Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Threonine--tRNA ligase (thrS), partial

What is the biological significance of Threonine--tRNA ligase in Leptospira interrogans?

Threonine--tRNA ligase (thrS) in L. interrogans is a critical enzyme involved in protein synthesis, specifically catalyzing the attachment of threonine to its cognate tRNA. This aminoacylation process is essential for accurate translation of genetic information and subsequent protein synthesis. In pathogenic Leptospira, proper protein synthesis is crucial for virulence factor expression, survival mechanisms, and adaptation to different environmental conditions. L. interrogans is the etiological agent of leptospirosis, a widespread zoonosis affecting over 1 million people annually with approximately 60,000 deaths . The proper functioning of thrS is essential for the bacterium's survival and pathogenicity, making it a potential target for antimicrobial development and understanding pathogenesis mechanisms.

How does L. interrogans serovar Copenhageni differ from serovar Icterohaemorrhagiae at the genomic level?

Despite their close genetic relationship, L. interrogans serovars Copenhageni and Icterohaemorrhagiae display distinct genomic differences. Phylogenetic analyses based on SNP datasets reveal that while both serovars cluster together with statistical support, they exhibit distinct spatial clustering patterns . The most significant distinguishing feature is a frameshift mutation within a homopolymeric tract of the lic12008 gene (related to LPS biosynthesis) present in all L. interrogans serovar Icterohaemorrhagiae strains but absent in Copenhageni strains . This internal indel can genetically distinguish between these serovars with high discriminatory power. Additionally, genome sequence variation analysis has identified 1,072 SNPs (276 in non-coding regions and 796 in coding regions) and 258 indels (191 in coding regions and 67 in non-coding regions) between these serovars . These genetic differences may influence thrS structure and expression patterns between the serovars, potentially affecting their virulence and host adaptation capabilities.

What is known about the expression patterns of thrS in L. interrogans during infection?

The expression of thrS in L. interrogans likely changes during different stages of infection, though specific expression data for thrS is limited in the available literature. Generally, pathogenic Leptospira modulate their gene expression in response to environmental changes they encounter during host infection. When transitioning from environmental survival to host infection, L. interrogans undergoes significant transcriptional changes to adapt to temperature shifts, pH changes, osmolarity differences, and host immune responses. During infection, L. interrogans has developed mechanisms to evade the human immune system, including the ability to escape from phagosomes into the cytosol in human macrophages, where they can proliferate and activate apoptosis . Essential genes involved in protein synthesis, including aminoacyl-tRNA synthetases like thrS, may be constitutively expressed but potentially upregulated during active replication phases within the host to support increased protein synthesis demands for virulence factor production.

What are the optimal conditions for expression and purification of recombinant thrS from L. interrogans serovar Copenhageni?

For optimal expression and purification of recombinant thrS from L. interrogans serovar Copenhageni, the following methodological approach is recommended:

Expression System Selection:

• The pRSET expression system (Invitrogen) has shown effectiveness for leptospiral proteins, as demonstrated with other recombinant Leptospira outer membrane proteins including LipL32, OmpL1, and LipL41 .

• E. coli BL21(DE3) or BL21(DE3)pLysS strains typically yield good expression levels for leptospiral proteins.

Expression Protocol:

• Clone the thrS gene or its catalytic domain into the expression vector with an N-terminal His-tag for purification

• Transform into expression host and grow cultures at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with 0.5-1.0 mM IPTG

• Lower the temperature to 18-25°C post-induction to enhance soluble protein production

• Continue expression for 16-18 hours

Purification Strategy:

• Harvest cells and lyse using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

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

• Wash with increasing imidazole concentrations (20-50 mM)

• Elute the recombinant protein with 250-300 mM imidazole

• Further purify using size exclusion chromatography in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

Protein Verification:

• Confirm protein identity by Western blot with anti-His antibodies and mass spectrometry

• Assess purity using SDS-PAGE (expected >95% purity)

• Verify enzymatic activity using aminoacylation assays

This approach typically yields 5-10 mg of purified recombinant thrS per liter of bacterial culture with >95% purity suitable for structural and functional studies.

How can researchers effectively evaluate the enzymatic activity of recombinant thrS?

To effectively evaluate the enzymatic activity of recombinant thrS from L. interrogans serovar Copenhageni, researchers should employ a multi-faceted approach:

1. ATP-PPi Exchange Assay:

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

• Reaction mixture: 100 mM HEPES-KOH (pH 7.5), 10 mM MgCl₂, 2 mM ATP, 2 mM threonine, 2 mM [³²P]PPi, and enzyme

• Incubate at 37°C, remove aliquots at time intervals, and quantify [³²P]ATP formation

2. tRNA Aminoacylation Assay:

• Measures the complete aminoacylation reaction

• Reaction mixture: 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl₂, 5 mM ATP, 20 μM [¹⁴C]threonine, 5-10 μM tRNA^Thr, and enzyme

• Incubate at 37°C, collect samples at various timepoints, precipitate charged tRNA with TCA, filter, and measure radioactivity

• Calculate aminoacylation rates as pmol of threonyl-tRNA^Thr formed per minute per μg enzyme

3. Pyrophosphate Release Assay:

• Alternative non-radioactive method

• Uses coupled enzymatic reactions to measure PPi release during aminoacylation

• Commercial kits (e.g., EnzChek Pyrophosphate Assay Kit) can be adapted for this purpose

4. Kinetic Parameter Determination:

• Measure initial reaction rates at varying concentrations of threonine (0.1-100 μM), ATP (0.1-5 mM), and tRNA^Thr (0.1-20 μM)

• Calculate Km, kcat, and kcat/Km values using Michaelis-Menten kinetics

5. Specificity Analysis:

• Test activity with non-cognate amino acids (serine, valine) to assess enzymatic specificity

• Evaluate activity with tRNA^Thr from different sources (E. coli vs. Leptospira)

Data Interpretation:

• Active thrS typically exhibits Km values in the low μM range for threonine and tRNA^Thr

• kcat values normally range from 1-10 s⁻¹

• Specificity constants (kcat/Km) for cognate substrates should be 100-1000 fold higher than for non-cognate substrates

These assays provide comprehensive evaluation of thrS activity, enabling researchers to assess both wild-type and mutant thrS properties for structure-function relationship studies.

What methods can be used to evaluate the structural properties of recombinant thrS?

Several complementary structural biology techniques can be employed to characterize recombinant thrS structure:

X-ray Crystallography:

• Most definitive method for obtaining atomic-resolution structure

• Protein concentration: 5-10 mg/ml in 20 mM HEPES pH 7.5, 150 mM NaCl

• Commercial crystallization screens (Hampton Research, Molecular Dimensions)

• Optimization of crystallization conditions based on initial hits

• Data collection at synchrotron radiation sources

• Structure determination by molecular replacement using homologous tRNA synthetase structures

Small-Angle X-ray Scattering (SAXS):

Circular Dichroism (CD) Spectroscopy:

• Evaluates secondary structure composition

• Far-UV CD (190-260 nm) measures α-helix and β-sheet content

• Near-UV CD (250-350 nm) assesses tertiary structure integrity

• Thermal denaturation studies provide stability information (melting temperature)

Differential Scanning Fluorimetry (DSF):

• Measures protein thermal stability

• Screens buffer conditions for optimal stability

• Protocol: 0.1-0.5 mg/ml protein with SYPRO Orange dye in 96-well format

• Temperature ramp from 25°C to 95°C with fluorescence monitoring

Native Mass Spectrometry:

• Determines oligomeric state and complex formation

• Sample preparation: buffer exchange to ammonium acetate (200 mM, pH 7.0)

• ESI-MS under native conditions

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Maps solvent accessibility and dynamics

• Identifies regions involved in substrate binding

• Analysis of deuterium uptake rates provides information on conformational changes

Integrating data from these complementary techniques provides a comprehensive understanding of thrS structure, enabling correlation with enzymatic function and facilitating rational design of inhibitors or substrate analogs for therapeutic development.

How can thrS be used as a potential target for novel antimicrobial development against Leptospira infections?

thrS represents a promising antimicrobial target against Leptospira due to several key properties:

Target Validation Rationale:

• Essential enzyme for protein synthesis and bacterial survival

• Sufficiently different from human threonyl-tRNA synthetase to enable selective targeting

• Involvement in a critical metabolic pathway (protein synthesis)

Screening Approaches for Inhibitor Discovery:

• High-Throughput Biochemical Assays:

  • Aminoacylation assay adapted to microplate format

  • Malachite green assay for detecting pyrophosphate release

  • Z-factor typically >0.7 when optimized

• Structure-Based Virtual Screening:

  • Molecular docking against thrS active site

  • Focus on ATP-binding pocket, threonine-binding site, and tRNA interaction interface

  • Prioritize compounds with predicted binding energy <-8.0 kcal/mol

• Fragment-Based Screening:

  • Thermal shift assays to identify fragments that stabilize thrS

  • NMR-based methods to confirm binding

  • Fragment growing/linking to develop higher-affinity inhibitors

Hit-to-Lead Optimization Strategy:

• Structure-activity relationship studies focusing on:

  • Binding affinity improvement (target IC₅₀ <1 μM)

  • Selectivity over human threonyl-tRNA synthetase (>100-fold)

  • Antimicrobial activity against Leptospira (MIC <10 μg/ml)

  • Favorable physicochemical properties for penetration of bacterial membranes

Validation Experiments:

• Enzyme inhibition assays with purified thrS

• Cell-based assays measuring growth inhibition of L. interrogans

• Confirmation of on-target activity via resistant mutant generation

• Animal model testing using hamster model of leptospirosis

Case Studies:
Recent research on aminoacyl-tRNA synthetase inhibitors has yielded promising compounds with MIC values in the range of 0.5-10 μg/ml against various bacterial pathogens. While specific thrS inhibitors for Leptospira are still in development, the structural differences between bacterial and human threonyl-tRNA synthetases offer a viable therapeutic window for selective targeting, potentially addressing the growing global health burden of leptospirosis that affects over 1 million people annually with approximately 60,000 deaths .

How does thrS contribute to the pathogenesis of L. interrogans serovar Copenhageni?

While thrS itself is not a classical virulence factor, it plays several critical roles in supporting L. interrogans pathogenesis:

Survival and Adaptation Mechanisms:

• As an essential enzyme for protein synthesis, thrS enables the production of virulence factors required for host infection and immune evasion

• L. interrogans must adapt to diverse environmental conditions during its lifecycle, transitioning from environmental water to mammalian hosts

• Proper protein synthesis is crucial for the bacterium's stress response to temperature shifts, pH changes, and osmotic stress encountered during infection

Connection to Virulence Factor Expression:

• L. interrogans pathogenesis involves multiple virulence factors, including outer membrane proteins (OMPs), lipopolysaccharides (LPS), and various enzymes

• The expression of these factors depends on functional translation machinery, where thrS plays an essential role

• Biofilm formation, which contributes to environmental persistence and possibly host colonization, requires intact protein synthesis pathways

Immune System Interactions:

• L. interrogans has developed sophisticated mechanisms to evade host immune responses

• In human macrophages, leptospires can escape from phagosomes into the cytosol, where they proliferate and activate apoptosis

• This survival strategy depends on proper protein synthesis mediated by functional aminoacyl-tRNA synthetases like thrS

Potential Regulatory Roles:

• Some aminoacyl-tRNA synthetases in bacteria have moonlighting functions beyond protein synthesis

• These include roles in transcriptional regulation, stress responses, and cellular signaling

• While not specifically documented for L. interrogans thrS, such non-canonical functions could contribute to pathogenesis

Experimental Evidence for thrS Role:
Indirect evidence for thrS importance comes from studies showing that:

• Inhibition of protein synthesis pathways severely impairs L. interrogans survival

• Genes involved in translation are consistently expressed during infection

• Mutants with defects in protein synthesis machinery show attenuated virulence

Understanding thrS contribution to pathogenesis provides valuable insights for developing targeted interventions against leptospirosis, a disease with significant global impact particularly affecting tropical low-income countries.

What are the challenges in developing specific antibodies against recombinant thrS for immunological studies?

Developing specific antibodies against recombinant L. interrogans thrS presents several technical challenges that researchers must address:

Antigenicity and Immunogenicity Considerations:

• thrS is a highly conserved protein across bacterial species, potentially limiting its immunogenicity

• Structural similarities with host (mammalian) threonyl-tRNA synthetases may lead to cross-reactivity

• The large size of thrS (~80 kDa) can result in multiple epitopes with varying immunogenicity

Strategic Approaches for Antibody Development:

• Epitope Selection Methods:

  • In silico prediction of antigenic regions using algorithms like Kolaskar-Tongaonkar, BepiPred, and IEDB

  • Selection of unique peptide sequences (15-25 amino acids) specific to L. interrogans thrS

  • Focus on surface-exposed regions based on structural modeling

  • Avoid regions with high sequence similarity to mammalian counterparts

• Immunization Protocols:

  • Multiple-host strategy: develop antibodies in both rabbits and mice to increase success probability

  • Prime-boost regimen: primary immunization with full-length protein followed by boosters with specific peptides

  • Adjuvant selection: complete Freund's for primary immunization, incomplete Freund's for boosters

  • Monitoring antibody titers by ELISA throughout immunization schedule

• Purification and Validation Steps:

  • Affinity purification using recombinant thrS coupled to solid support

  • Cross-absorption with E. coli lysates to remove antibodies against contaminating proteins

  • Extensive validation through:

    • Western blotting against recombinant thrS and L. interrogans lysates

    • Immunoprecipitation followed by mass spectrometry

    • Immunofluorescence microscopy to confirm specificity

Troubleshooting Common Problems:

Quality Control Criteria:

• Specificity: single band of expected size on Western blot of L. interrogans lysates

• Sensitivity: detection limit <10 ng of recombinant protein

• Background: minimal reactivity against mammalian cell lysates

• Reproducibility: consistent performance across immunoassays

These methodological considerations ensure development of high-quality antibodies for studying thrS expression, localization, and function in L. interrogans, advancing our understanding of this enzyme's role in leptospiral biology and pathogenesis.

How should researchers analyze sequence variations in thrS genes across different Leptospira serovars?

Comprehensive analysis of thrS sequence variations across Leptospira serovars requires a systematic bioinformatic approach:

Sequence Acquisition and Alignment:

• Obtain thrS sequences from multiple Leptospira serovars through genome databases (NCBI, UniProt) or targeted sequencing

• Perform multiple sequence alignment using tools like MUSCLE, MAFFT, or Clustal Omega with parameters optimized for nucleotide or protein sequences

• Visualize alignments using Jalview or similar software to identify regions of conservation and variation

Variation Analysis Methodology:

1. Single Nucleotide Polymorphism (SNP) Analysis:

• Identify SNPs using tools like SNP-sites or custom scripts

• Classify SNPs as synonymous or non-synonymous

• Calculate nucleotide diversity (π) and SNP density across the gene

• Compare SNP patterns between pathogenic and saprophytic Leptospira species

2. Structural Impact Assessment:

• Map sequence variations onto protein structural models

• Analyze if variants cluster in specific functional domains

• Predict impact on protein stability using tools like CUPSAT or I-Mutant

• Assess conservation at active site residues and tRNA binding interfaces

3. Phylogenetic Analysis:

• Construct maximum likelihood and Bayesian phylogenetic trees

• Apply appropriate substitution models (e.g., GTR+G+I for nucleotide sequences)

• Evaluate bootstrap support or posterior probabilities for branch reliability

• Compare thrS phylogeny with species phylogeny to detect horizontal gene transfer events

4. Selection Pressure Analysis:

• Calculate dN/dS ratios to detect selective pressure

• Use methods like PAML, FUBAR, or MEME to identify sites under positive selection

• Compare selection patterns between pathogenic and non-pathogenic species

5. Recombination Detection:

• Apply methods like RDP4 or GARD to detect recombination events

• Identify potential donor and recipient sequences

• Assess impact of recombination on phylogenetic inferences

Reference-Based Comparison Table:
Based on available data for L. interrogans isolates, the following pattern of variation might be expected:

This analytical framework provides a robust approach for characterizing thrS variations, contributing to our understanding of Leptospira evolution and potentially identifying serovar-specific markers relevant for diagnostic development and vaccine design.

What are the best approaches for comparing enzymatic activities of thrS from different L. interrogans serovars?

To effectively compare enzymatic activities of thrS from different L. interrogans serovars, researchers should implement a comprehensive biochemical characterization approach:

Standardized Expression and Purification Protocol:

• Express all thrS variants using identical expression systems and conditions

• Purify proteins using the same protocol to ensure comparable purity (>95%)

• Verify protein integrity by SDS-PAGE, Western blot, and mass spectrometry

• Quantify protein concentration using both Bradford assay and UV absorption (A280)

Comparative Kinetic Analysis Framework:

• Steady-State Kinetic Parameter Determination:

  • Measure initial reaction rates at various substrate concentrations

  • Determine Km and kcat for each substrate:

    • Threonine (0.1-100 μM range)

    • ATP (0.1-5 mM range)

    • tRNA^Thr (0.1-20 μM range)

  • Calculate catalytic efficiency (kcat/Km) for each substrate

  • Use identical buffer conditions (pH 7.5, 10 mM MgCl₂, 37°C)

• pH and Temperature Profiling:

  • Measure activity across pH range (5.5-9.0)

  • Determine temperature optima and stability (20-60°C)

  • Generate thermal inactivation curves for each variant

• Substrate Specificity Analysis:

  • Test activity with non-cognate amino acids (serine, valine)

  • Calculate specificity ratios (kcat/Km for threonine versus other amino acids)

  • Evaluate charging efficiency with heterologous tRNA^Thr sources

• Inhibitor Sensitivity Profiling:

  • Test sensitivity to known aminoacyl-tRNA synthetase inhibitors

  • Determine IC₅₀ values using standardized conditions

  • Analyze structure-activity relationships of inhibitors

Data Analysis and Visualization:

Statistical Considerations:

• Perform all measurements in triplicate (minimum)

• Apply appropriate statistical tests (t-test or ANOVA with post-hoc analysis)

• Set significance threshold at p<0.05

• Calculate 95% confidence intervals for all key parameters

Correlation with Structural Differences:

• Map sequence variations to structural models

• Correlate kinetic differences with specific amino acid substitutions

• Generate structure-function hypotheses based on observed variations

This systematic approach enables rigorous comparison of thrS enzymatic properties across different L. interrogans serovars, potentially revealing functional adaptations related to host specificity, environmental persistence, or pathogenicity that contribute to our understanding of the biology of these important pathogens.

How can researchers interpret contradictory data between in vitro and in vivo studies of recombinant thrS?

When facing contradictory results between in vitro and in vivo studies of recombinant thrS, researchers should implement a systematic approach to reconcile these discrepancies:

Methodological Analysis Framework:

• Verify Protein Identity and Integrity:

  • Confirm recombinant protein sequence by mass spectrometry

  • Assess post-translational modifications that might differ between systems

  • Evaluate protein folding using circular dichroism or thermal shift assays

  • Check for truncations or degradation products by Western blotting

• Compare Experimental Conditions:

  • Analyze buffer composition differences (pH, ionic strength, cofactors)

  • Consider temperature variations between in vitro assays and in vivo conditions

  • Evaluate the presence of potential inhibitors or activators in biological samples

  • Assess protein concentration differences between systems

• Examine Biological Context Factors:

  • Consider protein-protein interactions present in vivo but absent in vitro

  • Evaluate subcellular localization effects on enzyme activity

  • Assess regulatory mechanisms operating in vivo (feedback inhibition, allosteric regulation)

  • Analyze the impact of growth phase or environmental stress on thrS function

Resolution Strategies for Common Contradiction Scenarios:

Advanced Reconciliation Approaches:

• Cellular Fraction Studies:

  • Perform experiments with crude cell extracts from L. interrogans

  • Gradually purify components to identify missing cofactors or interacting partners

  • Reconstitute minimal systems with defined components

• Site-Directed Mutagenesis:

  • Create mutants affecting key residues to test mechanistic hypotheses

  • Compare mutant behavior in vitro and in vivo to pinpoint discrepancy sources

  • Use alanine-scanning mutagenesis to map functional surfaces

• Real-Time Monitoring:

  • Develop cellular reporters for thrS activity in vivo

  • Compare kinetics in cellular context versus purified system

  • Visualize enzyme localization during various cellular states

By systematically analyzing contradictions between in vitro and in vivo results, researchers can gain deeper insights into the physiological context of thrS function in L. interrogans. Such investigations often lead to discoveries of novel regulatory mechanisms or previously unknown protein interactions that enhance our understanding of bacterial aminoacyl-tRNA synthetases and their roles in pathogenesis.

What are the most promising approaches for using thrS in developing new diagnostics for leptospirosis?

Recombinant thrS offers several strategic advantages for developing next-generation leptospirosis diagnostics:

Serological Diagnostic Applications:

• ELISA-Based Detection Systems:

  • Develop indirect ELISA using purified recombinant thrS as antigen

  • Expected performance metrics based on similar recombinant protein assays:

    • Sensitivity: 85-95% for acute phase sera

    • Specificity: 90-97% compared to healthy controls

    • Cross-reactivity: Potential issues with Lyme disease patients (23% in similar studies)

  • Implementation as IgM-specific ELISA for acute diagnosis and IgG ELISA for epidemiological studies

• Multiplex Serological Panels:

  • Combine thrS with established antigens (LipL32, LipL41, OmpL1)

  • Create protein microarrays for simultaneous testing

  • Include controls for cross-reactive pathogens (Leptospira species, Treponema, Borrelia)

  • Improve specificity through machine learning algorithms analyzing reactivity patterns

Molecular Diagnostic Applications:

• PCR Target Development:

  • Design serovar-specific primers targeting thrS variable regions

  • Develop nested PCR or real-time PCR assays with sensitivity down to 10-100 leptospires/ml

  • Validate against diverse Leptospira strains and in clinical specimens

• LAMP (Loop-Mediated Isothermal Amplification):

  • Design thrS-targeted LAMP primers for point-of-care testing

  • Optimize for isothermal amplification (60-65°C)

  • Develop colorimetric detection systems for resource-limited settings

  • Target sensitivity of 100 leptospires/ml with 45-minute turnaround time

Innovative Approaches:

• Aptamer-Based Detection:

  • Select DNA/RNA aptamers with high affinity for thrS

  • Develop lateral flow assays using aptamer-conjugated nanoparticles

  • Engineer electrochemical biosensors with aptamer recognition elements

  • Target detection limit: 50-100 pg/ml thrS in biological fluids

• CRISPR-Cas Diagnostic Systems:

  • Design guide RNAs targeting thrS gene sequences

  • Combine with Cas12a or Cas13a for collateral cleavage-based detection

  • Develop paper-based visual readout systems

  • Achieve sensitivity comparable to PCR with faster turnaround time

Validation Strategy:

• Test against sera panel including:

  • Confirmed leptospirosis cases (n≥100)

  • Non-leptospirosis febrile illnesses (dengue, malaria, typhoid)

  • Healthy controls from endemic and non-endemic regions

  • Follow-up samples to establish seroconversion patterns

• Field validation in endemic settings with comparison to reference methods

The development of thrS-based diagnostics could address current limitations in leptospirosis diagnosis, potentially improving detection during the early phase of infection when antibody responses to commonly used antigens are still developing, and providing more specific identification of infecting serovars for enhanced epidemiological surveillance and treatment guidance.