Recombinant Francisella tularensis subsp. tularensis tRNA pseudouridine synthase A (truA)

What is the biological function of tRNA pseudouridine synthase A (truA) in Francisella tularensis?

Recent research indicates that RNA modification enzymes like truA may also contribute to virulence regulation networks in F. tularensis, similar to how the trmE gene (which encodes another tRNA modification enzyme) affects pathogenicity island gene expression. The trmE gene has been shown to significantly influence ppGpp accumulation, which is critical for virulence gene expression in F. tularensis .

How does truA relate to other virulence factors in the Francisella pathogenicity island (FPI)?

While direct evidence linking truA to the Francisella pathogenicity island (FPI) is limited in the provided literature, related RNA modification enzymes like those encoded by the trmE gene have been demonstrated to affect FPI gene expression. The FPI consists of 17 conserved open reading frames that are critical for F. tularensis virulence and intracellular survival .

Research has shown that mutations in RNA modification genes like trmE significantly reduce expression from the iglA promoter, which controls virulence genes in the FPI. When measured using an iglA-lacZ plasmid reporter, the trmE mutant showed only about 22% of the wild-type expression levels. This connection suggests that RNA modification systems, potentially including truA, may be integrated into the regulatory networks controlling virulence in this pathogen .

What are the optimal expression systems for recombinant F. tularensis truA production?

The optimal expression systems for recombinant F. tularensis truA production depend on research objectives and downstream applications. Based on available data for tRNA pseudouridine synthase A expression generally, the following systems have proven effective:

What purification strategy yields the highest activity for recombinant F. tularensis truA?

A multi-step purification strategy typically yields the highest activity for recombinant F. tularensis truA, although specific optimizations may be required for this particular enzyme:

• Initial capture using affinity chromatography:

  • His-tagged constructs can be purified using immobilized metal affinity chromatography (IMAC)

  • Optimal binding buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole

  • Elution with imidazole gradient (20-250 mM)

• Intermediate purification:

  • Ion exchange chromatography (IEX) using a MonoQ column

  • Buffer conditions: 20 mM Tris-HCl (pH 8.0), with NaCl gradient from 50-500 mM

• Polishing step:

  • Size exclusion chromatography (SEC) using a Superdex 200 column

  • Running buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT

Throughout the purification process, include protease inhibitors to prevent degradation, and maintain the protein at 4°C. After purification, assess enzyme activity using a pseudouridylation assay with appropriate tRNA substrates and analyze protein purity by SDS-PAGE and mass spectrometry .

How does truA contribute to F. tularensis virulence and survival within host cells?

While the specific contribution of truA to F. tularensis virulence is not directly addressed in the provided literature, inferences can be made based on related RNA modification enzymes studied in this pathogen:

RNA modification enzymes like those encoded by migR, trmE, and cphA genes have been shown to significantly affect F. tularensis virulence by modulating the stringent response, particularly through affecting ppGpp accumulation. The stringent response is a bacterial stress response that helps pathogens adapt to nutritional stress conditions encountered during infection .

In F. tularensis, the regulation of virulence genes is tightly linked to stress response pathways. Mutations in trmE (which encodes a tRNA modification enzyme related to truA) significantly reduce ppGpp accumulation to approximately 22% of wild-type levels. This reduction correlates with decreased expression of virulence genes from the Francisella pathogenicity island (FPI) .

Given these connections, truA likely contributes to F. tularensis virulence through:

• Maintaining translational fidelity under stress conditions

• Potentially participating in regulatory networks that control virulence gene expression

• Contributing to bacterial adaptation within different host cell environments

The differential replication capabilities observed in various mammalian cells for RNA modification mutants suggest that these enzymes help F. tularensis respond to distinct nutritional stresses encountered in different host cell types .

What experimental methods are most effective for studying truA's role in bacterial stress response?

To effectively study truA's role in bacterial stress response, particularly in relation to F. tularensis pathogenicity, a multi-faceted experimental approach is recommended:

• Genetic manipulation approaches:

  • Create precise gene knockouts or conditional mutants of truA using homologous recombination techniques as demonstrated with trmE and cphA mutations in F. tularensis LVS

  • Complement mutants with plasmid-expressed wild-type truA to confirm phenotype specificity

  • Use reporter systems (such as iglA-lacZ) to measure virulence gene expression in truA mutants

• Biochemical assays:

  • Measure ppGpp accumulation using thin-layer chromatography (TLC) assays to determine if truA affects the stringent response

  • Compare ppGpp levels between wild-type and truA mutant strains under various stress conditions

  • Quantify pseudouridylation at specific tRNA positions using techniques like HPLC or mass spectrometry

• Cellular infection models:

  • Assess intracellular replication capabilities in different mammalian cell types (macrophages, hepatocytes, etc.)

  • Monitor bacterial escape from the phagosome in wild-type versus truA mutant strains

  • Measure host cell cytokine responses to determine if truA affects immunomodulatory properties

• Transcriptomic and proteomic analyses:

  • Compare global gene expression profiles between wild-type and truA mutant strains using RNA-seq

  • Identify protein expression changes using quantitative proteomics

  • Map regulatory networks affected by truA mutation

These methods, particularly when used in combination, can provide comprehensive insights into how truA contributes to F. tularensis stress response and virulence regulation .

How can structural analysis of F. tularensis truA inform drug development against tularemia?

Structural analysis of F. tularensis truA offers significant potential for rational drug design against tularemia through several strategic approaches:

• Identification of unique structural features: F. tularensis is classified as a Tier 1 Select Agent due to its potential use as a bioterrorism agent. By resolving the three-dimensional structure of F. tularensis truA using X-ray crystallography or cryo-electron microscopy, researchers can identify unique structural elements that differ from human pseudouridine synthases .

• Active site targeting: Detailed mapping of the active site architecture can reveal distinct pocket geometries and catalytic residues. These features can guide the design of selective inhibitors that disrupt pseudouridylation activity essential for bacterial survival and virulence. Molecular docking studies can then be performed to screen for compounds that bind with high affinity to these sites.

• Allosteric site discovery: Beyond the active site, structural analysis may reveal allosteric sites unique to bacterial truA. These sites offer opportunities for developing inhibitors that alter enzyme conformation or dynamics without competing with substrate binding.

• Integration with virulence data: When structural insights are combined with functional data about truA's role in virulence (potentially through ppGpp regulation pathways similar to other RNA modification enzymes), more targeted approaches to disrupting pathogenicity can be developed .

• Structure-guided fragment-based drug discovery: Starting with small molecular fragments that show binding to specific regions of truA, researchers can iteratively optimize these compounds using structure-activity relationship studies to develop high-affinity, selective inhibitors.

This structural biology approach is particularly valuable considering the limitations of current tularemia treatments and the need for novel therapeutics against this highly infectious pathogen .

What are the technical challenges in studying the interaction between truA and the stringent response pathway in F. tularensis?

Studying the interaction between truA and the stringent response pathway in F. tularensis presents several significant technical challenges:

• Biosafety considerations: As F. tularensis is classified as a Tier 1 Select Agent, research requires specialized biocontainment facilities (BSL-3), which limits accessibility and increases experimental complexity .

• Genetic manipulation difficulties:

  • F. tularensis is fastidious and requires cysteine for growth

  • Transformation efficiencies are typically low

  • Genetic tools are more limited compared to model organisms

  • Creating precise genetic modifications without polar effects on adjacent genes requires careful design

• Functional redundancy: The stringent response involves multiple regulatory components, and potential redundancy between RNA modification enzymes may mask phenotypes in single gene knockouts.

• Temporal dynamics of ppGpp regulation: The transient nature of ppGpp accumulation makes capturing relevant interactions technically challenging. Research on related RNA modification enzymes shows that:

  • Wild-type F. tularensis LVS produces large amounts of ppGpp

  • Mutations in trmE reduce ppGpp to approximately 22% of wild-type levels

  • Similar reductions might occur with truA mutations, requiring sensitive detection methods

• Complex host-pathogen interactions: The relevance of truA to pathogenesis may differ across infection models and cell types, as seen with other RNA modification enzymes. Research shows different bacterial replication capabilities in various mammalian cells for RNA modification mutants, indicating that:

  • Different nutritional stresses exist in different host cells

  • The role of RNA modification enzymes may vary depending on the specific host environment

• Integration of multiple stress response pathways: The stringent response intersects with multiple stress adaptation mechanisms, making it difficult to isolate the specific contribution of truA.

Addressing these challenges requires multidisciplinary approaches combining genetics, biochemistry, structural biology, and infection models to fully elucidate truA's role in F. tularensis pathogenicity.

How does F. tularensis truA differ from homologous enzymes in other bacterial pathogens?

Comparative analysis of F. tularensis truA with homologous enzymes from other bacterial pathogens reveals important evolutionary adaptations that may contribute to its unique pathogenic properties:

The unique properties of F. tularensis truA likely reflect evolutionary adaptations to its highly specialized intracellular lifestyle. Given that F. tularensis can survive for several weeks at low temperatures in animal carcasses, soil, and water, its RNA modification systems may have evolved to maintain translational accuracy across diverse environmental conditions .

The integration of truA into stress response pathways appears to be a common feature across bacterial pathogens, but F. tularensis may have evolved specific regulatory connections between RNA modification and virulence gene expression. This is suggested by research on related RNA modification enzymes like those encoded by trmE, which significantly impacts ppGpp accumulation and FPI gene expression .

These evolutionary adaptations make F. tularensis truA a potential target for species-specific therapeutic interventions that would minimize off-target effects on the human microbiome.

What methodological approaches are most effective for studying evolutionary conservation of truA function across Francisella species?

To effectively study the evolutionary conservation of truA function across Francisella species, researchers should employ a multi-faceted methodological approach that integrates bioinformatics, genetic manipulation, and functional characterization:

• Comparative genomics and phylogenetics:

  • Sequence alignment of truA genes from various Francisella subspecies (tularensis, holarctica, novicida)

  • Identification of conserved domains versus variable regions

  • Construction of phylogenetic trees to trace evolutionary relationships

  • Analysis of selection pressures using dN/dS ratios to identify regions under positive selection

• Structural biology approaches:

  • Homology modeling of truA proteins from different Francisella species

  • Identification of conserved active site residues versus variable surface features

  • X-ray crystallography or cryo-EM of truA from representative species for direct structural comparison

• Complementation studies:

  • Creation of truA deletion mutants in different Francisella species

  • Cross-species complementation experiments to determine functional conservation

  • Quantification of phenotypic rescue to assess degree of functional equivalence

• Transcriptomics and regulatory network analysis:

  • RNA-seq comparison between wild-type and truA mutants across species

  • Identification of conserved versus species-specific regulatory targets

  • Mapping of potential species-specific adaptations in regulatory networks

• Biochemical characterization:

  • Purification of recombinant truA from multiple Francisella species

  • Comparative enzyme kinetics studies using identical substrates

  • Identification of species-specific substrate preferences or catalytic efficiencies

• Host-pathogen interaction models:

  • Testing truA mutants from different species in identical host cell models

  • Quantification of intracellular survival, replication, and virulence

  • Assessment of species-specific adaptations to host environments

These complementary approaches would provide comprehensive insights into how truA function has been conserved or adapted across the Francisella genus during evolution, potentially revealing important adaptations that contribute to the varied virulence potential observed between subspecies .

What are the optimal conditions for measuring F. tularensis truA enzymatic activity in vitro?

The optimal conditions for measuring F. tularensis truA enzymatic activity in vitro require careful consideration of multiple parameters to ensure maximum enzyme performance and reliable results:

Buffer Composition and pH:

• Optimal buffer: 50 mM HEPES or Tris-HCl

• pH range: 7.5-8.0 (optimal pH typically ~7.8)

• Salt concentration: 50-100 mM NaCl or KCl

• Reducing agent: 1-5 mM DTT or 2-5 mM β-mercaptoethanol to maintain cysteine residues

• Divalent cations: 2-5 mM MgCl₂ as cofactor

Substrate Considerations:

• Purified tRNA substrates (either total tRNA or specific tRNA species)

• Concentration: 0.5-5 μM tRNA

• Pre-treatment: Heat denaturation at 80°C for 2 minutes followed by slow cooling to ensure proper refolding

Reaction Conditions:

• Temperature: 30-37°C (optimal likely 35-37°C, consistent with F. tularensis growth temperature)

• Incubation time: 30-60 minutes for standard activity assays

• Enzyme concentration: 50-500 nM purified recombinant truA

• Reaction volume: 50-100 μL for analytical scale

Detection Methods:

• Radiochemical assay: Using ³H-labeled UTP in transcription reactions to generate substrate tRNAs, followed by quantification of ³H-pseudouridine formation

• HPLC analysis: Enzymatic digestion of tRNA followed by HPLC separation and detection of pseudouridine

• Mass spectrometry: LC-MS/MS analysis of pseudouridylated versus non-modified tRNAs

• CMC-derivatization: Treatment with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) which specifically modifies pseudouridine, followed by primer extension or sequencing

Controls:

• Positive control: E. coli truA with known activity

• Negative controls: Heat-inactivated enzyme and reaction without enzyme

• Substrate specificity controls: Various tRNA species to determine substrate preference

These optimized conditions should facilitate reliable measurement of F. tularensis truA activity, enabling detailed characterization of this enzyme's biochemical properties and potential role in bacterial virulence .

How can researchers distinguish between truA activity and other pseudouridine synthases in F. tularensis extracts?

Distinguishing between truA activity and other pseudouridine synthases in F. tularensis extracts requires strategic approaches that exploit the unique characteristics of each enzyme:

• Site-specific analysis:

  • truA typically modifies positions 38-40 in the anticodon stem-loop of tRNAs

  • Other pseudouridine synthases target different positions (e.g., truB modifies position 55)

  • Use position-specific primer extension assays after CMC derivatization to identify the exact sites of pseudouridylation

  • Employ site-specific reverse transcription stops to map modification sites precisely

• Substrate specificity:

  • Design substrate tRNAs lacking all pseudouridylation sites except those targeted by truA

  • Use synthetic tRNA constructs with site-specific mutations that prevent recognition by other pseudouridine synthases

  • Compare activity patterns between different tRNA species that have different patterns of modification

• Immunodepletion approach:

  • Develop specific antibodies against F. tularensis truA

  • Sequentially deplete extracts of truA and measure remaining pseudouridylation activity

  • Compare depletion patterns with those obtained using antibodies against other pseudouridine synthases

• Genetic approach:

  • Create specific gene knockouts of individual pseudouridine synthases

  • Compare pseudouridylation patterns in extracts from wild-type and mutant strains

  • Complement with recombinant enzymes to confirm specificity

• Biochemical separation:

  • Employ ion exchange chromatography to separate different pseudouridine synthases

  • Characterize fractions by mass spectrometry to confirm enzyme identity

  • Test each fraction for position-specific pseudouridylation activity

• Inhibitor profiling:

  • Identify specific inhibitors with differential effects on various pseudouridine synthases

  • Use selective inhibitors to block specific enzyme activities in complex extracts

  • Create inhibition profiles characteristic of each enzyme