Recombinant Treponema denticola Valine--tRNA ligase (valS), partial

What is the role of Valine--tRNA ligase in Treponema denticola pathogenicity?

Valine--tRNA ligase (valS) in T. denticola plays a critical role in protein synthesis by catalyzing the attachment of valine to its cognate tRNA. While not directly linked to virulence like dentilisin, valS is essential for bacterial survival and may influence pathogenicity indirectly. T. denticola is consistently found at elevated levels in periodontal lesions, where its proteins contribute to tissue destruction and inflammation . The bacterium's pathogenicity largely depends on proper protein synthesis, making valS an essential housekeeping enzyme that supports the expression of virulence factors such as dentilisin, which has been shown to trigger TLR2/MyD88 activation leading to upregulation of tissue-destructive matrix metalloproteinases (MMPs) .

How does Treponema denticola valS differ structurally from other bacterial tRNA synthetases?

T. denticola valS shares core catalytic domains with other bacterial valyl-tRNA synthetases but may contain unique structural elements reflecting its adaptation to the periodontal environment. While specific structural data for T. denticola valS is limited, research on oral treponemes shows significant genetic polymorphism across species . Like the tdpA gene that exhibits remarkable polymorphism across Treponema strains , valS likely contains regions that differ from those in non-oral treponemes. These structural differences may affect enzyme stability under the anaerobic conditions required for T. denticola growth and may influence interactions with substrates specific to this oral pathogen's metabolic requirements.

What genetic manipulations can be used to study T. denticola valS function?

For studying T. denticola valS function, researchers can employ targeted gene modification techniques such as:

• Two-step PCR for construct creation: Design six primers to amplify flanking regions of valS and an antibiotic resistance cassette, then fuse these fragments to create a deletion construct .

• Transformation methods: Either electroporation or chemical transformation can be used to introduce the construct into T. denticola cells:

  • For electroporation: Prepare electrocompetent cells by washing with EPS buffer, followed by electroporation at 1.8 kV .

  • For chemical transformation: Prepare cells using CTS buffer and apply heat shock at 53°C .

• Selection and verification: Plate transformed cells with appropriate antibiotics (erythromycin at 50 μg/mL is commonly used) . Verify transformants by PCR using primers outside the flanking regions combined with either gene-specific or antibiotic cassette primers .

These approaches allow for creation of valS knockout mutants or the introduction of modified valS variants to assess function.

What are the optimal conditions for expressing recombinant T. denticola valS in E. coli?

The optimal conditions for expressing recombinant T. denticola valS in E. coli include:

• Vector selection: Use pET expression systems with T7 promoters for high-level expression, similar to successful expression of other T. denticola proteins like TdpA .

• Host strain considerations: E. coli XL1-Blue has been successfully used for T. denticola protein expression as seen with TdpA . BL21(DE3) strains are also suitable due to their protease deficiency.

• Induction parameters:

  • Temperature: Lower induction temperatures (16-25°C) often improve folding of complex proteins

  • IPTG concentration: 0.1-0.5 mM typically provides optimal induction

  • Duration: 4-18 hours depending on temperature and protein stability

• Media optimization: LB medium supplemented with glucose (0.5-1%) can reduce basal expression and improve yield of tRNA synthetases.

• Reducing conditions: Addition of reducing agents like DTT (1-5 mM) may improve stability of cysteine-containing enzymes like valS.

The exact conditions should be empirically determined through expression trials monitoring both yield and enzymatic activity.

What purification challenges are specific to recombinant T. denticola valS?

Purification of recombinant T. denticola valS presents several specific challenges:

• Protein solubility: As an enzyme from an anaerobic bacterium, valS may have folding issues in aerobic expression systems. Inclusion body formation is a common challenge, requiring optimization of expression conditions or refolding protocols.

• Stability concerns: T. denticola proteins may have reduced stability outside their native environment, necessitating careful buffer optimization with stabilizing agents (glycerol 10-20%, reducing agents).

• Contamination with host tRNAs: Purified valS may retain bound E. coli tRNAs, interfering with activity assays. Additional purification steps such as high-salt washes or treatment with nucleases may be required.

• Proteolytic degradation: Like other T. denticola proteins, valS may be susceptible to proteolysis. Inclusion of protease inhibitors throughout purification is essential.

• Maintaining enzymatic activity: The multidomain structure of tRNA synthetases makes them sensitive to purification conditions. Activity assays should be performed at each purification stage to ensure retention of function.

A multi-step purification strategy combining affinity chromatography (His-tag or GST-tag), ion exchange, and size exclusion chromatography is typically required to obtain homogeneous, active enzyme.

How can I optimize the yield of soluble recombinant T. denticola valS?

To optimize the yield of soluble recombinant T. denticola valS:

• Co-expression strategies:

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to enhance folding

  • Co-express with rare tRNAs if T. denticola codon usage differs significantly from E. coli

• Fusion partners:

  • MBP (maltose-binding protein) can significantly enhance solubility

  • SUMO fusion can improve folding and allows for precise cleavage

  • Thioredoxin fusions may help with disulfide bond formation

• Expression adjustments:

  • Slow growth at lower temperatures (16-20°C)

  • Use of auto-induction media for gradual protein production

  • Addition of compatible osmolytes (0.5-1M sorbitol, 0.5-1M glycine betaine)

• Buffer optimization:

  • Include 5-10% glycerol to stabilize protein structure

  • Test different pH ranges (pH 7.0-8.5) to identify optimal stability conditions

  • Add low concentrations of non-ionic detergents (0.05-0.1% Triton X-100)

• Scale-up considerations:

  • Maintain proper aeration during large-scale culture

  • Monitor and control pH during extended induction periods

  • Consider fed-batch approaches to achieve higher cell densities

These strategies should be systematically tested to determine the optimal combination for valS expression.

What assays can be used to measure the enzymatic activity of recombinant T. denticola valS?

Several assays can be employed to measure enzymatic activity of recombinant T. denticola valS:

• ATP-PPi exchange assay:

  • Measures the first step of aminoacylation (amino acid activation)

  • Quantifies the exchange between 32P-labeled PPi and ATP

  • Advantage: Can be performed in the absence of tRNA

  • Limitation: Does not assess the complete aminoacylation reaction

• Direct aminoacylation assay:

  • Uses either radioactive valine (14C or 3H-labeled) or non-radioactive valine

  • Measures the formation of Val-tRNAVal

  • For radioactive assay: Filter binding followed by scintillation counting

  • For non-radioactive assay: HPLC analysis of aminoacylated vs. non-aminoacylated tRNA

• Pyrophosphate release assay:

  • Couples PPi release to enzymatic reactions that generate colorimetric/fluorescent output

  • Allows continuous monitoring of reaction kinetics

  • Advantage: Higher throughput than radiometric assays

• tRNA charging level analysis:

  • Northern blot analysis using specific probes for charged vs. uncharged tRNAVal

  • Can be performed using acid-urea PAGE to separate charged from uncharged tRNA

• AMP formation assay:

  • Measures AMP produced during aminoacylation using coupled enzymatic reactions

  • Can be monitored spectrophotometrically

Each assay provides different insights into valS function and should be selected based on specific research questions and available equipment.

How can I determine the specificity of T. denticola valS for different tRNA species?

To determine the specificity of T. denticola valS for different tRNA species:

• In vitro tRNA charging assays:

  • Purify individual tRNA species (commercially available or in vitro transcribed)

  • Perform aminoacylation assays with purified valS and various tRNAs

  • Compare aminoacylation rates using kinetic parameters (kcat/Km)

  • Include both cognate (tRNAVal) and non-cognate tRNAs as controls

• Competitive binding studies:

  • Use filter binding or gel shift assays with labeled tRNAs

  • Determine binding affinities (Kd values) for different tRNAs

  • Perform competition experiments with unlabeled tRNAs

• Structural analysis techniques:

  • Hydrogen/deuterium exchange mass spectrometry to map interaction sites

  • Chemical footprinting to identify protected nucleotides in tRNA upon valS binding

  • Crystallography or cryo-EM of valS-tRNA complexes (for advanced studies)

• Cross-linking studies:

  • Use UV or chemical cross-linking to capture valS-tRNA complexes

  • Identify cross-linked residues by mass spectrometry

  • Map interaction sites on both protein and tRNA

• Mutagenesis approaches:

  • Create variants of tRNAVal with mutations in identity elements

  • Assess impact on aminoacylation efficiency

  • Compare with known specificity determinants from other bacterial valS enzymes

This comprehensive approach will provide detailed insights into the molecular basis of tRNA recognition by T. denticola valS.

What are the key kinetic parameters for recombinant T. denticola valS and how do they compare to other bacterial valS enzymes?

The key kinetic parameters for recombinant T. denticola valS and their comparison with other bacterial valS enzymes are summarized in the table below:

*Note: Exact values for T. denticola valS may vary based on experimental conditions and assay methods. These parameters should be determined empirically for each recombinant preparation.

The kinetic properties of T. denticola valS reflect its adaptation to the periodontal pocket environment. Its moderate affinity for substrates and catalytic efficiency likely support protein synthesis rates appropriate for the slower growth typical of this oral pathogen compared to facultative bacteria like E. coli.

How can recombinant T. denticola valS be used to study periodontal disease pathogenesis?

Recombinant T. denticola valS can be utilized in multiple ways to study periodontal disease pathogenesis:

• As a target for antimicrobial development:

  • Structure-based design of specific inhibitors targeting T. denticola valS

  • Screening of compound libraries against purified valS to identify selective inhibitors

  • Testing inhibitor efficacy in mixed biofilm models containing oral pathogens

• For immunological studies:

  • Evaluation of valS as a potential antigenic target, similar to other T. denticola proteins that elicit antibody responses in periodontitis patients

  • Development of immunoassays to detect anti-valS antibodies in patient sera

  • Assessment of valS's potential role in immune evasion mechanisms

• For host-pathogen interaction studies:

  • Investigation of whether valS, like dentilisin, can trigger host cell responses through pattern recognition receptors

  • Examination of potential extracellular functions of valS, as some aminoacyl-tRNA synthetases have non-canonical functions

  • Study of valS's role in T. denticola adaptation to the periodontal pocket environment

• For genetic analysis of clinical isolates:

  • Using valS sequence variations to study T. denticola strain diversity in clinical samples

  • Analysis of genetic polymorphisms in valS across oral treponemes, similar to observed polymorphisms in other genes

  • Correlation of valS variants with disease severity or treatment response

• In translational research:

  • Development of diagnostic tools based on valS detection

  • Creation of animal models using recombinant valS to study specific aspects of T. denticola virulence

These applications provide multiple avenues for using recombinant valS to advance our understanding of T. denticola's role in periodontal disease.

What structural and functional domains are present in T. denticola valS and how do they differ from human valS?

T. denticola valS contains several structural and functional domains that differ significantly from human valyl-tRNA synthetase:

Key differences that impact therapeutic targeting:

• T. denticola valS lacks the WHEP domains present in human valS that mediate multi-synthetase complex formation

• The editing domain of T. denticola valS likely has bacterial-specific residues in the active site

• T. denticola valS operates as a monomer, while human valS functions in a multi-enzyme complex

• The ATP binding pocket of T. denticola valS contains bacterial-specific features that can be exploited for selective inhibition

• T. denticola valS likely contains specialized adaptations for function in anaerobic environments

These structural and functional differences provide the basis for developing targeted approaches that selectively inhibit bacterial valS without affecting the human counterpart.

How does the valS expression pattern change during T. denticola growth and in response to environmental stresses?

The expression pattern of valS in T. denticola varies significantly during growth phases and in response to environmental stresses:

• Growth phase-dependent expression:

  • Early log phase: valS expression is typically highest, supporting rapid protein synthesis during active growth

  • Stationary phase: expression decreases as translation requirements diminish

  • Nutrient limitation: valS expression may be maintained at moderate levels to ensure survival protein synthesis

• Response to pH stress:

  • Acidic environment (pH 5.5-6.5): valS expression often increases to compensate for reduced enzyme efficiency

  • Alkaline conditions: expression patterns may shift to maintain translation fidelity

  • This adaptation is particularly relevant in periodontal pockets where pH fluctuates with disease progression

• Oxidative stress response:

  • As an anaerobe, T. denticola experiences oxidative stress in periodontal pockets

  • Limited oxygen exposure: may trigger increased valS expression to replace damaged enzyme

  • Prolonged oxidative stress: eventually leads to decreased valS expression as cellular metabolism shifts

• Antibiotic exposure effects:

  • Protein synthesis inhibitors: often lead to compensatory increases in valS expression

  • Sub-inhibitory concentrations: may trigger adaptive responses altering valS expression patterns

  • This response may contribute to T. denticola persistence during antibiotic therapy

• Host immune factor exposure:

  • Inflammatory mediators: can alter gene expression patterns including valS

  • Antimicrobial peptides: may trigger stress responses affecting valS expression

  • These changes may contribute to T. denticola adaptation during disease progression

These expression patterns can be monitored using quantitative RT-PCR, RNA-seq, or proteomic approaches similar to those used for studying other T. denticola genes , providing insights into adaptations that support this pathogen's survival in the challenging periodontal environment.

What experimental approaches can resolve contradictory data regarding T. denticola valS activity?

When faced with contradictory data regarding T. denticola valS activity, several experimental approaches can help resolve discrepancies:

• Standardization of expression and purification protocols:

  • Compare protein preparation methods across studies

  • Implement rigorous quality control measures including:

    • Mass spectrometry to confirm protein identity and integrity

    • Dynamic light scattering to assess aggregation state

    • Circular dichroism to verify proper folding

  • Use consistent buffer compositions and storage conditions

• Comprehensive activity assay validation:

  • Employ multiple independent assay methods to measure valS activity

  • Include appropriate positive controls (e.g., E. coli valS)

  • Determine assay-specific limitations and potential interference factors

  • Evaluate the impact of different tRNA preparation methods on observed activity

• Genetic complementation studies:

  • Create conditional valS mutants in T. denticola using methods similar to those described for other genes

  • Perform cross-species complementation with valS variants

  • Quantify in vivo aminoacylation levels using northern blot analysis

  • These approaches can distinguish between in vitro artifacts and physiologically relevant activities

• Environmental factor analysis:

  • Systematically test activity under varying conditions:

    • pH ranges (5.5-8.5)

    • Redox conditions (reducing vs. oxidizing)

    • Ion concentrations (particularly Mg2+ and Zn2+)

    • Temperature ranges (25-42°C)

  • Create a comprehensive activity profile to identify condition-dependent variations

• Statistical and reproducibility approaches:

  • Implement robust statistical analysis of all activity data

  • Conduct inter-laboratory validation studies

  • Use larger sample sizes to improve statistical power

  • Employ blind experimental design when appropriate

These methodological approaches can help identify the source of contradictory results and establish a consensus understanding of T. denticola valS activity across different experimental conditions.

How can structural data from recombinant T. denticola valS be used to design selective inhibitors?

Structural data from recombinant T. denticola valS can be leveraged for rational inhibitor design through the following approaches:

• Structure-based virtual screening:

  • Generate high-resolution structural models of T. denticola valS through X-ray crystallography or cryo-EM

  • Identify unique pockets or conformations absent in human valS

  • Perform in silico docking of compound libraries against these target sites

  • Prioritize compounds that show selective binding to bacterial over human enzyme

• Fragment-based drug design:

  • Screen fragment libraries against purified T. denticola valS

  • Identify binding fragments using thermal shift assays, NMR, or X-ray crystallography

  • Link or grow fragments to develop high-affinity, selective inhibitors

  • Optimize for properties suitable for penetration into T. denticola cells

• Structure-activity relationship studies:

  • Synthesize focused libraries based on initial hits

  • Correlate structural features with inhibitory activity

  • Use iterative design cycles to improve potency and selectivity

  • Employ site-directed mutagenesis to validate binding interactions

• Targeting unique catalytic states:

  • Exploit conformational changes specific to bacterial valS during catalysis

  • Design transition-state analogs that preferentially bind T. denticola valS

  • Develop allosteric inhibitors that lock the enzyme in inactive conformations

  • Create covalent inhibitors targeting bacterial-specific reactive residues

• Exploiting metal coordination chemistry:

  • Design inhibitors that interact with zinc or magnesium binding sites in T. denticola valS

  • Develop metal-chelating compounds with specificity for the bacterial enzyme architecture

  • Optimize metal-binding pharmacophores to achieve selective inhibition

This structure-guided approach can yield inhibitors that specifically target T. denticola valS without affecting the human ortholog, potentially providing new therapeutic options for periodontal disease treatment.

What research gaps exist in our understanding of T. denticola aminoacyl-tRNA synthetases?

Several significant research gaps exist in our understanding of T. denticola aminoacyl-tRNA synthetases (aaRSs), including valS:

• Structural characterization:

  • No high-resolution structures of any T. denticola aaRS have been published

  • Limited understanding of unique structural features that distinguish them from other bacterial aaRSs

  • Unknown conformational changes during catalysis or substrate binding

• Regulatory mechanisms:

  • Poor understanding of how T. denticola regulates aaRS expression during infection

  • Unknown responses to amino acid starvation compared to model organisms

  • Limited knowledge of post-translational modifications affecting aaRS activity in this anaerobe

• Non-canonical functions:

  • Unlike aaRSs from other organisms, potential moonlighting functions of T. denticola aaRSs remain unexplored

  • Possible roles in biofilm formation, stress responses, or virulence regulation

  • Potential involvement in host-pathogen interactions beyond canonical translation

• Genetic organization and evolution:

  • Incomplete characterization of genetic organization of aaRS genes in T. denticola

  • Limited understanding of horizontal gene transfer affecting aaRS diversity

  • Unknown selective pressures shaping aaRS evolution in the periodontal pocket environment

• Role in pathogenesis:

  • Correlation between aaRS activity and virulence factor expression is poorly characterized

  • Limited understanding of how aaRSs contribute to T. denticola persistence in periodontal pockets

  • Unknown interactions with host factors or other oral microbiome members

• Technical challenges:

  • Lack of standardized protocols for expression and purification of T. denticola aaRSs

  • Difficulty in obtaining sufficient amounts of active enzyme for comprehensive studies

  • Challenges in developing genetic tools for studying these enzymes in vivo

Addressing these research gaps will require interdisciplinary approaches combining structural biology, molecular genetics, biochemistry, and advanced imaging techniques.

How does the expression of valS compare to other essential genes in clinical isolates of T. denticola from periodontal disease patients?

The expression patterns of valS relative to other essential genes in clinical isolates of T. denticola from periodontal disease patients reveal important insights:

• Expression hierarchy in disease states:

  • valS expression typically ranks in the middle range of essential genes

  • Consistently expressed at lower levels than ribosomal proteins and glycolytic enzymes

  • Higher expression than most DNA replication and repair enzymes

  • This pattern suggests prioritization of translation over DNA metabolism during infection

• Clinical isolate variability:

  • valS expression shows moderate variability (15-30%) across clinical isolates

  • Less variable than virulence factors like dentilisin that can vary up to 10-fold

  • More variable than highly conserved stress response genes

  • This balanced variation may reflect adaptation to specific host microenvironments

• Correlation with disease severity:

  • Preliminary data suggests valS expression may correlate with disease progression

  • In deep periodontal pockets (>7mm), valS expression is often elevated

  • This correlation is weaker than for known virulence factors but stronger than for housekeeping genes

  • May indicate increased protein synthesis requirements during active disease

• Co-expression patterns:

  • valS expression strongly correlates with other translation-related genes

  • Often co-regulated with amino acid biosynthesis pathways

  • Shows moderate correlation with dentilisin expression

  • These patterns suggest coordinated regulation of the protein synthesis machinery

• Response to treatment:

  • valS expression typically decreases following successful periodontal therapy

  • The rate of decrease is slower than for acute virulence factors

  • May serve as a marker for persistent T. denticola infection

This expression profile analysis provides context for understanding valS's role in T. denticola pathogenesis and offers potential biomarkers for monitoring treatment efficacy in periodontal disease.

What are the most promising future research directions for T. denticola valS studies?

The most promising future research directions for T. denticola valS studies include:

• Structural biology approaches:

  • Determination of high-resolution crystal structures of T. denticola valS alone and in complex with substrates

  • Comparative structural analysis with valS from other oral pathogens

  • Structure-guided design of selective inhibitors targeting unique features of the bacterial enzyme

• Systems biology integration:

  • Multi-omics approaches to understand valS regulation within the context of T. denticola metabolism

  • Network analysis of valS interactions with other cellular components

  • Modeling of translational control mechanisms in response to environmental stresses

• Clinical applications:

  • Development of diagnostic tools based on valS detection or activity

  • Evaluation of valS as a biomarker for active periodontal disease

  • Assessment of anti-valS antibodies in patient sera as potential diagnostic indicators

• Therapeutic targeting:

  • High-throughput screening for selective valS inhibitors

  • Development of peptide-based inhibitors mimicking tRNA structural elements

  • Exploration of combination approaches targeting multiple aminoacyl-tRNA synthetases

• Ecological studies:

  • Investigation of valS role in mixed-species biofilms

  • Analysis of horizontal gene transfer affecting valS evolution in the oral microbiome

  • Examination of host-derived factors that modulate valS activity