Recombinant Bordetella bronchiseptica tRNA pseudouridine synthase A (truA)

What is tRNA pseudouridine synthase A (truA) and what is its function in Bordetella bronchiseptica?

tRNA pseudouridine synthase A (truA) is an enzyme that catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon stem-loop (ASL) of tRNA molecules . In Bordetella bronchiseptica, as in other bacteria, truA plays a crucial role in post-transcriptional modification of tRNA, which is essential for accurate and efficient protein translation. The enzyme contains a conserved active site aspartate residue that is believed to be involved in the catalytic mechanism shared among the pseudouridine synthase family members . This modification helps stabilize the tertiary structure of tRNA and enhances codon-anticodon interactions during protein synthesis, ultimately affecting bacterial growth, survival, and potentially pathogenicity.

How does B. bronchiseptica truA compare structurally to truA from other bacterial species?

While the specific crystal structure of B. bronchiseptica truA has not been fully characterized in the provided search results, insights can be drawn from the structural features of truA from related bacteria. The crystal structure of TruA from Thermus thermophilus HB8 reveals remarkably flexible structural features in the tRNA-binding cleft that are likely responsible for primary tRNA interaction .

Like other pseudouridine synthases, B. bronchiseptica truA is expected to contain:

• A completely conserved active site aspartate residue critical for catalytic function

• A tRNA-binding cleft with charged residues that guide the tRNA to the active site

• Structural features that facilitate conformational changes in the substrate tRNA to access the active site

Given that B. bronchiseptica belongs to the "B. bronchiseptica cluster" of closely related species , its truA enzyme likely shares significant structural homology with truA from other members of this group, with potential species-specific adaptations that may reflect its particular tRNA modification requirements.

What expression systems are most effective for producing recombinant B. bronchiseptica truA?

Based on research with similar bacterial proteins, including other Bordetella recombinant proteins, several expression systems have proven effective:

For B. bronchiseptica proteins, E. coli expression systems are commonly used, as B. bronchiseptica has been shown to exchange genetic material with E. coli, suggesting compatibility of genetic elements . When expressing recombinant B. bronchiseptica proteins, it's important to consider that B. bronchiseptica has a genome size of approximately 5.34 Mb , which may contain specific regulatory elements that differ from common expression hosts.

How does truA contribute to B. bronchiseptica pathogenesis and host adaptation?

While direct evidence linking truA to B. bronchiseptica pathogenesis is not explicitly stated in the search results, several connections can be inferred based on the role of tRNA modifications in bacterial physiology and pathogenesis:

tRNA modifications, including those catalyzed by truA, likely contribute to B. bronchiseptica's ability to adapt to different environmental conditions during infection. B. bronchiseptica is known to colonize a broad range of mammalian hosts and demonstrates different phenotypic phases (Bvg+, Bvgi, and Bvg-) in response to environmental stimuli . These phases are controlled by the BvgAS master regulator system, which affects the expression of virulence factors .

The accurate translation of proteins involved in these regulatory networks may depend on properly modified tRNAs. Since B. bronchiseptica can establish persistent infections without causing damage to some hosts , truA-mediated tRNA modifications may be involved in modulating protein expression patterns that contribute to this balance. In swine, B. bronchiseptica causes severe bronchopneumonia in young pigs and is a primary agent of atrophic rhinitis , suggesting that translation efficiency of virulence factors may be crucial for host-specific pathogenesis.

What are the methodological approaches for studying the impact of truA mutations on B. bronchiseptica virulence?

Researchers studying the impact of truA mutations on B. bronchiseptica virulence should consider the following methodological approaches:

• Generation of truA mutants:

  • Construct in-frame deletion mutants similar to the approach used for FHA and PRN virulence factors

  • Create point mutations in the conserved aspartate residue of the active site

  • Develop complementation strains to verify phenotypes

• In vitro phenotypic characterization:

  • Growth kinetics under various environmental conditions

  • Protein synthesis rate measurements

  • tRNA modification analysis using mass spectrometry

  • Biofilm formation assays

• Animal infection models:

  • Mouse models with varied immune statuses (as used for Bordetella virulence studies)

  • Natural host models, particularly swine

  • Measurement of colonization at different respiratory tract sites

  • Assessment of disease severity and antibody responses

• Gene expression analysis:

  • RNA-seq to identify global expression changes in truA mutants

  • Quantification of virulence factor expression in different Bvg phases

B. bronchiseptica mutants can be compared to wild-type strains for their ability to colonize and cause disease, as was done with FHA and PRN mutants in swine . The intranasal 50% infective dose for various animal models (rabbits, rats, and mice) is less than 200, 25, and 5 CFU, respectively, indicating these model systems accurately reflect characteristics of naturally occurring infection .

How does recombinant truA protein purification differ from other B. bronchiseptica enzymes?

Purification of recombinant B. bronchiseptica truA presents unique challenges compared to other B. bronchiseptica enzymes:

Unlike some other B. bronchiseptica proteins, truA's function in tRNA modification means that its activity assays require intact tRNA substrates and specific analytical methods to detect pseudouridine formation. The tRNA-binding cleft with charged residues that guide tRNA to the active site must remain structurally intact during purification, which may necessitate different buffer conditions than those used for virulence factors like FHA or PRN .

What are the optimal conditions for assaying recombinant B. bronchiseptica truA activity in vitro?

The optimal conditions for assaying recombinant B. bronchiseptica truA activity require careful consideration of multiple factors:

Reaction Components:

• Purified recombinant truA enzyme (10-100 nM)

• tRNA substrates (preferably native B. bronchiseptica tRNAs or in vitro transcribed tRNAs)

• Buffer system maintaining pH 7.0-8.0 (typically HEPES or Tris)

• Divalent cations (Mg²⁺, 5-10 mM)

• Reducing agents (DTT, 1-5 mM)

• Appropriate incubation temperature (30-37°C reflecting B. bronchiseptica growth conditions)

Detection Methods:

• Tritium release assay using [³H]-labeled tRNA

• HPLC analysis of nucleosides after complete tRNA digestion

• Mass spectrometry-based approaches for precise quantification of pseudouridine formation

• CMC (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate) modification coupled with primer extension

Controls:

• Heat-inactivated enzyme negative control

• Known pseudouridine synthase (such as TruA from E. coli) as positive control

• tRNA lacking target uridine positions as specificity control

Since B. bronchiseptica can grow in different environmental conditions corresponding to its Bvg+/Bvg- phases , assaying truA activity under varying pH and temperature conditions may provide insights into how tRNA modification responds to environmental changes during infection.

How can researchers effectively study the interaction between recombinant B. bronchiseptica truA and its tRNA substrates?

To effectively study the interaction between recombinant B. bronchiseptica truA and its tRNA substrates, researchers should employ multiple complementary approaches:

• Structural Analysis:

  • X-ray crystallography of truA-tRNA complexes, similar to the approach used for T. thermophilus TruA

  • Cryo-electron microscopy for visualizing larger complexes

  • NMR spectroscopy for dynamic interaction studies

• Binding Kinetics:

  • Surface plasmon resonance (SPR) to determine association/dissociation constants

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Fluorescence anisotropy with labeled tRNA substrates

• Functional Interaction Studies:

  • Site-directed mutagenesis of charged residues in the tRNA-binding cleft

  • Analysis of tRNA conformational changes during binding using chemical probing

  • Cross-linking studies to identify specific contact points

• Computational Approaches:

  • Molecular dynamics simulations of truA-tRNA interactions

  • Homology modeling based on the T. thermophilus TruA structure

  • Docking studies to predict binding interfaces

Based on insights from the T. thermophilus TruA structure, researchers should pay particular attention to the "remarkably flexible structural features in the tRNA-binding cleft" and the charged residues that "may lead the tRNA to the active site for catalysis" . The data also suggest that "a conformational change of the substrate tRNA is necessary to facilitate access to the active site aspartate residue, deep within the cleft" , making it important to study the dynamic aspects of this interaction.

How should researchers interpret differences in truA activity across Bordetella species?

When interpreting differences in truA activity across Bordetella species, researchers should consider several important factors:

Evolutionary Context:
The Bordetella genus shows remarkable limited genetic diversity among B. pertussis, B. parapertussis, and B. bronchiseptica strains, which have been reclassified as "subspecies" of a single species with different host adaptations . B. bronchiseptica is considered the evolutionary progenitor, while B. pertussis and B. parapertussis are human-adapted lineages of B. bronchiseptica . These evolutionary relationships should inform interpretation of enzymatic differences.

Genome Comparison Framework:

• B. bronchiseptica strain RB50 has a genome size of 5.34 Mb

• B. pertussis strain Tohama 1 has a genome size of 4.09 Mb

• B. parapertussis hu strain 12822 has a genome size of 4.77 Mb

These differences reflect genomic streamlining during host adaptation, which may affect truA regulation and function.

Methodological Considerations:
When comparing truA activity across species, researchers should:

• Use consistent substrates and assay conditions

• Account for differences in optimal growth conditions for each species

• Consider the impact of gene expression regulation specific to each species

• Examine sequence variations in the catalytic domain and tRNA-binding regions

Interpretation Framework:
Differences in truA activity may reflect:

• Host-specific adaptation of tRNA modification patterns

• Changes in translation requirements for different environmental niches

• Co-evolution with species-specific tRNA populations

• Selection pressures related to pathogenicity mechanisms

What technical challenges exist in comparing in vitro and in vivo functions of recombinant B. bronchiseptica truA?

Researchers face several technical challenges when attempting to compare in vitro and in vivo functions of recombinant B. bronchiseptica truA:

In Vitro vs. In Vivo Environmental Conditions:

• In vitro experiments typically use purified components under defined conditions

• In vivo, truA functions within the complex bacterial cytoplasm with varying ion concentrations, macromolecular crowding, and potential interacting partners

• B. bronchiseptica can exist in different phenotypic phases (Bvg+, Bvgi, and Bvg-) in response to environmental stimuli , which may affect truA activity

Substrate Accessibility:

• In vitro studies often use synthetic or in vitro transcribed tRNAs

• In vivo, tRNAs may have additional modifications or be bound to other factors

• The pool of available tRNAs differs between laboratory culture and during host infection

Methodological Limitations:

• Detection sensitivity for pseudouridine modifications in vivo

• Difficulty in distinguishing truA-specific modifications from those made by other pseudouridine synthases

• Challenges in maintaining physiologically relevant enzyme concentrations in vitro

Analysis Framework:
To address these challenges, researchers should:

• Develop in vivo tRNA modification profiling methods specific for B. bronchiseptica

• Compare truA mutant and wild-type strains under various growth conditions

• Use ribosome profiling to assess the impact of truA on translation in vivo

• Develop reporter systems to monitor truA activity during infection

Given that B. bronchiseptica can infect a broad range of mammalian hosts and shows varying levels of virulence in different models , researchers should also consider host-specific effects on truA function during infection.

What are the future research directions for B. bronchiseptica truA studies?

The study of Bordetella bronchiseptica tRNA pseudouridine synthase A (truA) represents an emerging field with several promising research directions:

• Structural and Functional Characterization:

  • Determination of the crystal structure of B. bronchiseptica truA, similar to the T. thermophilus TruA structure

  • Mapping of B. bronchiseptica-specific tRNA modification patterns

  • Identification of potential moonlighting functions beyond tRNA modification

• Role in Pathogenesis:

  • Investigation of truA's contribution to B. bronchiseptica's ability to colonize diverse hosts

  • Analysis of truA's impact on virulence factor expression across different Bvg phases

  • Examination of truA's role in persistent infection establishment

• Comparative Studies:

  • Analysis of truA evolution across the "B. bronchiseptica cluster"

  • Investigation of host-specific adaptations in truA function

  • Comparison with truA from other respiratory pathogens

• Therapeutic Potential:

  • Evaluation of truA as a potential drug target for B. bronchiseptica infections

  • Development of specific inhibitors for bacterial pseudouridine synthases

  • Assessment of truA's potential as a component in next-generation vaccines, similar to other B. bronchiseptica antigens

• Technological Advancements:

  • Development of high-throughput methods for analyzing tRNA modifications in vivo

  • Creation of reporter systems to monitor truA activity during infection

  • Application of CRISPR-Cas techniques for precise genomic manipulation of truA