Recombinant Haemophilus ducreyi tRNA pseudouridine synthase A (truA)

What is Haemophilus ducreyi tRNA pseudouridine synthase A (truA) and how does it compare to other pseudouridine synthases?

Haemophilus ducreyi tRNA pseudouridine synthase A (truA) belongs to the TruA family of pseudouridine synthases that catalyze the conversion of uridine to pseudouridine at specific positions in tRNA molecules. Based on comparative studies with TruA from other bacteria, H. ducreyi TruA likely modifies positions 38, 39, and/or 40 in the anticodon stem-loop (ASL) of multiple tRNAs . Unlike other pseudouridine synthases such as TruB that target conserved sequences, TruA exhibits remarkable substrate "promiscuity" by modifying tRNAs with highly divergent sequences .

H. ducreyi contains several pseudouridine synthases including TruB, RluA, and TruC, which have been characterized to varying degrees . TruA differs from these enzymes in three key aspects:

• Substrate range: TruA modifies multiple tRNAs with divergent sequences, whereas other pseudouridine synthases like TruB typically have more restricted target sites

• Target flexibility: TruA can modify nucleotides that are as far as 15 Å apart using a single active site

• Structural organization: While all pseudouridine synthases contain a conserved catalytic domain with an essential aspartate residue, they differ in their RNA recognition domains

These distinctive features make TruA an intriguing subject for studying RNA modification mechanisms in pathogenic bacteria like H. ducreyi.

What are the optimal conditions for expressing and purifying recombinant H. ducreyi truA?

Based on experimental protocols used for similar H. ducreyi pseudouridine synthases, the following optimized methodology is recommended:

Expression System:

E. coli is the preferred heterologous expression system for H. ducreyi pseudouridine synthases, as demonstrated with RluA and TruC . For truA, a pET-based expression vector with an N-terminal His-tag facilitates purification.

Culture Conditions:

• Growth medium: LB broth supplemented with appropriate antibiotics

• Induction: 0.5-1.0 mM IPTG when cultures reach OD₆₀₀ = 0.6-0.8

• Post-induction growth: 16-18 hours at 16-18°C (reduced temperature minimizes inclusion body formation)

Purification Protocol:

• Cell lysis using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography for final purification

Quality Control:

Assess purity by SDS-PAGE (expect >85% purity similar to other H. ducreyi pseudouridine synthases) . Verify identity by mass spectrometry and Western blotting.

Researchers should note that expression levels may be enhanced by codon optimization of the H. ducreyi truA gene for E. coli expression, as bacterial codon usage biases can affect heterologous protein production.

How can the enzymatic activity of recombinant H. ducreyi truA be measured in vitro?

Measuring the enzymatic activity of H. ducreyi TruA requires specific approaches to detect pseudouridine formation. The following methodological approaches are recommended:

Substrate Preparation:

For H. ducreyi TruA, suitable substrates include in vitro transcribed tRNAs containing uridines at positions 38-40 in the anticodon stem-loop. Based on TruA's known substrate promiscuity, preparing multiple tRNA substrates with different sequences is advisable .

Activity Assay Methods:

• Tritium Release Assay:

  • Label substrate RNA with [³H]UTP during in vitro transcription

  • Incubate labeled RNA with purified TruA

  • Measure released tritium using scintillation counting

  • Calculate enzyme activity based on the amount of tritium released

• CMC-Primer Extension Assay:

  • Treat RNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) after TruA reaction

  • Perform reverse transcription (RT stops at pseudouridine-CMC adducts)

  • Analyze RT products by gel electrophoresis

  • Quantify band intensity to determine pseudouridylation efficiency

• Mass Spectrometry:

  • Digest RNA enzymatically after TruA reaction

  • Analyze by LC-MS/MS to detect pseudouridine-containing oligonucleotides

  • Compare with control samples to quantify pseudouridylation

Reaction Conditions:

• Buffer: 50 mM Tris-HCl (pH 8.0), 100 mM NH₄Cl, 5 mM MgCl₂, 1 mM DTT

• Temperature: 37°C (optimal for most bacterial pseudouridine synthases)

• Reaction time: 30-60 minutes (time course recommended to determine kinetics)

Data Analysis:

Enzyme kinetics should be determined by measuring initial velocities at various substrate concentrations. Using the Michaelis-Menten equation, calculate Km and kcat values to characterize the enzyme's catalytic efficiency.

What structural features are essential for the catalytic activity of truA and how can they be investigated?

The catalytic activity of TruA depends on several key structural features that can be investigated through various experimental approaches:

Essential Structural Features:

• Catalytic Aspartate Residue: All pseudouridine synthases, including TruA, contain a completely conserved active site aspartate that is essential for catalysis . In H. ducreyi TruA, this residue can be identified through sequence alignment with other TruA proteins.

• RNA-Binding Cleft: TruA contains flexible structural features in the tRNA-binding cleft that are responsible for primary tRNA interaction . Charged residues in intermediate positions within this cleft likely guide the tRNA to the active site.

• Conformational Flexibility: Based on studies of TruA from other organisms, the enzyme likely undergoes conformational changes to facilitate access of the target uridine to the active site aspartate .

Investigative Approaches:

• Site-Directed Mutagenesis:

  • Mutate the conserved catalytic aspartate to asparagine or alanine (D→N or D→A)

  • Create mutations in predicted RNA-binding residues

  • Assess effects on enzymatic activity using assays described in Question 3

• Structural Analysis:

  • X-ray crystallography of H. ducreyi TruA alone and in complex with tRNA

  • Homology modeling based on existing TruA structures (e.g., from T. thermophilus)

  • Molecular dynamics simulations to study conformational changes

• RNA-Protein Interaction Studies:

  • Electrophoretic mobility shift assays (EMSA) to measure binding affinity

  • Fluorescence resonance energy transfer (FRET) to study dynamic interactions

  • Footprinting assays to identify RNA contact points

Case Study from Related Enzymes:

Studies on TruB1 demonstrated that mutants with inactivated enzyme activity (mutations in conserved catalytic residues) retained RNA binding ability, while mutants with suppressed RNA-binding ability lost both RNA binding and enzymatic function . Similar approaches can be applied to H. ducreyi TruA to distinguish between catalytic and binding residues.

How does truA substrate recognition differ from other pseudouridine synthases in H. ducreyi?

TruA exhibits unique substrate recognition characteristics compared to other pseudouridine synthases in H. ducreyi, which has important implications for experimental design and analysis:

Comparative Substrate Recognition:

Key Differences in Recognition Mechanism:

• Sequence vs. Structure Recognition:
  TruA primarily recognizes structural features of the anticodon stem-loop rather than specific sequences, explaining its ability to modify multiple tRNAs with divergent sequences . In contrast, TruB recognizes a conserved sequence in the T-stem loop.

• Substrate Flexibility Requirements:
  TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and appears to select against intrinsically stable tRNAs to avoid overstabilization through pseudouridylation . This suggests a balanced approach to maintaining RNA structural dynamics.

• Multi-Site Recognition:
  Unlike other pseudouridine synthases, TruA can modify nucleotides that are spatially distant (up to 15 Å apart) using a single active site , indicating a unique binding mode that allows repositioning of the substrate.

Experimental Implications:

When designing experiments to study H. ducreyi TruA, researchers should:

• Use multiple tRNA substrates with varying sequences in the ASL region

• Compare folding energies and structural flexibility of target RNAs

• Consider the potential impact of pseudouridylation on RNA stability and function

These distinctive recognition mechanisms make TruA particularly interesting for studying the evolution of RNA modification enzymes and their roles in bacterial physiology.

What role might truA play in H. ducreyi pathogenesis and survival in the host environment?

While direct evidence for TruA's role in H. ducreyi pathogenesis is limited, we can make informed inferences based on H. ducreyi's lifecycle and known functions of pseudouridine modifications:

Potential Roles in Pathogenesis:

• Stress Adaptation:
  H. ducreyi encounters various stresses during infection, including nutrient limitation, oxidative stress, and host defense mechanisms . Transcriptomic analysis of H. ducreyi during human infection revealed upregulation of genes involved in alternative carbon pathways, heat shock response, and growth arrest response . TruA-mediated tRNA modifications likely contribute to translational efficiency under these stress conditions.

• Regulation of Virulence Factor Expression:
  H. ducreyi expresses multiple virulence factors essential for infection, including hemoglobin receptors (HgbA), serum resistance proteins (DsrA), and adhesins . Optimal translation of these virulence factors may depend on TruA-modified tRNAs.

• Survival in Abscess Environment:
  H. ducreyi resides in abscesses during infection, where it must adapt to microaerophilic or anaerobic conditions . RNA modifications may enhance translational accuracy under these conditions.

Evidence from Related Systems:

Studies in other bacteria have shown that tRNA modifications, including pseudouridylation, can affect:

• Translational fidelity and efficiency

• Codon biases and synonymous codon choice

• Stress response and adaptive capacity

Experimental Approaches to Test These Hypotheses:

• Comparative Transcriptomics:

  • Generate an isogenic truA mutant in H. ducreyi

  • Compare transcriptomes and proteomes of wild-type and mutant strains

  • Identify changes in gene expression patterns, particularly of virulence factors

• Infection Models:

  • Test truA mutant in the human challenge model of chancroid

  • Assess survival, persistence, and virulence compared to wild-type

  • Measure immune response to truA mutant infection

• Stress Response Assays:

  • Expose wild-type and truA mutant to various stresses (oxidative, nutrient limitation, pH)

  • Measure survival, growth rates, and protein synthesis under stress conditions

  • Analyze tRNA modification profiles using mass spectrometry

Understanding TruA's role in H. ducreyi pathogenesis could potentially reveal new targets for therapeutic intervention against this sexually transmitted pathogen.

How can researchers design true experimental studies to assess the function of H. ducreyi truA?

Designing true experimental studies for H. ducreyi TruA requires careful consideration of control groups, variables, and randomization to establish causal relationships 12. The following methodological framework is recommended:

Construct Generation and Validation:

• Generate an isogenic truA deletion mutant in H. ducreyi

• Create a complemented strain by reintroducing truA on a plasmid

• Confirm constructs by sequencing and expression analysis

Core Experimental Design:

• Treatment Group: H. ducreyi truA mutant

• Control Groups: Wild-type H. ducreyi and complemented strain

• Replicate Structure: Minimum 3 biological replicates with 3 technical replicates each

• Randomization: Randomize the order of sample processing and analysis to minimize bias

Phenotypic Characterization Experiments:

Growth Studies:

• Measure growth rates in standard media and under various stress conditions

• Variables to test: temperature, pH, nutrient limitation, oxidative stress

• Data collection: OD₆₀₀ measurements, viable cell counts

RNA Modification Analysis:

• Isolate total tRNA from all strains

• Quantify pseudouridine at positions 38-40 using mass spectrometry

• Compare modification profiles across strains and growth conditions

Protein Synthesis Assays:

• Measure global protein synthesis rates using pulse-chase experiments

• Assess translation fidelity using reporter constructs

• Quantify specific virulence factor expression levels

Infection Models:

• Human challenge model or swine model of H. ducreyi infection

• Randomized inoculation sites

• Blinded assessment of lesion development and bacterial recovery

Data Analysis Approach:

• Apply appropriate statistical tests based on data distribution

• Use multiple comparison corrections for significance testing

• Implement controls for potential confounding variables

Avoiding Common Pitfalls:

This true experimental design approach will provide robust evidence for the causal relationship between TruA function and observed phenotypes in H. ducreyi.

How do the structural mechanisms of tRNA pseudouridylation by truA differ from other pseudouridine synthases?

The structural mechanisms of tRNA pseudouridylation by TruA exhibit several distinctive features compared to other pseudouridine synthases, with implications for experimental approaches:

TruA (based on studies in other bacteria):

• Regional Modification:

  • TruA can modify three different positions (38, 39, 40) in the anticodon stem-loop using a single active site

  • This requires a unique binding mode that allows repositioning of the substrate

• Conformational Changes:

  • TruA likely induces melting of base pairs to access target uridines

  • The enzyme utilizes the intrinsic flexibility of the ASL region for site recognition

• Catalytic Mechanism:

  • Uses a conserved aspartate residue that attacks the ribose of the target uridine

  • Forms a glycal intermediate during the reaction

Other H. ducreyi Pseudouridine Synthases:

TruB:

• Specifically modifies U55 in the T-loop of nearly all tRNAs

• High-resolution structure available (pLDDT score: 94.95)

• Recognizes a conserved sequence in the T-stem loop

RluA:

• Has dual specificity for both 23S rRNA and certain tRNAs

• Contains conserved domains for substrate recognition

TruC:

• Modifies position 65 in tRNA

• Different substrate recognition compared to TruA and TruB

Structural Basis for Mechanistic Differences:

The structural basis for TruA's unique mechanism likely involves:

• Flexible RNA-Binding Cleft:
  TruA contains remarkably flexible structural features in the tRNA-binding cleft that accommodate various tRNA sequences .

• Charged Residue Positioning:
  Charged residues occupying intermediate positions in the cleft guide the tRNA to the active site for catalysis .

• Conformational Selection:
  TruA appears to select substrates based on their intrinsic flexibility, avoiding overstabilization of tRNAs through pseudouridylation .

Experimental Approaches to Study Structural Mechanisms:

• Crystallography with Different Substrates:

  • Co-crystallize H. ducreyi TruA with different tRNA substrates

  • Compare binding modes and conformational changes

  • Identify key interaction residues

• Molecular Dynamics Simulations:

  • Model the interaction between TruA and various tRNA substrates

  • Simulate conformational changes during binding and catalysis

  • Predict the effects of mutations on substrate recognition

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Map protein dynamics during substrate binding

  • Identify regions with altered solvent accessibility

  • Compare flexibility changes in different enzyme-substrate complexes

Understanding these structural mechanisms is essential for developing targeted approaches to modulate TruA activity in pathogenic bacteria.

What site-directed mutagenesis approaches are most informative for studying H. ducreyi truA catalytic mechanism?

Site-directed mutagenesis provides powerful insights into the catalytic mechanism of H. ducreyi TruA. Based on structural and functional studies of pseudouridine synthases, the following strategic approaches are recommended:

Catalytic Core Residues:

• Conserved Aspartate: The catalytic aspartate is essential for all pseudouridine synthases . Mutation to asparagine (D→N) preserves size but eliminates nucleophilic capacity, while mutation to alanine (D→A) creates a more drastic change.

• Stabilizing Arginine: A conserved arginine stabilizes the catalytic aspartate. Mutation to lysine (R→K) maintains positive charge but alters geometry.

• Aromatic Residue: TruA family typically contains a conserved tyrosine in the active site, whereas TruD family contains phenylalanine . Interchanging these residues (Y→F or F→Y) can provide insights into their specific roles.

RNA-Binding Residues:

• Positively Charged Residues: Lysines and arginines in the RNA-binding cleft mediate interactions with the RNA phosphate backbone. Systematic alanine substitutions can identify critical binding residues.

• Recognition Loop Residues: Residues in flexible loops often mediate specific RNA contacts. Mutations or small deletions can alter substrate specificity.

Interdomain Interfaces:

• Hinge Region Residues: Mutations at domain interfaces can affect conformational dynamics essential for substrate binding and catalysis.

Mutagenesis Strategy and Workflow:

• Sequence Alignment and Structural Modeling:

  • Align H. ducreyi TruA with characterized pseudouridine synthases

  • Model the structure based on available crystal structures (e.g., E. coli or T. thermophilus TruA)

  • Identify conserved and variable residues

• Mutant Design Strategy:

  Mutation Type	Purpose	Examples
  Conservative	Test specific chemical properties	D→N, K→R, Y→F
  Non-conservative	Eliminate function	D→A, K→A, Y→A
  Charge reversal	Test electrostatic interactions	K→E, R→E
  Double mutants	Test compensatory effects	D→N + R→K

• Functional Assessment:

  • Measure enzymatic activity using assays described in Question 3

  • Determine RNA binding affinities

  • Assess structural changes by circular dichroism or thermal stability assays

Case Study Approach:

The TruB1 study demonstrated that mutations affecting enzyme activity (D48N, D90N) did not impact RNA binding, whereas a mutation affecting RNA binding (K64A) eliminated both binding and enzymatic function . This approach separates catalytic and binding functions and can be applied to H. ducreyi TruA.

Advanced Applications:

• Chimeric Enzymes: Create chimeras between H. ducreyi TruA and other pseudouridine synthases to test domain-specific functions

• Systematic Alanine Scanning: Comprehensively map the functional surface of the enzyme

• Suppressor Mutations: Identify secondary mutations that restore function to primary mutants

These mutagenesis approaches will provide mechanistic insights into how H. ducreyi TruA recognizes and modifies its substrates, potentially revealing unique features of this pathogen's RNA modification machinery.

How can transcriptomic and genomic approaches enhance our understanding of H. ducreyi truA function during infection?

Transcriptomic and genomic approaches offer powerful tools for understanding H. ducreyi TruA function in the context of infection, particularly given the challenges of studying this obligate human pathogen:

RNA-Seq Analysis During Infection:

Previous studies have successfully performed RNA-Seq on H. ducreyi isolated from human pustules, revealing significant adaptations to the host environment . Similar approaches can be applied to study truA expression and its potential regulators:

• Methodology:

  • Collect biopsy specimens from H. ducreyi-infected tissues

  • Isolate bacterial RNA using selective approaches

  • Perform RNA-Seq and compare to in vitro growth conditions

  • Identify co-regulated genes and potential regulatory networks

• Key Findings from Existing Research:
  H. ducreyi transcriptome in vivo shows upregulation of genes involved in alternative carbon utilization, stress response, and anaerobic adaptation . Understanding how truA expression correlates with these changes could reveal its role in adaptation.

Differential Expression Analysis:

Compare wild-type and truA mutant transcriptomes to identify genes affected by TruA-mediated tRNA modification:

• Experimental Design:

  • Culture wild-type and truA mutant under various conditions (standard, stress, host-mimicking)

  • Perform RNA-Seq and compare transcriptomes

  • Identify differentially expressed genes, particularly those involved in virulence and stress response

• Analysis Framework:
  Focus on genes with altered codon usage patterns that might be sensitive to changes in tRNA modification status

Comparative Genomics:

Analyze truA sequence conservation across different H. ducreyi strains and related pathogens:

• Strain Comparison:
  H. ducreyi isolates fall into two classes that differ in several extracellular or secreted proteins and LOS structure . Comparing truA sequence and surrounding genomic context across these classes could reveal selective pressures.

• Phylogenetic Analysis:
  Determine if truA evolution correlates with adaptation to specific host niches or virulence characteristics

Genome-Wide Association Studies (GWAS):

For pathogens with sufficient genomic diversity and phenotypic data, GWAS can identify genetic variants associated with specific phenotypes:

• Approach:

  • Sequence truA and surrounding regions from diverse clinical isolates

  • Correlate genetic variants with clinical outcomes or in vitro phenotypes

  • Identify potential functional variants in truA or its regulatory elements

Integration with Structural Data:

Combining transcriptomic/genomic data with structural analyses provides a comprehensive view of TruA function:

• Map sequence conservation onto structural models to identify functionally important surfaces

• Correlate expression changes with structural features to understand regulation

• Use structural predictions to interpret the impact of natural variants

Practical Example:

The RpoE sigma factor in H. ducreyi regulates multiple RNA modification enzymes, including pseudouridine synthases like RsuA and RluA . By analyzing the transcriptomic data from RpoE overexpression experiments, researchers identified 180 RpoE-dependent genes, 98% of which were upregulated . Similar approaches could reveal regulatory networks governing truA expression and link them to specific stress responses or virulence mechanisms.