Recombinant Bartonella quintana Phenylalanine--tRNA ligase alpha subunit (pheS)

What is the Phenylalanine-tRNA ligase alpha subunit (pheS) and its function in Bartonella quintana?

The Phenylalanine-tRNA ligase alpha subunit (pheS) is a critical component of the enzymatic machinery responsible for aminoacylation in Bartonella quintana. This protein functions as part of the phenylalanyl-tRNA synthetase complex (PheRS), which catalyzes the attachment of phenylalanine to its cognate tRNA molecules during protein synthesis. The enzyme has an EC designation of 6.1.1.20 and is essential for bacterial survival as it ensures the correct incorporation of phenylalanine into growing polypeptide chains during translation . In pathogenic bacteria like B. quintana, the causative agent of trench fever, this protein plays a vital role in maintaining protein synthesis under the varying environmental conditions encountered during the infectious cycle.

What expression systems are most effective for producing recombinant B. quintana pheS protein?

E. coli expression systems are generally most effective for producing recombinant B. quintana pheS, as demonstrated by successful expression of the homologous protein from B. bacilliformis . When designing expression constructs, researchers should consider using a full-length protein approach (amino acids 1-361 based on B. bacilliformis homolog) with appropriate fusion tags to aid purification . Expression vectors containing T7 or similar strong promoters with inducible control provide optimal results. Temperature modulation during expression is crucial, as B. quintana naturally transitions between 37°C (human host) and 28°C (body louse vector), with significant gene expression changes occurring at these different temperatures . Expression at lower temperatures (16-28°C) often improves protein solubility and proper folding for this type of bacterial protein.

What purification strategies yield the highest purity for recombinant B. quintana pheS protein?

A multi-step purification strategy typically yields the highest purity for recombinant B. quintana pheS. Based on protocols used for similar proteins:

• Initial capture: Affinity chromatography using appropriate tags (His, GST, or other fusion tags determined during the manufacturing process)

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing step: Size exclusion chromatography to remove aggregates and achieve final purity

This approach typically yields purity levels >85% as measured by SDS-PAGE . For analytical applications requiring higher purity, additional steps such as hydroxyapatite chromatography may be necessary. The purified protein should be stored with 5-50% glycerol at -20°C/-80°C to maintain stability, with shelf life typically extending to 6 months for liquid formulations and 12 months for lyophilized preparations .

What role might pheS play in B. quintana's adaptation to high hemin concentrations encountered in the louse vector?

While pheS is primarily involved in protein synthesis, it may indirectly contribute to B. quintana's adaptation to the hemin-rich environment of the body louse gut. B. quintana has the highest reported in vitro hemin requirement of any bacterium and possesses specific adaptations for hemin acquisition . The relationship between protein synthesis machinery and hemin adaptation may involve several mechanisms:

• Stress Response Coordination: Hemin toxicity triggers stress responses that require rapid protein synthesis adaptation, potentially involving pheS regulation.

• Specialized Protein Expression: The expression of hemin-handling proteins (such as HbpA) increases in high-hemin environments, placing demands on the translation machinery including pheS .

• Regulatory Network Integration: Transcription factors like RpoE, which are upregulated in response to high hemin concentrations, may indirectly influence pheS expression or activity .

Experimental approaches to investigate this relationship should include comparative proteomic analysis of B. quintana grown under varying hemin concentrations, assessment of translation fidelity under hemin stress, and investigation of potential physical interactions between pheS and hemin-binding proteins .

What experimental approaches can identify potential inhibitors of B. quintana pheS as antimicrobial targets?

B. quintana pheS represents a potential antimicrobial target due to its essential role in protein synthesis. Several experimental approaches can identify effective inhibitors:

• High-throughput Screening (HTS):

  • In vitro aminoacylation assays using purified recombinant pheS and appropriate tRNA substrates

  • Fluorescence-based assays measuring ATP consumption or pyrophosphate release

  • Thermal shift assays to identify compounds that alter protein stability

• Structure-based Drug Design:

  • X-ray crystallography or cryo-EM to determine B. quintana pheS structure

  • Molecular docking studies using the active site or allosteric pockets

  • Fragment-based screening focused on the catalytic domain

• Validation Approaches:

  • Growth inhibition assays using B. quintana cultures

  • Time-kill assays to assess bactericidal versus bacteriostatic effects

  • Mutation frequency studies to assess resistance development

• Selectivity Assessment:

  • Comparative inhibition studies using human phenylalanyl-tRNA synthetase

  • Cytotoxicity testing in mammalian cell lines

  • In silico analysis of structural differences between bacterial and human orthologs

Successful inhibitors would likely target unique structural features of the bacterial pheS while minimizing interaction with the human ortholog.

What are the optimal storage conditions for maintaining the stability and activity of purified recombinant B. quintana pheS?

The optimal storage conditions for maintaining stability and activity of purified recombinant B. quintana pheS require careful consideration of buffer components, temperature, and handling protocols. Based on information from similar recombinant proteins:

Storage Buffer Components:

• Base buffer: 20-50 mM Tris-HCl or phosphate buffer (pH 7.0-8.0)

• Salt: 100-300 mM NaCl to maintain solubility

• Glycerol: 5-50% (final concentration) to prevent freeze damage and maintain stability

• Reducing agent: 1-5 mM DTT or 2-mercaptoethanol to protect thiol groups

• Optional additives: 0.1-1 mM EDTA to chelate metal ions that could promote oxidation

Temperature Considerations:

• Long-term storage: -80°C (preferred) or -20°C

• Working aliquots: 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly decrease protein activity

Handling Recommendations:

• Centrifuge vials briefly before opening to bring contents to the bottom

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

• Divide into single-use aliquots before freezing

• Document preparation date and number of freeze-thaw cycles

The typical shelf life is approximately 6 months for liquid preparations at -20°C/-80°C and 12 months for lyophilized formulations .

How can researchers optimize enzymatic assays to measure B. quintana pheS aminoacylation activity?

Optimizing enzymatic assays for B. quintana pheS aminoacylation activity requires careful consideration of multiple parameters:

Assay Components:

• Enzyme preparation: Purified recombinant pheS (concentration range: 10-500 nM)

• Substrates:

  • L-phenylalanine (typical range: 0.1-5 mM)

  • ATP (typical range: 1-5 mM)

  • tRNA^Phe (either purified from E. coli or in vitro transcribed, 0.5-10 μM)

• Buffer conditions:

  • pH range: 7.0-8.0 (optimal typically around 7.5)

  • Divalent cations: Mg^2+ (5-20 mM) is essential

  • Monovalent cations: K^+ (50-100 mM) often enhances activity

  • Temperature: Test both 28°C and 37°C to reflect host environments

Detection Methods:

• Radioactive assays:

  • [^14C] or [^3H]-labeled phenylalanine incorporation into tRNA

  • Filter-binding assay followed by scintillation counting

• Non-radioactive alternatives:

  • Pyrophosphate release coupled to colorimetric/fluorescent detection

  • HPLC analysis of aminoacylated vs. non-aminoacylated tRNA

  • Mass spectrometry to detect charged tRNA

Optimization Tips:

• Conduct initial enzyme concentration and time-course experiments to establish linear range

• Include controls lacking individual components (enzyme, ATP, amino acid, tRNA)

• Consider the influence of temperature on enzyme kinetics, as B. quintana experiences different temperatures in its life cycle

• Test for potential inhibition by high concentrations of substrates or byproducts

What techniques are most effective for studying protein-protein interactions involving B. quintana pheS in its native context?

Studying protein-protein interactions involving B. quintana pheS in its native context requires approaches that can detect both stable and transient interactions while maintaining physiological relevance:

In vivo Approaches:

• Bacterial Two-Hybrid (B2H) System:

  • Modified for use in Bartonella or in heterologous hosts like E. coli

  • Allows detection of direct binary interactions

  • Can be adapted for screening interaction partners from genomic libraries

• Cross-linking coupled with Mass Spectrometry (XL-MS):

  • Chemical cross-linkers applied to intact B. quintana cells

  • Preserves native interactions before cell disruption

  • MS/MS analysis identifies cross-linked peptides and interaction sites

• Co-immunoprecipitation from B. quintana lysates:

  • Requires antibodies against B. quintana pheS or epitope tags

  • Can identify components of larger complexes

  • Western blotting or MS to identify co-precipitated proteins

In vitro Approaches:

• Surface Plasmon Resonance (SPR):

  • Real-time binding kinetics between purified pheS and potential partners

  • Quantitative KD, kon, and koff measurements

  • Requires purified, functional proteins

• Microscale Thermophoresis (MST):

  • Detects interactions based on changes in thermal mobility

  • Minimal protein consumption

  • Can be performed in complex buffers mimicking cellular conditions

• Analytical Ultracentrifugation (AUC):

  • Characterizes complex formation in solution

  • Provides stoichiometry information

  • No immobilization required

Specialized Approaches for Bartonella:

• Consider temperature effects (28°C vs 37°C) on interactions

• Evaluate the impact of hemin concentration on protein complexes

• Account for membrane association when studying interactions involving membrane-proximal proteins

For pheS specifically, researchers should focus on interactions with the beta subunit (pheT), other translation machinery components, and potential regulatory factors that might respond to environmental signals encountered by B. quintana during its lifecycle.

What are the most promising future research directions for B. quintana pheS in pathogenesis and therapeutic development?

Future research on B. quintana pheS holds significant promise in several key areas:

• Pathogenesis Understanding:

  • Investigating how translation fidelity via pheS contributes to B. quintana adaptation in diverse host environments

  • Determining whether pheS activity is modulated during infection stages

  • Exploring potential moonlighting functions beyond aminoacylation, as observed with some tRNA synthetases in other pathogens

• Therapeutic Development:

  • Structure-guided design of selective inhibitors targeting unique features of B. quintana pheS

  • Development of attenuated vaccine strains through engineered pheS modifications

  • Exploration of antimicrobial peptides derived from pheS structural analysis

• Diagnostic Applications:

  • Development of serological tests based on immunogenic epitopes of pheS

  • Design of nucleic acid amplification tests targeting the pheS gene region

  • Exploration of pheS as a biomarker for active B. quintana infection

• Biotechnology Applications:

  • Engineering B. quintana pheS for incorporation of non-canonical amino acids

  • Development of in vitro translation systems optimized for extreme conditions

  • Exploration of pheS as a potential tool for directed evolution experiments