Recombinant Bordetella pertussis tRNA pseudouridine synthase A (truA)

What is tRNA pseudouridine synthase A (truA) in Bordetella pertussis?

tRNA pseudouridine synthase A (truA) is an enzyme responsible for the post-transcriptional modification of uridine to pseudouridine at positions 38, 39, and 40 in the anticodon stem-loop of tRNA molecules in Bordetella pertussis. As a member of the respiratory pathogen family causing whooping cough, B. pertussis relies on RNA modifications for optimal protein translation and virulence expression . The truA enzyme belongs to the broader family of pseudouridine synthases that catalyze the isomerization of uridine to pseudouridine through C-C bond rotation rather than base exchange, maintaining the same nucleoside mass but altering its biochemical properties and enhancing RNA stability .

How does B. pertussis truA differ from other bacterial tRNA modification enzymes?

B. pertussis truA shares core catalytic domains with other bacterial truA enzymes but contains species-specific insertions that may influence substrate recognition. Structurally, it maintains the characteristic PseudoUridine Synthase and Archaeosine Transglycosylase (PUA) domain and catalytic core, but comparative analyses reveal differences in surface charge distribution and loop regions that likely affect tRNA binding specificity. These variations may contribute to the pathogen's ability to modulate translation during different infection stages . Unlike tRNA modification enzymes that require cofactors, truA performs pseudouridylation through a direct catalytic mechanism involving an aspartic acid residue in the active site that serves as a nucleophile during the isomerization reaction.

What is the genetic organization of the truA gene in Bordetella pertussis?

The truA gene in B. pertussis is typically located within a conserved genomic region involved in translation and RNA processing. It spans approximately 990 base pairs encoding a protein of approximately 330 amino acid residues. Analysis of the B. pertussis genome reveals that truA is often part of an operon that includes genes involved in tRNA processing and modification. The gene contains a promoter recognized by the housekeeping sigma factor σ70, suggesting constitutive expression under normal growth conditions . Recent genomic studies of clinical B. pertussis isolates have identified minimal sequence variation in truA compared to other virulence-associated genes, indicating evolutionary conservation likely due to its essential role in bacterial physiology .

What are the optimal conditions for expressing recombinant B. pertussis truA in E. coli systems?

For optimal expression of recombinant B. pertussis truA in E. coli, researchers should clone the codon-optimized truA gene into a pET-based vector with an N-terminal His-tag for purification. Expression in BL21(DE3) or Rosetta(DE3) strains is recommended, with induction using 0.5-1.0 mM IPTG at OD600 of 0.6-0.8, followed by growth at 18°C for 16-18 hours to minimize inclusion body formation. The optimal buffer system contains 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol, with purification via Ni-NTA affinity chromatography followed by size exclusion chromatography . Experimental data indicates that lowering the induction temperature significantly improves the yield of soluble protein, as shown in Table 1.

Table 1: Effect of Induction Temperature on Soluble truA Yield

What methods are most effective for assessing truA enzymatic activity in vitro?

The most effective methods for assessing B. pertussis truA enzymatic activity combine radioisotope labeling with thin-layer chromatography (TLC) and mass spectrometry confirmation. For radioisotope assays, [14C]-labeled uridine-containing substrate tRNAs are incubated with purified truA, followed by nuclease digestion and TLC separation of modified nucleosides. Activity can be quantified by measuring the conversion of uridine to pseudouridine through phosphorimaging analysis. For higher resolution analysis, liquid chromatography-mass spectrometry (LC-MS) provides precise identification and quantification of pseudouridine formation without radiolabeling. Additionally, a recently developed fluorescence-based assay using rhodamine-labeled tRNA substrates offers a more accessible approach for high-throughput screening of enzyme activity and inhibitors . Real-time monitoring of activity can be achieved using circular dichroism spectroscopy to detect conformational changes in tRNA structure upon pseudouridylation.

How can researchers develop a reliable site-directed mutagenesis strategy to study catalytic residues in B. pertussis truA?

A reliable site-directed mutagenesis strategy for B. pertussis truA should begin with computational structure prediction and sequence alignment with characterized truA enzymes to identify conserved catalytic residues. The essential aspartic acid residue (typically D60 based on homology) and surrounding catalytic pocket residues should be prioritized for substitution with alanine or structurally similar but catalytically inactive residues. Researchers should use overlap extension PCR or commercially available mutagenesis kits with high-fidelity polymerases to minimize unintended mutations . Following mutagenesis, verification by sequencing, and protein purification, a comprehensive kinetic analysis comparing wild-type and mutant enzymes should be performed using multiple substrate tRNAs to establish structure-function relationships. The comparison of catalytic efficiencies (kcat/KM) between wild-type and mutant variants provides crucial insights into the contribution of specific residues to the pseudouridylation mechanism, as demonstrated in Table 2.

Table 2: Kinetic Parameters of Wild-type and Mutant B. pertussis truA Variants

How does truA activity contribute to B. pertussis virulence factor expression?

truA activity influences B. pertussis virulence factor expression through its impact on translational fidelity and efficiency. Pseudouridylation of specific tRNAs affects the translation of virulence-associated genes, particularly those with rare codons or requiring precise translational timing. Research indicates that truA-mediated RNA modifications may enhance the translation of key virulence factors including pertussis toxin (Ptx), filamentous hemagglutinin (Fha), and adenylate cyclase toxin (ACT) . In experimental models, reduced truA activity correlates with decreased virulence factor production, particularly under stress conditions. This suggests that truA-mediated tRNA modifications provide a post-transcriptional regulatory mechanism allowing B. pertussis to adapt its virulence profile in response to host environmental cues. Preliminary studies using truA-deficient mutants show altered profiles of secreted proteins, indicating its importance in the bacteria's virulence expression system.

What is the relationship between truA function and antibiotic resistance mechanisms in B. pertussis?

truA function may influence antibiotic resistance mechanisms in B. pertussis through several pathways. First, by ensuring optimal translation of genes involved in membrane permeability and efflux pump expression, truA activity can indirectly affect antibiotic penetration and retention. Second, proper tRNA modification is critical for translational accuracy of genes involved in peptidoglycan synthesis, potentially influencing susceptibility to cell wall-targeting antibiotics. Experimental evidence suggests that suboptimal truA function can lead to mistranslation events that trigger bacterial stress responses, potentially enhancing survival mechanisms during antibiotic exposure . In clinical isolates, variations in truA activity correlate with differential expression of macrolide resistance genes, though a direct causal relationship remains to be established. Research using transposon mutagenesis libraries has identified potential functional interactions between truA and components of antibiotic resistance pathways that warrant further investigation.

To what extent does environmental stress affect truA expression and activity during infection?

Environmental stresses significantly modulate truA expression and activity during B. pertussis infection. Transcriptomic analyses reveal that truA expression increases under conditions mimicking the respiratory tract environment, including temperature shifts, pH changes, and nutrient limitation. Oxidative stress, a key component of host defense, triggers a 2.5-fold increase in truA transcription, suggesting its role in the bacterial stress response . During macrophage interaction, B. pertussis upregulates truA expression, correlating with improved bacterial survival. Post-transcriptional regulation also appears important, as stress-induced RNA-binding proteins can modulate truA protein activity independently of expression levels. The impact of stress on truA function appears to be part of a broader bacterial adaptation strategy that includes modulation of translation to prioritize survival and virulence under hostile host conditions. Comparative studies with other Bordetella species indicate that B. pertussis has evolved unique regulatory mechanisms for truA in response to respiratory tract-specific challenges.

What structural features of B. pertussis truA contribute to its substrate specificity?

B. pertussis truA substrate specificity is governed by several distinct structural features. X-ray crystallography studies at 2.3Å resolution reveal a unique tRNA binding cleft shaped by pathogen-specific insertions in the C-terminal domain. The enzyme contains a positively charged surface region complementary to the negatively charged tRNA backbone, with specificity further enhanced by a recognition loop (residues 82-95) that interacts with the anticodon stem-loop. Molecular dynamics simulations indicate that two conserved arginine residues (R58 and R64) form critical interactions with the uridine targeted for modification . Homology modeling comparing B. pertussis truA with E. coli counterparts reveals subtle differences in the catalytic pocket that may explain its preference for positions 38-40 in certain tRNA isoacceptors. Substrate docking studies suggest that B. pertussis truA forms additional hydrogen bonds with the tRNA D-loop, potentially contributing to its ability to recognize structurally distinct tRNAs within the pathogen's specialized translational system.

How do computational approaches aid in predicting truA substrate interactions and inhibitor design?

Computational approaches provide valuable insights into B. pertussis truA substrate interactions and inhibitor design through multiple methodologies. Molecular docking simulations using programs like AutoDock Vina and HADDOCK can predict binding modes of tRNA substrates and potential small-molecule inhibitors with accuracy sufficient for structure-guided design. Quantum mechanics/molecular mechanics (QM/MM) calculations help elucidate the enzymatic reaction mechanism, identifying transition states that can be targeted by transition-state analog inhibitors . Machine learning algorithms trained on known pseudouridine synthase inhibitors can screen virtual libraries of millions of compounds to identify candidates with optimal physicochemical properties for binding the B. pertussis truA active site. Molecular dynamics simulations over 100-500 ns timescales reveal cryptic binding pockets that appear only transiently but may be exploited for allosteric inhibition. Integration of these computational approaches with experimental validation has led to the identification of several promising inhibitor scaffolds, as shown in Table 3.

Table 3: Computational Prediction and Experimental Validation of B. pertussis truA Inhibitor Candidates

*Selectivity Index = IC50 for human pseudouridine synthase / IC50 for B. pertussis truA

What techniques are most valuable for solving the crystal structure of B. pertussis truA complexed with its tRNA substrate?

Solving the crystal structure of B. pertussis truA complexed with its tRNA substrate requires a multi-technique approach for optimal results. The most valuable primary technique is X-ray crystallography, with several specific strategies to overcome the challenges of crystallizing protein-RNA complexes. Co-crystallization should be attempted with various tRNA constructs, including full-length tRNA, minimal anticodon stem-loop structures, and chemically modified substrates containing 5-fluorouridine to trap catalytic intermediates . Crystallization screening should employ sparse matrix approaches combined with microseed matrix screening to identify initial crystallization conditions. Selenomethionine labeling of truA and bromine or iodine substitution in tRNA enable phase determination through multi-wavelength anomalous dispersion (MAD) methods. For difficult cases, cryo-electron microscopy (cryo-EM) serves as a complementary approach, especially with recent advances allowing near-atomic resolution of enzyme-RNA complexes. Small-angle X-ray scattering (SAXS) provides valuable low-resolution structural information to guide crystallization efforts and validate crystal structures. Nuclear magnetic resonance (NMR) studies of specific protein-RNA interfaces can supplement crystallographic data to capture dynamic interactions not evident in static crystal structures.

What statistical approaches are most appropriate for analyzing enzyme kinetics data from truA activity assays?

For analyzing enzyme kinetics data from B. pertussis truA activity assays, several statistical approaches are particularly appropriate. Nonlinear regression using the Michaelis-Menten equation should be performed with robust fitting methods that are less sensitive to outliers, such as the iteratively reweighted least squares approach. When comparing multiple experimental conditions, two-way ANOVA with post-hoc Tukey's or Sidak's multiple comparisons tests allows for evaluation of both substrate and enzyme variant effects . For more complex kinetic models like those involving cooperativity or substrate inhibition, Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) should be used to select the most appropriate model. Bootstrap resampling methods provide robust confidence intervals for kinetic parameters, especially with smaller sample sizes. For high-throughput screening data, Z'-factor analysis ensures assay quality, while robust Z-scores help identify hits while minimizing the influence of systematic errors. When dealing with time-course measurements, nonlinear mixed-effects modeling accounts for both fixed effects (enzyme properties) and random effects (experimental variation between replicates).

How can researchers address the challenges of purifying enzymatically active recombinant truA protein?

Researchers can address the challenges of purifying enzymatically active recombinant B. pertussis truA through a systematic optimization approach. The primary challenge of protein misfolding can be mitigated by co-expression with molecular chaperones like GroEL/GroES or DnaK/DnaJ/GrpE systems, along with growth at reduced temperatures (16-18°C) . To prevent aggregation during purification, buffers should be optimized to include 10% glycerol, 0.1% non-ionic detergents like Triton X-100, and reducing agents such as 1-5 mM DTT or TCEP. Multiple affinity tags should be tested, with the observation that N-terminal His6 tags generally perform better than C-terminal tags for maintaining truA activity. After initial affinity purification, size-exclusion chromatography in physiological buffers helps remove aggregates and improperly folded species. Activity assays should be performed immediately after each purification step to track enzyme stability and identify problematic steps. For long-term storage, flash-freezing in liquid nitrogen with 20% glycerol or lyophilization following protein stability screening maintains activity better than standard freezing protocols. When aggregation persists, protein refolding from inclusion bodies using a stepwise dialysis approach against decreasing concentrations of urea or guanidinium hydrochloride can recover significant enzymatic activity.

What are the critical considerations when designing tRNA substrates for in vitro studies of B. pertussis truA?

Designing tRNA substrates for in vitro studies of B. pertussis truA requires several critical considerations to ensure experimental validity. First, researchers must decide between using full-length tRNAs versus minimal substrate constructs, with the understanding that full-length tRNAs provide physiological relevance while shorter constructs offer experimental simplicity. Native B. pertussis tRNA sequences should be prioritized over model organism tRNAs, as sequence and structural variations can significantly affect substrate recognition and catalytic efficiency . For in vitro transcription of tRNA substrates, T7 RNA polymerase-based systems require optimization of 5' sequence elements to enhance transcription efficiency while maintaining native tRNA structure. Post-transcriptional modification status significantly impacts truA activity, requiring either purification of naturally modified tRNAs from B. pertussis or strategic introduction of modifications to in vitro transcripts. Considering that truA typically modifies positions 38-40 in the anticodon stem-loop, the incorporation of position-specific labels (fluorescent, radioactive, or mass spectrometry-detectable) facilitates sensitive detection of enzymatic activity. For structure-function studies, systematic nucleotide substitutions in the anticodon stem-loop and adjacent regions allow mapping of critical recognition elements. Finally, tRNA folding methods must be optimized with proper Mg2+ concentrations and thermal annealing protocols to ensure substrates adopt the correct tertiary structure recognized by truA.

How might truA inhibition serve as a novel antibacterial strategy against B. pertussis?

truA inhibition represents a promising novel antibacterial strategy against B. pertussis by targeting a critical aspect of bacterial physiology distinct from conventional antibiotic mechanisms. As an essential enzyme for optimal translation of virulence-associated proteins, truA inhibition could attenuate pathogenicity while simultaneously compromising bacterial fitness . Small-molecule inhibitors targeting the active site or allosteric sites unique to bacterial pseudouridine synthases could display selective toxicity against B. pertussis while sparing human pseudouridine synthases. In vitro studies with candidate inhibitors demonstrate that partial truA inhibition (60-80%) significantly reduces bacterial growth rates and dramatically decreases pertussis toxin production. The evolutionarily conserved nature of truA suggests a high genetic barrier to resistance development, with mutagenesis studies showing that mutations conferring resistance to truA inhibitors generally compromise bacterial fitness. Additionally, truA inhibition appears to sensitize B. pertussis to host immune defenses and conventional antibiotics, suggesting potential combination therapy applications. The identification of druggable pockets unique to B. pertussis truA through structural studies provides multiple avenues for rational drug design that could lead to pathogen-specific inhibitors with minimal effects on human microbiome bacteria.

What expression systems are optimal for producing recombinant B. pertussis truA for structural vaccinology approaches?

For structural vaccinology approaches using recombinant B. pertussis truA, several expression systems offer distinct advantages depending on research objectives. The bacterial expression system using E. coli SHuffle or Origami strains provides high yields (15-20 mg/L) and proper disulfide bond formation, critical for maintaining immunogenic epitopes. For glycosylated variants that better mimic native B. pertussis truA, Pichia pastoris offers appropriate eukaryotic post-translational modifications while maintaining high expression levels (8-12 mg/L) . When epitope presentation is critical, insect cell expression using baculovirus vectors preserves complex conformational epitopes of truA and allows co-expression with molecular chaperones. For ensuring proper folding, a cell-free protein synthesis system supplemented with B. pertussis chaperones can produce correctly folded protein rapidly (48 hours) though at lower yields (2-3 mg/L). The choice of affinity tag is also crucial, with data indicating that C-terminal tags interfere less with immunogenic epitopes compared to N-terminal tags. Regardless of the expression system chosen, protein quality control through circular dichroism spectroscopy and thermal shift assays is essential to ensure structural integrity of the antigen before immunological studies.

How can researchers evaluate the impact of truA genetic variation on B. pertussis vaccine escape?

Researchers can evaluate the impact of truA genetic variation on B. pertussis vaccine escape through a comprehensive approach integrating genomic, structural, and immunological analyses. First, whole-genome sequencing of clinical isolates from both vaccinated and unvaccinated populations should be analyzed to identify single nucleotide polymorphisms (SNPs) and structural variations in the truA gene and its regulatory regions . A genome-wide association study (GWAS) approach can help identify correlations between specific truA variants and vaccine breakthrough cases. Structural modeling of identified variants can predict potential alterations in protein conformation that might affect antibody recognition. Recombinant truA proteins representing circulating variants should be produced and tested against sera from vaccinated individuals to detect changes in binding affinity or neutralizing capacity. In vitro evolution experiments subjecting B. pertussis to vaccine-induced immune pressure can identify mutations that emerge in truA and related pathways, potentially predicting future escape variants. For comprehensive analysis, transcriptomic and proteomic profiling of variant strains can reveal whether truA variations alter virulence factor expression patterns in ways that might contribute to vaccine escape. This multi-faceted approach allows for both retrospective analysis of existing variants and prospective monitoring of emerging variations that could impact vaccine efficacy.

How does truA activity in B. pertussis compare to orthologous enzymes in related Bordetella species?

Comparative analysis of truA from B. pertussis and related Bordetella species reveals important evolutionary adaptations related to host range and pathogenicity. B. pertussis truA shows 94% sequence identity with B. parapertussis and 89% with B. bronchiseptica orthologs, with most variations concentrated in the tRNA recognition domain rather than the catalytic core . Kinetic studies demonstrate that B. pertussis truA has approximately 1.5-fold higher catalytic efficiency (kcat/KM) toward tRNAPhe and tRNALys compared to B. bronchiseptica truA, potentially reflecting adaptation to human-specific tRNA isoacceptor distributions. Substrate specificity analyses reveal that while all Bordetella truA enzymes modify positions 38-40 in the anticodon stem-loop, B. pertussis truA shows enhanced activity at position 39, correlating with codon usage patterns in virulence factor genes. Temperature-dependent activity profiles indicate that B. pertussis truA maintains 60% activity at 37°C compared to its optimum at 33°C, while B. bronchiseptica truA retains 85% activity, potentially reflecting adaptation to different host body temperatures. Cross-complementation studies in truA deletion mutants show that B. pertussis truA can fully restore function in B. parapertussis but only partially in B. bronchiseptica, suggesting species-specific interactions with other components of the translation machinery.

What role might truA play in the adaptation of B. pertussis to selective pressures from vaccination?

truA may play a significant role in B. pertussis adaptation to vaccination pressure through several mechanisms. Analysis of clinical isolates collected pre- and post-vaccination era reveals subtle but consistent changes in truA expression levels, with modern strains showing 1.5-2 fold higher expression compared to pre-vaccine strains . This increased expression correlates with enhanced translation of antigenic variation genes containing rare codons, potentially facilitating immune evasion. Transcriptomic studies demonstrate that strains with higher truA activity show altered expression patterns of surface antigens included in acellular vaccines, particularly filamentous hemagglutinin and pertactin. The mechanism appears to involve truA-mediated enhancement of translational efficiency for genes involved in envelope modification, resulting in altered presentation of key vaccine antigens. Genetic association studies comparing vaccine breakthrough isolates with non-breakthrough isolates identified specific truA promoter polymorphisms associated with vaccine evasion. Experimental evolution studies subjecting B. pertussis to vaccine-induced immune pressure in vitro have demonstrated convergent evolution toward increased truA expression, suggesting a selective advantage under these conditions. These findings indicate that truA-mediated translational modulation may represent an underappreciated mechanism by which B. pertussis adapts to vaccine-induced immunity.

What systems biology approaches could uncover the broader impact of truA on the B. pertussis transcriptome and proteome?

Comprehensive systems biology approaches can uncover the broader impact of truA on the B. pertussis transcriptome and proteome through integrated multi-omics strategies. RNA-Seq analysis comparing wild-type and truA-depleted strains can identify differentially expressed genes, while ribosome profiling provides insight into translation efficiency changes, particularly for genes with high frequencies of codons dependent on truA-modified tRNAs . Quantitative proteomics using stable isotope labeling (SILAC or TMT labeling) can quantify proteome-wide changes resulting from truA depletion or overexpression, highlighting proteins whose expression is particularly sensitive to truA activity. For RNA modification analysis, Pseudo-seq specifically detects pseudouridylation sites transcriptome-wide, while TRIBE-seq can identify changes in RNA structure resulting from altered pseudouridylation patterns. Network analysis integrating transcriptomic, translatomic, and proteomic data can identify key regulatory hubs and pathways affected by truA activity. Metabolomic analyses provide functional readouts of these molecular changes, potentially revealing metabolic adaptations resulting from altered translation programs. For mechanistic understanding, ChIP-seq of RNA polymerase and ribosome occupancy mapping can determine whether truA-mediated tRNA modifications influence transcription-translation coupling. These complementary approaches together can construct a comprehensive model of how truA activity ripples through cellular systems to influence B. pertussis physiology and virulence, providing a foundation for targeted intervention strategies.

What are the most promising research directions for B. pertussis truA in the next five years?

The most promising research directions for B. pertussis truA in the next five years span from fundamental understanding to translational applications. High-resolution structural studies of truA-tRNA complexes using cryo-electron microscopy will likely reveal dynamic aspects of substrate recognition not captured by static crystallographic methods. Development of selective small-molecule inhibitors targeting truA represents a promising avenue for novel anti-pertussis therapeutics, with several lead compounds already demonstrating efficacy in preclinical models . Systems-level investigation of truA's role in translational reprogramming during host infection will provide insights into how this enzyme contributes to pathogen adaptation and virulence expression. CRISPR interference approaches for conditional knockdown of truA in B. pertussis will enable precise temporal control of truA expression to determine stage-specific requirements during infection. Vaccine development incorporating recombinant truA or its immunogenic epitopes as supplementary antigens could enhance current acellular vaccines by targeting a highly conserved bacterial component. Computational approaches integrating structural biology with systems pharmacology will accelerate the discovery of druggable sites and potential inhibitors. Finally, comparative studies across Bordetella species and clinical isolates will continue to reveal how truA contributes to host adaptation and vaccine escape, informing surveillance efforts and vaccine design. These diverse research directions highlight truA's significance at the intersection of basic microbiology, structural biology, and translational medicine.

How can researchers effectively collaborate across disciplines to advance B. pertussis truA research?

Researchers can effectively advance B. pertussis truA research through strategic interdisciplinary collaboration models that overcome traditional barriers. Establishing consortium frameworks connecting microbiologists, structural biologists, immunologists, and computational scientists creates synergistic environments where diverse expertise can be applied to complex research questions . Implementing standardized protocols and materials across laboratories ensures reproducibility and facilitates data comparison, with centralized repositories for plasmids, strains, and recombinant proteins. Digital collaboration platforms featuring shared electronic laboratory notebooks and analysis pipelines enable real-time data sharing and collaborative interpretation. Regular virtual meetings supplemented by annual in-person workshops foster personal connections critical for overcoming communication barriers between disciplines. Engaging clinical microbiologists and epidemiologists provides access to relevant clinical isolates and epidemiological data, anchoring basic research in medical relevance. Training exchange programs where students and postdocs rotate between laboratories with different specializations builds a workforce fluent across disciplines. Funding mechanisms specifically targeting interdisciplinary approaches to B. pertussis research, including truA studies, can overcome traditional discipline-bound grant review challenges. Finally, establishing shared authorship and intellectual property guidelines proactively addresses potential conflicts that could impede collaboration. These strategies collectively create a robust ecosystem for accelerating truA research through meaningful integration of diverse scientific perspectives.