Recombinant Mycobacterium abscessus Ribosome-recycling factor (frr)

What is the Ribosome-recycling factor (frr) in Mycobacterium abscessus and what is its primary function?

The Ribosome-recycling factor (RRF or frr gene product) in M. abscessus is a specialized protein that plays an essential role in the translation cycle by dissociating post-termination ribosomal complexes. During protein synthesis, RRF functions to separate the 70S ribosome into its constituent 30S and 50S subunits after the completion of protein synthesis, thereby making these components available for new rounds of translation. This process is critical for efficient protein synthesis and cellular function.

RRF operates by binding to the A-site of the ribosome following translation termination, and in conjunction with elongation factor G (EF-G) and GTP hydrolysis, facilitates the dissociation of the ribosomal subunits and the release of the mRNA and deacylated tRNA. This recycling step ensures the continuous availability of translation machinery components for subsequent protein synthesis events .

How does the expression of recombinant M. abscessus RRF differ from other mycobacterial species?

The expression of recombinant M. abscessus RRF presents unique challenges compared to other mycobacterial species due to several factors:

• M. abscessus has evolved specialized ribosomal components that contribute to its intrinsic antibiotic resistance, which may affect RRF interactions and function.

• The optimal expression conditions for M. abscessus RRF differ from those of other mycobacterial species, requiring specific temperature, pH, and induction parameters.

• The solubility and stability profiles of M. abscessus RRF may necessitate specialized buffer conditions to maintain proper folding and activity during purification.

Expression systems for M. abscessus RRF typically employ E. coli BL21(DE3) strains with T7 promoter-driven expression vectors, with optimal induction occurring at lower temperatures (16-18°C) to enhance protein solubility. Purification often requires a combination of affinity chromatography (typically Ni-NTA for His-tagged constructs) followed by size exclusion chromatography to achieve high purity.

What experimental methods are most effective for assessing the ribosome-splitting activity of recombinant M. abscessus RRF?

The evaluation of M. abscessus RRF ribosome-splitting activity can be effectively assessed through several complementary approaches:

• Sucrose Density Gradient Analysis: This technique allows for the separation and quantification of 70S ribosomes versus 30S and 50S subunits, providing direct evidence of RRF-mediated ribosome splitting. The analysis of ribosome profiles from wild-type versus ΔhflX strains upon erythromycin exposure has demonstrated an increased population of 70S ribosomes in the absence of HflX, suggesting a similar approach would be valuable for studying RRF function .

• Light Scattering Assays: Real-time monitoring of ribosome dissociation can be performed using light scattering techniques, where decreases in scattering intensity correlate with the conversion of 70S ribosomes to 30S and 50S subunits.

• In vitro Translation Assays: Measuring the ability of RRF to rescue translation of reporter proteins in the presence of translation inhibitors provides functional evidence of its activity.

• Complementation Studies: As demonstrated with HflX, the ability of RRF to partially restore antibiotic sensitivity in deletion mutants provides valuable insights into its functional role in vivo .

How does the Ribosome-recycling factor interact with antibiotic resistance mechanisms in M. abscessus?

M. abscessus employs multiple strategies for antibiotic resistance, with ribosomal protection mechanisms playing a significant role. While erm41 has been considered the primary mechanism of intrinsic macrolide resistance in M. abscessus, recent research has identified additional significant factors, including HflX .

The Ribosome-recycling factor appears to function in a complementary manner to HflX in mediating resistance to macrolide-lincosamide antibiotics. Research has demonstrated that constitutive expression of M. smegmatis RRF partially restored antibiotic sensitivity in a ΔMs_hflX mutant strain, suggesting that RRF can partially compensate for HflX function . This supports the hypothesis that ribosome splitting represents a significant mechanism of antibiotic resistance.

What is the relationship between RRF and other ribosome-associated factors in M. abscessus antibiotic resistance?

M. abscessus employs multiple ribosome-associated factors that collectively contribute to its robust antibiotic resistance profile. Key relationships include:

• RRF and HflX: While both factors facilitate ribosome splitting, they appear to target different ribosomal substrates. HflX strongly associates with ribosomal subunits in bacteria exposed to erythromycin or clindamycin and dissociates 70S ribosomes independent of GTP hydrolysis . RRF can partially complement HflX function in antibiotic resistance, suggesting overlapping but distinct mechanisms .

• RRF and HelR: HelR (MAB_3189c) represents another significant resistance determinant in M. abscessus that interacts with RNA polymerase (RNAP) to confer rifamycin resistance . While HelR functions through RNAP protection rather than ribosome recycling, both mechanisms represent strategies to overcome antibiotic-mediated stalling of essential cellular machinery.

• RRF and Arr: The ADP-ribosyltransferase Arr inactivates rifampicin through chemical modification . Unlike RRF, which rescues stalled ribosomes, Arr directly neutralizes the antibiotic. Both genes are upregulated upon rifamycin exposure, suggesting coordinated resistance responses .

This network of ribosome-associated factors highlights the sophisticated resistance mechanisms employed by M. abscessus, with RRF contributing to a broader system of protective responses against translation-targeting antibiotics.

How do genomic variations in the frr gene impact functional characteristics across M. abscessus subspecies?

The M. abscessus complex comprises three subspecies (M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii) that share 96.6-99.8% average nucleotide identity . This genetic diversity extends to genes involved in antibiotic resistance mechanisms, including the frr gene.

Analysis of genomic variations suggests:

• Sequence Conservation: The core functional domains of RRF are generally well-conserved across subspecies, reflecting the essential nature of ribosome recycling.

• Subspecies-Specific Adaptations: Subtle variations in frr gene sequences may contribute to differences in antibiotic susceptibility profiles observed between subspecies.

• Horizontal Gene Transfer Impact: Recent studies have identified horizontal gene transfer (HGT) events in M. abscessus, particularly affecting the rpoB gene . While the search results don't specifically mention HGT affecting the frr gene, the prevalence of genetic exchange in this species suggests potential for functional diversification of ribosome-associated factors.

The impact of these variations includes potential differences in:

• RRF binding affinity to ribosomes

• Efficiency of ribosome splitting

• Interaction with other translation factors

• Contribution to antibiotic resistance profiles

What methodological challenges exist in studying the interaction between RRF and stalled ribosomes in M. abscessus?

Investigating RRF interactions with stalled ribosomes in M. abscessus presents several significant methodological challenges:

• Slow Growth and Pathogenicity: As a pathogenic mycobacterium, M. abscessus requires specialized containment facilities (BSL-2), complicating experimental workflows and limiting laboratory access.

• Ribosome Heterogeneity: Stalled ribosomes represent heterogeneous populations with different mRNA and tRNA occupancies, complicating structural and functional analyses.

• Low Yield of Native Components: Isolation of sufficient quantities of native M. abscessus ribosomes for biochemical studies is challenging due to the organism's slow growth and relatively low biomass production.

• Reconstitution of Physiological Conditions: Recreating the precise conditions under which RRF interacts with drug-stalled ribosomes in vitro requires careful optimization of buffer conditions, ion concentrations, and other parameters.

• Transient Interactions: The dynamic nature of RRF-ribosome interactions often involves transient states that are difficult to capture using traditional biochemical approaches.

Advanced methodological approaches to overcome these challenges include:

• Cryo-electron microscopy for structural analysis of RRF-ribosome complexes

• Single-molecule fluorescence techniques to observe transient interactions

• Ribosome profiling to identify RRF binding sites on stalled ribosomes

• Crosslinking mass spectrometry to map protein-protein interactions between RRF and ribosomal components

How might targeting the interaction between RRF and stalled ribosomes serve as a strategy to overcome antibiotic resistance?

The emerging understanding of RRF's role in antibiotic resistance opens promising avenues for novel therapeutic strategies:

• Selective Inhibition: Developing compounds that selectively inhibit M. abscessus RRF while sparing human ribosome-recycling mechanisms could potentiate the efficacy of existing antibiotics. This approach would prevent the rescue of drug-stalled ribosomes, increasing the effectiveness of translation-targeting antibiotics.

• Combination Therapies: Identifying synergistic combinations of antibiotics that simultaneously target translation and inhibit rescue mechanisms could overcome the robust resistance profile of M. abscessus.

• Structure-Based Drug Design: Structural determination of M. abscessus RRF in complex with stalled ribosomes could guide the rational design of inhibitors that specifically interfere with this interaction.

• Allosteric Modulators: Compounds that bind to allosteric sites on RRF could alter its conformational dynamics, thereby modifying its interaction with ribosomes without completely inhibiting essential cellular functions.

The research supporting this approach includes observations that Ms-HflX (another ribosome splitting factor) strongly associates with ribosomal subunits in vivo in bacteria exposed to erythromycin or clindamycin . This suggests that targeting ribosome recycling factors could be a viable strategy for enhancing antibiotic efficacy.

What comparative insights can be gained from studying RRF across different Mycobacterium species?

Comparative analysis of RRF across different Mycobacterium species offers valuable insights into both fundamental biology and potential therapeutic applications:

• Evolutionary Conservation and Divergence: Comparing RRF sequences and structures across pathogenic (M. tuberculosis, M. abscessus) and non-pathogenic (M. smegmatis) mycobacteria reveals conserved functional domains and species-specific adaptations.

• Differential Antibiotic Susceptibility: Species-specific variations in RRF function may contribute to differences in intrinsic antibiotic resistance profiles. For example, while rifampicin is effective against M. tuberculosis, it is ineffective against M. abscessus despite the absence of mutations in the rifampicin resistance determining region .

• Host-Pathogen Interaction Impacts: Differences in RRF function may influence bacterial survival under host-imposed stresses, particularly in macrophages where translation inhibition is a key defense mechanism.

• Model System Development: Insights from more tractable mycobacterial species (like M. smegmatis) can inform experimental approaches for studying RRF in more challenging pathogenic species.

Research has demonstrated that expression of M. abscessus resistance factors in M. tuberculosis can confer rifamycin tolerance, suggesting functional conservation despite evolutionary divergence . This approach of heterologous expression represents a valuable tool for comparative functional studies of RRF across mycobacterial species.