Recombinant Legionella pneumophila subsp. pneumophila Phenylalanine--tRNA ligase beta subunit (pheT), partial

What is the biological role of Phenylalanine--tRNA ligase beta subunit in Legionella pneumophila?

Phenylalanine--tRNA ligase beta subunit (pheT) is an essential component of the heterodimeric enzyme phenylalanyl-tRNA synthetase (PheRS), which catalyzes the attachment of phenylalanine to its cognate tRNA during protein synthesis. In L. pneumophila, this process is critical for bacterial survival and virulence. The enzyme ensures translational fidelity by correctly charging tRNA molecules with phenylalanine, preventing misincorporation of amino acids during protein synthesis. This function is particularly important in L. pneumophila as the bacterium must adapt to different environments during its lifecycle, including survival within host cells. Unlike many bacterial proteins that may serve dual roles as virulence factors, pheT primarily functions in maintaining proper protein synthesis, though its activity is indirectly essential for the expression of virulence factors.

What are the recommended expression systems for producing recombinant L. pneumophila pheT?

For successful expression of recombinant L. pneumophila pheT, several expression systems have proven effective. E. coli-based expression systems remain the most widely used due to their efficiency and scalability. When expressing L. pneumophila proteins in E. coli, researchers should consider using strains optimized for expression of proteins from organisms with different codon usage patterns, such as BL21(DE3)pLysS or Rosetta strains. Expression can be induced using IPTG (500 μM) for approximately 3 hours, similar to protocols used for other L. pneumophila recombinant proteins . For maintaining proper folding, co-expression with chaperones may be beneficial, particularly when working with larger proteins or those prone to inclusion body formation.

Alternative systems include expression in L. pneumophila itself using inducible plasmids, which provides the native cellular environment but typically yields lower protein amounts. When higher eukaryotic post-translational modifications are suspected to be important, yeast or insect cell systems may be more appropriate. For functional studies requiring native conformation, expression in the original host is preferable, though this approach presents technical challenges due to L. pneumophila's specialized growth requirements.

What purification strategies are most effective for L. pneumophila pheT?

Purification of recombinant L. pneumophila pheT typically employs a multi-step approach. The most effective initial purification step is affinity chromatography using a fusion tag. Histidine tags (6xHis) are commonly employed due to their small size and minimal interference with protein function. Following cell lysis using methods such as sonication or French press, the clarified lysate should undergo nickel or cobalt affinity chromatography.

For higher purity requirements, a secondary purification step is recommended, typically ion exchange chromatography based on pheT's theoretical isoelectric point. Size exclusion chromatography serves as an excellent final polishing step to remove aggregates and obtain homogeneous protein. Throughout the purification process, it's crucial to maintain reducing conditions (typically with DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues. Purification buffers should be optimized to maintain stability, often containing 10-20% glycerol and protease inhibitors.

After purification, protein identity should be confirmed via mass spectrometry and Western blotting, while activity can be assessed through aminoacylation assays. Researchers should note that unlike some L. pneumophila proteins that have been reported to have dual functions (such as certain toxin-antitoxin system components), pheT functions primarily as an enzyme rather than as a virulence factor translocated into host cells .

How can researchers assess the functional activity of purified recombinant pheT?

Assessing the functional activity of purified recombinant L. pneumophila pheT requires verification of its aminoacylation activity. The standard approach involves an aminoacylation assay measuring the enzyme's ability to charge tRNAPhe with [14C]- or [3H]-labeled phenylalanine. This reaction typically contains purified pheT (along with the alpha subunit if testing the heterodimeric complex), L. pneumophila tRNAPhe (or commercial tRNAPhe), ATP, labeled phenylalanine, and appropriate buffers with magnesium.

After incubation at optimal temperature (usually 37°C), the reaction is stopped using trichloroacetic acid precipitation. The precipitated aminoacylated tRNA is collected on filter disks, washed, and quantified by scintillation counting. Alternatively, researchers can employ a pyrophosphate release assay, which measures the inorganic pyrophosphate produced during the aminoacylation reaction using coupled enzymatic reactions.

For more detailed mechanistic studies, pre-steady-state kinetics using rapid quench techniques can provide insights into the individual steps of the aminoacylation process. Circular dichroism spectroscopy should be used to verify proper folding of the recombinant protein, while thermal shift assays can assess stability under different buffer conditions.

What strategies can overcome solubility and stability challenges with recombinant pheT?

Recombinant L. pneumophila pheT, like many bacterial enzymes, may present solubility and stability challenges during expression and purification. Several strategies can address these issues:

• Co-expression with molecular chaperones: Chaperones like GroEL/GroES (similar to L. pneumophila's own HtpB chaperonin) can significantly improve proper folding and solubility . L. pneumophila HtpB has been shown to facilitate proper protein folding and could potentially be co-expressed with pheT.

• Fusion partners: Solubility-enhancing fusion tags such as MBP (maltose-binding protein), GST (glutathione S-transferase), or SUMO can dramatically improve solubility. These can be cleaved post-purification using specific proteases.

• Buffer optimization: Systematic screening of buffer conditions (pH, salt concentration, additives) is crucial. Typically, pheT stability benefits from the inclusion of 10-20% glycerol, reducing agents (1-5 mM DTT), and moderate salt concentrations (100-300 mM NaCl).

• Expression temperature: Lowering the expression temperature to 16-20°C significantly reduces inclusion body formation, though it extends expression time to 16-24 hours.

• Refolding protocols: If inclusion bodies persist, denaturation followed by controlled refolding can recover active protein. Gradual dialysis against decreasing concentrations of denaturants while maintaining reducing conditions often yields positive results.

• Co-purification with the alpha subunit: As pheT naturally functions as part of a heterodimeric complex, co-expression and co-purification with the alpha subunit (pheS) may enhance stability and solubility.

How can researchers investigate potential moonlighting functions of L. pneumophila pheT during infection?

Investigating potential moonlighting functions of L. pneumophila pheT during infection requires sophisticated experimental approaches to distinguish its canonical enzymatic role from possible non-canonical functions:

• Translocation assays: To determine whether pheT is translocated into host cells during infection, researchers should employ TEM-1 β-lactamase translocation assays. By creating a pheT-TEM-1 fusion protein expressed in L. pneumophila, researchers can monitor translocation into host cells using the fluorescent substrate CCF2/AM. As demonstrated with other L. pneumophila proteins, a blue/green fluorescence ratio > 1 would indicate translocation .

• Site-directed mutagenesis: Create separation-of-function mutants by introducing mutations that disrupt the aminoacylation activity without affecting potential protein-protein interactions. This approach helps differentiate between phenotypes resulting from loss of aminoacylation versus loss of potential moonlighting functions.

• Protein-protein interaction studies: Employ pull-down assays, co-immunoprecipitation, or bacterial two-hybrid systems to identify potential interaction partners within host cells. Follow up with proximity labeling techniques like BioID or APEX to validate interactions in the cellular context.

• Conditional expression systems: Develop strains where pheT expression can be modulated during different infection stages to determine temporal requirements for pheT functions.

• Subcellular localization: Use immunofluorescence microscopy with anti-pheT antibodies to track the localization of pheT within host cells at different infection timepoints, comparing with markers for various subcellular compartments.

Unlike some L. pneumophila proteins that have been conclusively shown to have dual functions inside and outside bacterial cells, careful controls should be included to distinguish genuine moonlighting functions from experimental artifacts .

What approaches are recommended for studying pheT genetic variations across clinical L. pneumophila isolates?

Studying genetic variations in pheT across clinical L. pneumophila isolates requires comprehensive sequencing and comparative analysis approaches:

This approach has proven effective for obtaining sequence information directly from respiratory secretions of patients, circumventing the need for bacterial isolation and culture .

What experimental designs effectively assess the impact of pheT inhibition on L. pneumophila virulence?

Assessing the impact of pheT inhibition on L. pneumophila virulence requires carefully designed experimental approaches:

• Gene knockdown/conditional expression: Since pheT is likely essential, complete deletion may not be viable. Instead, implement an inducible antisense RNA system or CRISPR interference (CRISPRi) to achieve tunable repression of pheT expression. Alternatively, create temperature-sensitive mutants that maintain function under permissive conditions but lose function when shifted to non-permissive temperatures.

• Chemical inhibition: Screen for small molecule inhibitors specific to L. pneumophila pheT using high-throughput enzymatic assays. Validated inhibitors can then be tested in infection models, with appropriate controls to ensure specificity.

• Infection models: Utilize established infection models including:

  • U937-derived macrophages, which can be differentiated into adherent, macrophage-like cells with phorbol 12-myristate 13-acetate (PMA)

  • Murine models of L. pneumophila pneumonia, which allow assessment of neutrophil recruitment and cytokine responses

  • Acanthamoeba castellanii, a natural host for L. pneumophila that provides insights into environmental persistence

• Virulence parameters to monitor include:

  • Intracellular replication rates

  • Phagosome biogenesis modifications

  • Cytokine profiles (particularly IFN-γ and IL-12 levels)

  • Recruitment of organelles such as mitochondria to phagosomes

  • Host cell death mechanisms

• Genetic complementation: To confirm phenotypes result from pheT inhibition rather than off-target effects, perform genetic complementation with wild-type pheT or heterologous aminoacyl-tRNA synthetases.

• Temporal assessment: Examine the effects of pheT inhibition at different stages of infection to distinguish between roles in invasion, intracellular survival, replication, and cell-to-cell spread.

This multi-faceted approach provides comprehensive insights into the relationship between aminoacyl-tRNA synthetase activity and bacterial virulence, potentially revealing new therapeutic targets.

How can researchers differentiate between host responses to L. pneumophila pheT versus other bacterial components?

Differentiating host responses specifically triggered by L. pneumophila pheT versus other bacterial components requires sophisticated experimental designs:

• Purified protein studies: Compare host cell responses to purified recombinant pheT versus other L. pneumophila proteins using:

  • Bead coating approach: Coat latex beads with purified pheT and track their fate within host cells, as has been done with other L. pneumophila proteins like HtpB

  • Microinjection/transfection: Deliver purified pheT directly into host cytoplasm and monitor cellular responses

• Targeted mutants: Create specific point mutations in pheT that affect different functional domains and assess their impact on host responses. Compare these to control strains with mutations in other L. pneumophila proteins.

• Transcriptomic/proteomic profiling: Employ RNA-Seq or proteomics to compare host response profiles between:

  • Cells exposed to wild-type L. pneumophila versus pheT-depleted strains

  • Cells exposed to purified pheT versus other purified L. pneumophila proteins

  • Cells expressing recombinant pheT versus control proteins

• Ectopic expression models: Generate stable transfected cell lines expressing L. pneumophila pheT, similar to the CHO-htpB cells used to study HtpB effects . This allows observation of pheT effects in isolation from other bacterial factors.

• Cytoskeletal and organelle tracking: Monitor specific host cell responses such as:

  • Mitochondrial recruitment to phagosomes (observed with some L. pneumophila proteins)

  • Actin microfilament rearrangements

  • Endoplasmic reticulum association

• Immunological assays: Measure specific immune responses including:

  • Cytokine production profiles (with particular attention to T1/T2 polarization)

  • Neutrophil recruitment and activation

  • Inflammasome activation

By employing these approaches systematically, researchers can delineate the specific contributions of pheT to host-pathogen interactions while controlling for effects mediated by other L. pneumophila components.

What cutting-edge approaches can reveal pheT's role in L. pneumophila stress adaptation and antibiotic resistance?

Investigating pheT's role in stress adaptation and antibiotic resistance requires integrating several advanced methodological approaches:

• Stress-responsive expression analysis: Monitor pheT expression levels using qRT-PCR or reporter constructs under various stress conditions relevant to L. pneumophila's lifecycle:

  • Nutrient limitation

  • Oxidative stress

  • Temperature shifts

  • Exposure to host defense molecules

  • Antimicrobial compounds

• Post-translational modification mapping: Employ mass spectrometry to identify stress-induced modifications of pheT, focusing on phosphorylation, acetylation, and other regulatory modifications. This approach has successfully identified phosphorylation targets of bacterial kinases like those in toxin-antitoxin systems .

• Mistranslation as adaptive mechanism: Investigate whether pheT accuracy changes under stress conditions by:

  • Pulse-labeling experiments with misincorporation-sensitive reporters

  • Mass spectrometry analysis of the proteome to detect phenylalanine misincorporation rates

  • In vitro aminoacylation assays under different pH, salt, and oxidative conditions

• Genetic approaches for stress resistance:

  • Create strains with altered pheT expression levels and test their survival under various stressors

  • Screen for suppressor mutations that restore growth to pheT-compromised strains

  • Implement CRISPR-based screens to identify genetic interactions with pheT under stress conditions

• Structural biology approaches:

  • Determine crystal structures of pheT under different conditions to identify conformational changes

  • Use hydrogen-deuterium exchange mass spectrometry to identify dynamic regions responsive to stress conditions

• Antibiotic interactions:

  • Assess minimum inhibitory concentration shifts for various antibiotics when pheT expression is altered

  • Investigate synergistic effects between aminoacyl-tRNA synthetase inhibitors and other antimicrobials

  • Explore potential connections between mistranslation rates and acquisition of antibiotic resistance

By integrating these approaches, researchers can develop a comprehensive understanding of how pheT contributes to L. pneumophila's remarkable adaptability across diverse environmental niches and in response to therapeutic interventions.