approaches to test these hypotheses include:

• Developing conditional RRF knockdown systems

• Assessing virulence in appropriate animal models

• Transcriptomic analysis under varying tryptophan concentrations

• Measurement of persistence duration in cell culture models

What are the implications of RRF function for translational coupling in C. abortus operons?

Translational coupling is a process where translation of downstream genes in an operon depends on the translation of upstream genes. Research in E. coli has shown that RRF depletion does not significantly affect coupling efficiency in reporter assays or ribosome density genome-wide , suggesting re-initiation is not a major mechanism of translational coupling.

For C. abortus, investigation of translational coupling in its operons would be valuable, particularly for:

• Gene pairs with overlapping stop/start codons: Over 30% of gene pairs in E. coli have overlapping stop and start sites . Similar analysis of the C. abortus genome would identify potential translationally coupled genes.

• Virulence-associated operons: Pmps and other virulence factors may be regulated through translational coupling.

• Developmental cycle-specific operons: Genes required for transitions between EB and RB forms.

Experimental approach:

• Construct reporter systems with C. abortus intergenic regions between the reporter genes

• Measure coupling efficiency with and without RRF depletion

• Analyze ribosome density ratios for neighboring genes in polycistronic transcripts

• Identify potential re-initiation sites in the C. abortus genome

How does the C. abortus RRF interact with rescue factors like tmRNA and ArfA?

In E. coli, RRF depletion has dramatic effects on ribosome rescue factors tmRNA and ArfA, with ArfA protein synthesis increased 39-fold after 60 minutes of RRF depletion . This suggests a compensatory response when recycling is impaired.

For C. abortus, researchers should:

• Confirm the presence of rescue systems: Identify tmRNA and ArfA homologs in the C. abortus genome

• Characterize expression patterns: Measure expression levels throughout the developmental cycle

• Study response to RRF depletion: Determine if similar upregulation occurs when recycling is impaired

• Investigate functional relationships: Test whether these systems can compensate for each other

This is particularly relevant because C. abortus exists in various metabolic states during its lifecycle and faces host defense mechanisms that may disrupt translation, requiring efficient rescue systems.

Could recombinant C. abortus RRF be useful as a vaccine antigen?

While using RRF directly as a vaccine antigen hasn't been extensively studied, its essential nature and conservation provide both advantages and challenges:

Potential as vaccine antigen:

Current successful approaches for C. abortus vaccine development have focused on membrane proteins such as the polymorphic membrane protein Pmp18D (specifically the N-terminal portion) . The rVCG-Pmp18.3 vaccine provided complete protection against neonatal mortality in mice .

To evaluate RRF as a vaccine candidate, researchers should:

• Identify immunogenic epitopes specific to C. abortus RRF

• Test immune responses in animal models

• Assess protection against challenge in pregnant animal models

• Consider combination with established antigens like Pmp18.3

What methodological approaches are most effective for studying interactions between C. abortus RRF and host factors?

Investigating interactions between C. abortus RRF and host factors presents unique challenges due to the organism's obligate intracellular lifestyle. Effective methodological approaches include:

• Proximity labeling techniques: Use BioID or APEX2 fused to RRF to identify proximal host proteins during infection

• Pull-down assays: Express tagged recombinant RRF and identify interacting host proteins by mass spectrometry

• Yeast two-hybrid screening: Screen against human or ovine cDNA libraries to identify potential interactors

• Computational prediction: Use machine learning approaches to predict potential host-pathogen protein interactions

• Fluorescence microscopy: Monitor RRF localization during infection using fluorescent tags or antibodies

Key host factors to investigate:

• Components of the host translation machinery

• Innate immune sensors of bacterial translation

• Tryptophan metabolism pathway components

• Placental-specific factors that might influence C. abortus persistence and reactivation

This research could reveal how C. abortus modulates host translation or immune responses to establish persistent infection.

What are the optimal conditions for preserving and assaying recombinant C. abortus RRF activity?

Based on studies with other bacterial RRFs, the following guidelines are recommended for preserving and assaying recombinant C. abortus RRF:

Storage and stability recommendations:

• Store purified protein at -80°C in buffer containing 50mM Tris-HCl (pH 7.5), 100mM KCl, 10mM MgCl2, 7mM β-mercaptoethanol, and 10% glycerol

• Avoid repeated freeze-thaw cycles; prepare single-use aliquots

• For longer-term storage, lyophilization may be considered

Activity assay protocol:

• Ribosome recycling assay: Measure the ability of RRF to dissociate post-termination complexes in conjunction with EF-G and IF3

  • Use purified ribosomes, mRNA with stop codon, and necessary factors

  • Monitor dissociation by light scattering or by separating ribosomal subunits on sucrose gradients

• ATPase stimulation assay: Measure the stimulation of EF-G's GTPase activity in the presence of RRF

  • Use purified EF-G, RRF, and ribosome complexes

  • Monitor GTP hydrolysis through colorimetric phosphate detection or using labeled GTP

• Ribosome binding assay: Use fluorescence anisotropy or surface plasmon resonance to measure direct binding of labeled RRF to ribosomes

How can researchers distinguish between direct RRF effects and secondary consequences in C. abortus studies?

Distinguishing direct RRF effects from secondary consequences requires careful experimental design:

• Temporal analysis: Monitor changes at multiple timepoints after RRF depletion or inhibition to establish the sequence of events

• Complementation studies: Use controlled expression of wild-type or mutant RRF to determine if observed phenotypes can be reversed

• Directed mutagenesis: Create specific mutations in RRF that affect particular functions (e.g., ribosome binding without affecting protein stability)

• Ribosome profiling: Compare the immediate changes in ribosome position following RRF depletion with later transcriptional and translational changes

• Mathematical modeling: Develop models that predict the cascade of effects following RRF inhibition to identify direct vs. secondary consequences

Example of analysis framework:

This approach allows researchers to establish a clear timeline distinguishing immediate consequences of RRF dysfunction from downstream adaptations.