Recombinant Legionella pneumophila Ribosome-recycling factor (frr)

What is the functional role of ribosome-recycling factor in Legionella pneumophila pathogenesis?

Ribosome-recycling factor (frr) in Legionella pneumophila serves as a critical component in the translation machinery, responsible for disassembling the post-termination complex after protein synthesis completion. This process is essential for efficient protein production during intracellular replication within host cells such as macrophages. The protein works synergistically with elongation factor G (EF-G) to release ribosomes, mRNA, and tRNA, enabling their participation in subsequent rounds of translation. In the context of L. pneumophila pathogenesis, efficient protein synthesis is particularly vital during the bacteria's intracellular lifecycle within bone marrow-derived macrophages (BMDMs), as evidenced by studies examining L. pneumophila's intracellular growth requirements . The efficiency of ribosomal processes likely impacts the expression of virulence factors, including Dot/Icm effectors like SulF that are crucial for establishing the Legionella-containing vacuole (LCV) and subsequent bacterial replication.

How does the structure of L. pneumophila frr compare to that of other bacterial species?

L. pneumophila frr maintains the conserved structural domains characteristic of bacterial ribosome-recycling factors while exhibiting species-specific variations that may reflect adaptation to its unique intracellular lifestyle. The protein typically consists of approximately 185 amino acids, as indicated by commercially available recombinant versions . Structural analyses of frr proteins across bacterial species reveal a conserved three-domain architecture: an N-terminal domain involved in binding to ribosomal protein S12 (RpsL), a central domain that interacts with 16S rRNA, and a C-terminal domain that facilitates interaction with EF-G. The RpsL interaction is particularly noteworthy in L. pneumophila research, as mutations in the rpsL gene (encoding ribosomal protein S12) significantly affect bacterial replication in mouse macrophages . Comparative structural studies between L. pneumophila frr and its counterparts in other intracellular pathogens may reveal adaptations specific to intracellular replication strategies, which could explain differences in host range and tissue tropism.

What expression systems are most effective for producing recombinant L. pneumophila frr?

For optimal expression of recombinant L. pneumophila frr, E. coli-based systems using BL21(DE3) or similar strains with T7 promoter-driven expression vectors have proven most efficient for laboratory-scale production. When implementing this system, researchers should optimize induction conditions by testing IPTG concentrations (typically 0.1-1.0 mM) and induction temperatures (18-37°C), with lower temperatures (18-25°C) often yielding higher proportions of soluble protein. Including a hexahistidine or other affinity tag facilitates purification using immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography to achieve high purity. For studying protein-protein interactions involving frr, such as potential interactions with host factors identified in CRISPR screens , maintaining the native protein conformation is crucial, which may require tag removal using specific proteases like TEV or thrombin. Expression in L. pneumophila itself using complementation systems can provide insights into in vivo function, though yields are typically lower than heterologous expression systems.

What are the known post-translational modifications of frr in L. pneumophila?

While specific post-translational modifications (PTMs) of frr in L. pneumophila have not been extensively characterized, research on other bacterial species and the importance of PTMs in L. pneumophila's interaction with host cells suggest several potential modifications. Phosphorylation sites may exist on specific serine, threonine, or tyrosine residues, potentially regulating frr activity during different growth phases or environmental conditions. Unlike some bacterial proteins involved in host interactions, such as Rab7 which undergoes SUMOylation for recruitment to the Legionella-containing vacuole , frr has not been documented to undergo SUMOylation. Mass spectrometry-based approaches, including bottom-up proteomics with enrichment strategies for phosphopeptides, can be employed to identify potential PTMs. Targeted mutagenesis of predicted modification sites followed by functional assays provides a methodology to determine the biological significance of identified PTMs. Understanding these modifications could reveal regulatory mechanisms that coordinate translation with other cellular processes during L. pneumophila's intracellular lifecycle.

How can CRISPR/Cas9 screening be adapted to study frr interactions with host factors?

CRISPR/Cas9 screening offers a powerful approach to identify host factors that interact with L. pneumophila frr during infection, building on methodologies established for studying other L. pneumophila proteins. To implement this approach, researchers should first generate a genome-wide sgRNA library targeting all genes in the host cell model of interest, such as the mouse bone marrow-derived macrophages (BMDMs) described in recent L. pneumophila research . These cells should express Cas9, which can be achieved by crossing Cas9+/+ mice with appropriate strains, such as the A/J mice used for L. pneumophila permissive infections . The experimental design should include a selection strategy based on a phenotype relevant to frr function, such as altered bacterial replication rates or protein synthesis efficiency in infected cells. After infection and selection, next-generation sequencing should be employed to identify enriched or depleted sgRNAs, indicating host genes that potentially interact with frr. Validation of hits requires targeted gene knockout or knockdown followed by infection experiments and direct protein-protein interaction studies using co-immunoprecipitation or proximity labeling approaches.

What techniques are most effective for studying the role of frr in L. pneumophila translation during intracellular growth?

Investigating frr's role in L. pneumophila translation during intracellular growth requires approaches that preserve the integrity of the host-pathogen interface while enabling detailed molecular analyses. Ribosome profiling (Ribo-seq) adapted for intracellular bacteria provides a powerful method to capture genome-wide translational dynamics with nucleotide resolution. This technique involves isolating infected host cells, treatment with translation inhibitors to freeze ribosomes on mRNAs, followed by selective lysis and isolation of bacterial ribosomes, RNase digestion to leave only ribosome-protected fragments, and next-generation sequencing. Complementing this approach, selective ribosome profiling using epitope-tagged ribosomes can distinguish between host and bacterial translation. To directly assess frr function, temperature-sensitive or inducible frr mutants can be created, allowing controlled modulation of frr activity during infection. Time-course experiments comparing wild-type and frr-mutant strains can reveal how translation efficiency influences the expression of virulence factors and other proteins required for intracellular survival, similar to studies conducted with RpsL variants .

How does frr contribute to antibiotic susceptibility in L. pneumophila?

The relationship between frr function and antibiotic susceptibility in L. pneumophila likely involves both direct and indirect mechanisms that can be investigated through multiple experimental approaches. Since frr interacts with ribosomal protein S12 (encoded by rpsL), which is the target of streptomycin, there may be functional interplay between frr and antibiotic resistance mutations in rpsL. This connection is particularly relevant given observations that different rpsL mutations (K43N versus K88R) in L. pneumophila confer distinct phenotypes regarding intracellular growth despite providing similar levels of streptomycin resistance . To investigate this relationship, researchers should generate L. pneumophila strains with varying levels of frr expression (overexpression, depletion, or mutation) and determine minimum inhibitory concentrations (MICs) for translation-targeting antibiotics such as streptomycin, gentamicin, and macrolides. Time-kill assays comparing wild-type and frr-modified strains can reveal differences in bactericidal rates. Ribosome structural studies using cryo-electron microscopy can elucidate how frr and antibiotics compete for or cooperatively bind to ribosomal components.

What biophysical methods best characterize the interaction between frr and bacterial ribosomes?

Characterizing the interaction between L. pneumophila frr and bacterial ribosomes requires a multi-faceted biophysical approach to capture both structural details and binding dynamics. Cryo-electron microscopy (cryo-EM) stands as the gold standard for visualizing frr-ribosome complexes at near-atomic resolution, enabling detailed mapping of interaction interfaces between frr and ribosomal components including RpsL. Surface plasmon resonance (SPR) and bio-layer interferometry (BLI) provide quantitative measurements of binding kinetics and affinity constants, with one partner (typically purified ribosomes) immobilized on a sensor chip and recombinant frr as the analyte in solution. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into conformational changes upon binding by measuring the rate of hydrogen-deuterium exchange in various protein regions. Microscale thermophoresis (MST) requires minimal sample amounts and can characterize interactions under near-physiological conditions. Complementary to these approaches, fluorescence anisotropy using fluorescently labeled frr can track binding events in real-time, while analytical ultracentrifugation provides information about complex stoichiometry and shape.

How can researchers optimize purification of recombinant L. pneumophila frr to maintain functional activity?

Optimizing purification of recombinant L. pneumophila frr requires careful consideration of buffer conditions and purification strategies to preserve the protein's native conformation and functional activity. Begin with lysis buffer optimization, testing various pH ranges (7.0-8.0) and salt concentrations (100-500 mM NaCl), while including stabilizing agents such as glycerol (5-10%) and reducing agents like DTT or β-mercaptoethanol (1-5 mM) to prevent disulfide bond formation. For affinity chromatography, if using His-tagged frr, employ gradient elution with imidazole (20-500 mM) rather than step elution to achieve higher purity. Following IMAC, implement size exclusion chromatography using analytical-grade columns (Superdex 75 or 200) to remove aggregates and contaminants while simultaneously assessing oligomeric state. Throughout purification, monitor protein stability using dynamic light scattering (DLS) to detect aggregation and thermal shift assays (TSA) to identify optimal buffer conditions. Activity assays should be established based on frr's known function in ribosome recycling, such as measuring the release of mRNA from post-termination complexes or assessing polysome breakdown in the presence of purified frr and EF-G.

What controls are essential when studying frr's role in L. pneumophila virulence?

When investigating frr's contribution to L. pneumophila virulence, a comprehensive set of controls is essential to ensure experimental validity and accurate interpretation of results. Genetic complementation controls should include both a negative control (frr deletion strain) and a positive control (deletion strain complemented with wild-type frr) to confirm phenotype specificity. Additionally, complementation with catalytically inactive frr mutants can distinguish between structural and enzymatic functions. When performing infection studies, include appropriate bacterial strain controls such as Lp02 rpsL WT and Lp02 rpsL K88R strains that display differential growth in macrophages , alongside your frr experimental strains. Host cell controls should encompass both permissive and restrictive cell types, such as BMDMs from wild-type and Hmg20a-deficient mice . For molecular interaction studies, incorporate controls for non-specific binding, including irrelevant proteins of similar size and charge characteristics to frr. Time-course experiments are crucial when assessing intracellular growth, with sampling at multiple time points (e.g., 24, 48, and 72 hours post-infection) to capture dynamic effects, as demonstrated in studies of L. pneumophila replication in BMDMs .

How can researchers address contradictory results in frr functional studies?

Addressing contradictory results in L. pneumophila frr functional studies requires systematic investigation of experimental variables and careful consideration of biological context. First, examine strain background differences, as L. pneumophila isolates vary in virulence properties, and laboratory strains may contain mutations affecting frr function or its interaction partners. For instance, the rpsL allele status (WT, K43N, or K88R) dramatically influences L. pneumophila's ability to replicate in BMDMs . Second, evaluate differences in experimental conditions, particularly culture media composition, growth phase of bacterial cultures, and host cell types used for infection studies. The detection methods employed can also contribute to discrepancies, so compare results obtained using different techniques such as CFU counting, microscopy-based vacuole size measurement, and flow cytometry. Genetic approaches should include complementation with frr variants containing point mutations in functional domains to pinpoint specific activity requirements. When contradictions persist, consider strain-specific genetic modifiers or host factors that may influence frr function, such as Hmg20a or Nol9 which were identified as restrictors of L. pneumophila growth in BMDMs .

How might frr interact with Dot/Icm effectors during L. pneumophila infection?

The potential interaction between frr and the Dot/Icm type IV secretion system effectors represents an unexplored frontier in understanding L. pneumophila pathogenesis. Ribosome-recycling factor may influence the translation efficiency of specific Dot/Icm effectors, creating a regulatory layer that coordinates effector production with different stages of intracellular infection. To investigate this relationship, researchers should employ ribosome profiling to examine translation efficiency of Dot/Icm effector mRNAs in wild-type versus frr-depleted or mutant L. pneumophila strains during infection. Particular attention should be paid to effectors with identified roles in manipulating host endosomal trafficking, such as SulF (Lpg2832), which was shown to recruit SUMOylated Rab7 to the Legionella-containing vacuole (LCV) . Co-immunoprecipitation experiments using epitope-tagged frr can identify direct interactions with Dot/Icm effectors or components of the secretion apparatus. Proximity labeling approaches using BioID or APEX2 fused to frr could capture transient interactions within the bacterial cell. Functional studies comparing the secretion efficiency and intracellular localization of selected Dot/Icm effectors between wild-type and frr-mutant strains would reveal whether frr influences the Dot/Icm system beyond translational control.

What role might frr play in stress adaptation during L. pneumophila's environmental-host transition?

The transition of L. pneumophila between environmental reservoirs and mammalian hosts represents a significant stress adaptation that likely involves translational reprogramming mediated in part by frr. During this transition, bacteria encounter temperature shifts, nutrient availability changes, and immune defenses that necessitate rapid proteomic adjustments. To investigate frr's role in this process, researchers should analyze frr expression and activity under conditions mimicking different phases of the L. pneumophila lifecycle, including various growth temperatures (25°C for environmental, 37°C for mammalian hosts), nutrient limitations, and exposure to host cell lysates. Ribosome profiling comparing environmental and host-like conditions can reveal translational shifts potentially mediated by frr. Creating L. pneumophila strains with inducible or temperature-sensitive frr variants would allow experimental manipulation of frr activity during host transition. Particular attention should be paid to translational efficiency of genes involved in the stringent response and those encoding proteins critical for intracellular survival, such as those involved in recruiting SUMOylated Rab7 to establish replication-permissive vacuoles .

How can structural information about L. pneumophila frr inform antimicrobial development?

Leveraging structural information about L. pneumophila frr offers promising avenues for developing targeted antimicrobials that could disrupt the bacterial translation cycle with minimal impact on host processes. X-ray crystallography and cryo-electron microscopy should be employed to determine the three-dimensional structure of L. pneumophila frr, both in isolation and in complex with its binding partners, including the bacterial ribosome and elongation factor G. Structure determination should focus on identifying unique structural features or binding pockets that differ from human translation factors, providing potential targets for selective inhibition. In silico molecular docking studies using virtual compound libraries can screen for molecules that bind to these unique sites, followed by biochemical validation using thermal shift assays and activity inhibition measurements. Structure-activity relationship studies of promising compounds can guide medicinal chemistry optimization. Given the importance of translation in bacterial virulence, targeting frr might offer advantages over targeting other components like RpsL, mutations in which can confer both antibiotic resistance and altered virulence properties as observed with the K43N and K88R variants .

How might genome-wide approaches reveal broader roles of frr in L. pneumophila biology?

Genome-wide approaches offer powerful tools to uncover the extensive network of interactions and functions involving frr in L. pneumophila biology beyond its canonical role in ribosome recycling. Transposon sequencing (Tn-seq) comparing wild-type and frr-depleted conditions can identify genes with synthetic lethal or suppressive genetic interactions with frr, revealing unexpected functional connections. RNA-seq and ribosome profiling can provide comprehensive views of transcriptional and translational landscapes altered by frr mutation or depletion, potentially uncovering regulatory roles in specific gene expression programs. Interactome mapping using proximity-dependent biotinylation followed by mass spectrometry can reveal the protein interaction network of frr within the bacterial cell. Chromatin immunoprecipitation sequencing (ChIP-seq) could investigate whether frr has moonlighting functions in transcriptional regulation, as has been discovered for other translation factors. These approaches should be applied across different growth conditions relevant to L. pneumophila's lifecycle, including growth in laboratory media, biofilm formation, and intracellular replication in host cells like BMDMs, where distinct host factors such as Hmg20a have been shown to restrict bacterial growth .

What are the implications of frr conservation across Legionella species for host range determination?

The conservation pattern of frr across different Legionella species presents an intriguing avenue for understanding host range determination and species-specific virulence characteristics. Comparative genomic analysis should examine frr sequence conservation, focusing on species-specific variations in domains that interact with the ribosome or other partners. Particular attention should be paid to correlations between frr sequence variations and known host range differences between Legionella species. Functional complementation experiments exchanging frr genes between Legionella species with different host ranges, followed by infection studies in various host cell types, could reveal whether frr contributes to host specificity. Structural biology approaches comparing frr proteins from different Legionella species bound to their cognate ribosomes may uncover species-specific interaction interfaces. Given that L. pneumophila strain variation in ribosomal protein RpsL significantly impacts intracellular replication capacity in BMDMs , investigating whether frr exhibits similar strain-specific adaptations could provide insights into evolutionary pressures shaping Legionella-host interactions.