Recombinant Aeromonas salmonicida tRNA pseudouridine synthase A (truA)

What are the optimal conditions for expression and purification of recombinant truA?

Optimal expression of recombinant Aeromonas salmonicida truA can be achieved using an E. coli expression system with appropriate vectors containing strong promoters such as T7 or CMV. Based on current protocols, the following methodology is recommended:

• Expression system: Use E. coli BL21(DE3) or similar strains optimized for recombinant protein expression.

• Vector selection: pET-based vectors for bacterial expression or pAd-easy-cmv for mammalian expression (if testing interaction with eukaryotic systems).

• Induction conditions: For IPTG-inducible systems, induce at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG at 25-30°C for 4-6 hours to reduce inclusion body formation.

• Purification strategy:

  • Initial capture using affinity chromatography (His-tag or GST-tag depending on the construct)

  • Intermediate purification using ion exchange chromatography

  • Polishing step with size exclusion chromatography to ensure high purity (>85% as verified by SDS-PAGE)

• Buffer optimization: Use buffers containing 20-50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, 5% glycerol, and potentially 1-5 mM DTT or 2-mercaptoethanol to maintain stability.

Final purity should exceed 85% as assessed by SDS-PAGE, with expected yield of 2-5 mg per liter of bacterial culture. After purification, the protein should be stored with 5-50% glycerol at -20°C or -80°C to maintain stability and prevent repeated freeze-thaw cycles .

How can researchers assess the enzymatic activity of purified truA protein?

Assessment of truA enzymatic activity requires specialized assays that detect the conversion of uridine to pseudouridine in tRNA substrates. The following methodologies are recommended:

• Tritium release assay: This traditional method measures the release of tritium from [5-³H]uridine-labeled tRNA when converted to pseudouridine. The reaction mixture typically contains:

  • Purified truA enzyme (0.1-1 μM)

  • [5-³H]uridine-labeled tRNA substrate (1-5 μM)

  • Buffer: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 100 mM NH₄Cl

  • Incubation at 37°C for 30-60 minutes

• CMC-primer extension analysis: This method uses N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) to modify pseudouridine residues, followed by primer extension to detect modification sites.

• LC-MS/MS analysis: Liquid chromatography coupled with tandem mass spectrometry can be used to directly detect and quantify pseudouridine formation in digested tRNA samples.

• HPLC analysis: Nucleoside analysis by HPLC after nuclease digestion of modified tRNA can quantitatively measure pseudouridine levels.

Activity data should be analyzed using standard enzyme kinetics approaches to determine parameters such as Km, Vmax, and catalytic efficiency (kcat/Km). Typical assays should include positive controls (known active pseudouridine synthases) and negative controls (catalytically inactive mutants or heat-inactivated enzyme) .

How does truA contribute to pathogenicity mechanisms in Aeromonas salmonicida?

The relationship between truA and Aeromonas salmonicida pathogenicity involves several complex mechanisms:

• Translational fidelity in stress conditions: truA's pseudouridine modifications enhance translational accuracy under stress conditions encountered during host infection. This adaptation is crucial for proper expression of virulence factors in changing host environments.

• Co-regulation with virulence factors: Genomic analyses of Aeromonas species reveal that RNA modification enzymes like truA may be co-regulated with virulence-associated genes. In Aeromonas salmonicida, the genome contains numerous virulence factors including hemolysins, toxins like aerolysin (aerA), and various secretion systems components that work in concert with basic cellular machinery where truA functions .

• Persistence mechanisms: Proper tRNA modification is essential for bacterial stress responses and adaptation to hostile environments. truA-mediated modifications may enhance bacterial persistence during infection through improved translational control of stress response genes.

• Immunomodulation potential: While direct evidence is limited, bacterial tRNA modifications may influence host immune recognition patterns. In comparative studies of Aeromonas virulence, secretion system components and toxins are major contributors to pathogenicity, with potential functional connections to translation-related factors .

Research approaches to investigate these connections include:

• Creating targeted truA gene knockouts in Aeromonas salmonicida and assessing virulence in fish infection models

• Transcriptomic and proteomic profiling of wild-type versus truA-deficient strains

• Comparative analysis of pseudouridylation patterns under host-mimicking stress conditions

• Monitoring expression of known virulence factors in truA mutants

These investigations would complement existing research on other immunogenic Aeromonas components like the VapA protein, which has shown significant promise as a vaccine candidate against A. salmonicida infections .

What experimental approaches can be used to investigate the structural basis of truA substrate recognition?

Investigating the structural basis of truA substrate recognition requires a multifaceted approach combining biochemical, biophysical, and computational methods:

• X-ray crystallography:

  • Co-crystallization of truA with tRNA substrates or substrate analogs

  • Optimal conditions: 20-25% PEG 3350/4000, pH 6.5-8.0, 100-200 mM salt (NaCl or ammonium acetate)

  • Resolution target: 2.0-2.5 Å to visualize RNA-protein interactions

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful for capturing different conformational states of truA-tRNA complexes

  • Sample preparation: 3-5 μl of truA-tRNA complex (5-10 μM) on glow-discharged grids

  • Data collection parameters: 300 kV microscope, 0.5-1.0 e⁻/Ų per frame, 40 frames total

• Site-directed mutagenesis studies:

  • Systematic mutation of conserved residues identified through sequence alignment

  • Key targets include:

    • Catalytic site residues (particularly the conserved aspartate)

    • Residues in the putative RNA-binding pocket

    • Interface residues involved in potential dimerization

• RNA footprinting assays:

  • SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension)

  • Hydroxyl radical footprinting

  • Dimethyl sulfate (DMS) probing

• Molecular dynamics simulations:

  • All-atom simulations of truA-tRNA complexes (100-500 ns)

  • Analysis of hydrogen bonding, electrostatic interactions, and conformational changes

The combined data from these approaches would enable construction of a comprehensive model of substrate recognition and catalysis, potentially revealing unique features of Aeromonas salmonicida truA compared to homologs in other bacterial species .

What are the critical storage and handling considerations for maintaining truA stability and activity?

Proper storage and handling of recombinant Aeromonas salmonicida truA are crucial for maintaining its stability and enzymatic activity:

Storage recommendations:

• Short-term storage (1-7 days): Store working aliquots at 4°C in appropriate buffer (typically 20-50 mM Tris-HCl pH 7.5, 100-150 mM NaCl, 1 mM DTT, 5-10% glycerol).

• Medium-term storage (1-6 months): Store at -20°C with 20-30% glycerol as a cryoprotectant.

• Long-term storage (>6 months): Store at -80°C with 50% glycerol in small aliquots (50-100 μl) to minimize freeze-thaw cycles.

Critical handling considerations:

• Avoid repeated freeze-thaw cycles: Each freeze-thaw cycle can reduce activity by 10-15%. Prepare single-use aliquots during initial purification.

• Temperature sensitivity: Maintain samples on ice during experiments and avoid exposure to temperatures above 25°C for extended periods.

• Oxidation sensitivity: Include reducing agents (1-5 mM DTT, 2-mercaptoethanol, or TCEP) in all buffers.

• Protein concentration: Maintain concentration between 0.1-1.0 mg/mL for optimal stability.

• pH stability range: Optimal stability is typically observed at pH 7.0-8.5.

Reconstitution protocol:

• Centrifuge vials briefly before opening to collect contents at the bottom.

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (default recommendation is 50%).

• Allow complete rehydration before use (15-30 minutes at 4°C).

Stability indicators:

• Appearance: Clear to slightly opalescent solution without visible precipitates

• Activity retention: >80% of initial activity after storage under recommended conditions for 3 months

• Purity maintenance: >85% as assessed by SDS-PAGE

How can researchers troubleshoot low activity or instability issues with recombinant truA?

When encountering issues with recombinant Aeromonas salmonicida truA activity or stability, researchers should systematically address potential problems:

Troubleshooting low enzymatic activity:

Stability enhancement strategies:

• Additive screening: Systematically test various additives known to enhance protein stability:

  • Polyols (5-10% glycerol, 0.5-2 M sorbitol)

  • Amino acids (50-100 mM arginine, proline)

  • Sugars (0.5-1 M sucrose, trehalose)

  • Non-ionic detergents (0.05-0.1% Triton X-100, NP-40)

• Buffer optimization: Test various buffer systems beyond the standard Tris-HCl:

  • HEPES (pH 7.0-8.0)

  • Sodium phosphate (pH 6.5-7.5)

  • MOPS (pH 6.5-7.5)

• Protein engineering approaches:

  • Introduce stabilizing mutations based on homology models

  • Create fusion constructs with stabilizing partners (MBP, thioredoxin)

  • Consider truncation constructs that remove flexible regions while preserving the catalytic core

When assessing activity improvements, it's essential to use consistent assay conditions and include appropriate controls to accurately quantify changes in enzyme performance across different optimization conditions .

How can truA research contribute to developing novel antimicrobial strategies against Aeromonas infections?

Research on Aeromonas salmonicida truA offers several promising avenues for developing novel antimicrobial strategies:

• Structure-based inhibitor design: The crystal structure of truA can serve as a template for rational design of small molecule inhibitors that specifically target the active site or RNA-binding interface. Computational approaches including molecular docking, virtual screening, and fragment-based drug design can identify lead compounds with potential to disrupt truA function with minimal impact on host enzymes.

• RNA modification as virulence attenuator: Creating attenuated Aeromonas strains with modified truA activity could potentially serve as live attenuated vaccine candidates. The connection between tRNA modification and bacterial adaptation to host environments suggests that truA-attenuated strains might maintain immunogenicity while displaying reduced virulence .

• Combination therapy approaches: Research indicates that targeting RNA modification pathways may sensitize bacteria to existing antibiotics by reducing translational fidelity under stress. Experimental designs should test combinations of truA inhibitors with conventional antibiotics like tetracyclines or fluoroquinolones against Aeromonas isolates, particularly those showing increased resistance traits .

• Biofilm disruption strategies: tRNA modifications have been implicated in bacterial biofilm formation. Investigating the role of truA in Aeromonas biofilm development could reveal targets for anti-biofilm therapies, which are particularly relevant for aquaculture settings where biofilms facilitate persistence.

• Diagnostic applications: Understanding truA sequence variation across Aeromonas species and strains could support development of molecular diagnostic tools for rapid identification of pathogenic Aeromonas in aquaculture facilities.

These approaches complement existing vaccine development efforts, such as the recombinant adenovirus vaccine harboring the Vapa gene, which has shown promise in reducing Aeromonas salmonicida mortality in rainbow trout (40% mortality in vaccinated fish compared to 76.6% in control groups) . Integration of these strategies could address the continuing challenge of furunculosis in global fisheries.

What comparative genomics approaches can reveal the evolutionary significance of truA in Aeromonas species?

Comparative genomics approaches provide powerful tools for understanding the evolutionary significance of truA across Aeromonas species:

• Phylogenetic analysis methodologies:

  • Multiple sequence alignment of truA sequences from diverse Aeromonas species/strains

  • Maximum likelihood or Bayesian inference phylogenetic tree construction

  • Selection pressure analysis using dN/dS ratios to identify positively selected residues

  • Ancestral sequence reconstruction to trace evolutionary changes

• Synteny analysis:

  • Examination of the genomic context of truA across Aeromonas species

  • Identification of conserved gene neighborhoods and potential operonic structures

  • Investigation of horizontal gene transfer events affecting truA or adjacent genes

• Structure-function correlation:

  • Mapping of conserved versus variable regions onto protein structure models

  • Identification of species-specific insertions/deletions with potential functional significance

  • Correlation of structural variations with host range or environmental adaptations

• Experimental validation approaches:

  • Cross-species complementation studies with truA orthologs

  • Activity assays comparing substrate specificity and catalytic efficiency

  • Generation of chimeric enzymes to identify domains responsible for species-specific functions

Recent comparative genomics studies of Aeromonas virulence factors have revealed significant insights into pathogenicity mechanisms, including the identification of type III secretion system components (AscF, AscG, AscV) and toxins like ADP-ribosyltransferase (AexT) . Similar approaches applied to truA could establish connections between RNA modification capabilities and virulence potential across the genus.

A comprehensive comparative analysis would ideally include both pathogenic and non-pathogenic Aeromonas species, with particular attention to:

• A. salmonicida (fish pathogen)

• A. hydrophila (opportunistic human pathogen)

• A. dhakensis (emerging pathogen)

• Environmental Aeromonas isolates

This evolutionary framework would provide context for understanding species-specific adaptations in RNA modification pathways and their relationship to host adaptation and virulence mechanisms .

How does truA research intersect with experimental design approaches for vaccine development against Aeromonas salmonicida?

The intersection of truA research with vaccine development against Aeromonas salmonicida reveals several promising research directions:

• True experimental design applications:
  Modern vaccine development requires rigorous experimental designs to establish causal relationships between specific antigens and protective immunity. True experimental designs, characterized by random assignment, control groups, and manipulation of independent variables, are essential for evaluating truA-based vaccine candidates . Key experimental design elements include:

  • Random assignment: Ensuring fish are randomly allocated to experimental groups to minimize selection bias

  • Appropriate controls: Including both negative controls (unvaccinated, empty vector) and positive controls (established vaccine formulations)

  • Pre-test and post-test measurements: Assessing immune parameters before and after vaccination

  • Standardized challenge models: Using consistent pathogen doses and challenge methods

• truA as a potential vaccine component:
  While traditional Aeromonas vaccines have focused on surface antigens like VapA, exploring conserved metabolic enzymes like truA could offer several advantages:

  • Sequence conservation across strains may provide broader protection

  • Essential metabolic function reduces likelihood of escape mutations

  • Potential for T-cell mediated responses in addition to antibody production

• Adjuvant effects on truA immunogenicity:
  Research should investigate how different adjuvant formulations affect the immunogenicity of truA-based vaccines:

  • Oil-based adjuvants (typical for fish vaccines)

  • Aluminum salts

  • TLR agonists

  • Cytokine adjuvants

• Delivery system optimization:
  Similar to successful approaches with VapA gene delivery, recombinant viral vectors expressing truA could be explored:

  • Adenovirus vectors (as used for VapA with 36.6% improved survival)

  • DNA vaccines

  • mRNA-based approaches

  • Virus-like particles as carriers

• Immune response assessment:
  Comprehensive evaluation of truA-based vaccines would require:

  • Measurement of specific antibody titers in peripheral blood

  • Quantification of IgM and IgT levels in key immune tissues (head kidney, hindgut)

  • T-cell response analysis

  • Challenge studies to determine relative percent survival

The application of true experimental designs in this context would ensure that any observed protection can be confidently attributed to the truA component of the vaccine, rather than to confounding variables. This methodological rigor is essential for advancing from preliminary findings to practical vaccine candidates for aquaculture applications .

What methodological considerations are important when investigating truA interactions with host immune systems?

Investigating interactions between Aeromonas salmonicida truA and host immune systems requires careful methodological considerations:

• In vitro immunological assays:

  • Macrophage activation studies: Assess the ability of purified truA to stimulate fish macrophages by measuring:

    • Nitric oxide production

    • Cytokine expression (IL-1β, TNF-α, IL-6)

    • Phagocytic activity

  • Lymphocyte proliferation assays: Measure proliferative responses of B and T cells isolated from fish lymphoid organs when exposed to truA

  • Dendritic cell maturation: Evaluate expression of co-stimulatory molecules and MHC presentation in antigen-presenting cells

• Ex vivo tissue explant cultures:

  • Maintain head kidney, spleen, or gill tissue in culture and expose to truA

  • Measure immune gene expression profiles using qRT-PCR

  • Assess tissue-specific responses that may not be captured in isolated cell systems

• Immunogenicity testing protocols:

  • Dose optimization: Test multiple concentrations (1-100 μg) of truA protein

  • Adjuvant selection: Compare oil-based, aluminum-based, and molecular adjuvants

  • Route of administration: Compare injection (intraperitoneal, intramuscular) vs. immersion approaches

  • Sampling timeline: Collect samples at 7, 14, 28, and 56 days post-immunization

• Antibody analysis techniques:

  • ELISA development: Establish protocols for detecting anti-truA antibodies in fish serum

  • Western blot confirmation: Verify antibody specificity using recombinant and native truA

  • Antibody subclass determination: Differentiate between IgM and IgT responses using specific antibodies

• Immune memory assessment:

  • Primary vs. secondary responses: Compare antibody titers and affinity maturation between initial exposure and boosters

  • Memory B-cell ELISpot assays: Quantify truA-specific memory B cells in immunized fish

These methodological approaches should build upon established protocols developed for other Aeromonas antigens, such as those used in VapA vaccine studies that demonstrated significant protection in rainbow trout. Those studies showed that recombinant adenovirus vaccines expressing VapA led to increased antibody levels in peripheral serum and enhanced expression of IgM and IgT in the head kidney and hindgut .

When designing these experiments, researchers should employ true experimental design principles including proper randomization, adequate sample sizes for statistical power, appropriate controls, and blinded assessment of outcomes to ensure robust and reproducible results .