Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Phenylalanine--tRNA ligase beta subunit (pheT), partial

What is the functional role of Phenylalanine--tRNA ligase beta subunit (PheT) in Leptospira interrogans and its significance in pathogenicity?

Phenylalanine--tRNA ligase beta subunit (PheT) in Leptospira interrogans serves a critical function in protein biosynthesis by catalyzing the attachment of phenylalanine to its cognate tRNA molecules. This aminoacylation process is essential for accurate translation of the genetic code and subsequent protein synthesis. While no direct pathogenicity role has been specifically documented for PheT in L. interrogans, recent research in related organisms provides valuable insights.

The significance of PheT in bacterial pathogenicity has been demonstrated in a 2025 study focusing on Mycobacterium abscessus, where genetic disruption of PheT led to clear growth inhibitory phenotypes both in vitro and in vivo . Transcriptome analysis revealed differential expression of host genes in response to PheT gene silencing, including genes involved in cell cycle, apoptotic signaling, and inflammatory responses .

For Leptospira interrogans, which causes over a million human cases and approximately 60,000 deaths annually worldwide , the role of aminoacyl-tRNA synthetases like PheT may be particularly important during infection. Global proteome analyses of L. interrogans have demonstrated significant proteome alterations under conditions that mimic in vivo infection . These studies help identify proteins that may be differentially expressed upon exposure to host components and environmental conditions such as iron limitation.

Research methodology for studying PheT function:

• Comparative transcriptomics/proteomics between virulent and avirulent strains

• CRISPR interference to evaluate gene essentiality

• Structural biology approaches to characterize enzyme-substrate interactions

• In vivo infection models to assess virulence in gene knockout strains

What expression systems and purification methods are most effective for producing recombinant PheT from Leptospira interrogans?

Recombinant PheT from Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni can be produced using several expression systems, each with distinct advantages depending on research requirements. According to available product information, PheT can be expressed in:

• Escherichia coli

• Yeast systems

• Baculovirus-infected insect cells

• Mammalian cell expression systems

The choice of expression system significantly impacts protein yield, solubility, and post-translational modifications. For basic structural studies where glycosylation is not critical, E. coli systems offer cost-effective, high-yield production. Commercial preparations report purification to ≥85% purity as determined by SDS-PAGE .

For purification of recombinant PheT, affinity chromatography using tags is the preferred initial approach. Available recombinant products utilize:

• N-terminal tags (commonly His6-tag)

• Potential C-terminal tags (determined based on tag-protein stability)

A methodological workflow for recombinant PheT production:

• Gene optimization: Codon optimization for the selected expression system

• Vector selection: pRSET or pDEST17 vectors have been successfully used for Leptospira proteins

• Expression optimization: Temperature, induction conditions, and media composition

• Purification protocol:

  • Initial capture: IMAC (Immobilized Metal Affinity Chromatography)

  • Secondary purification: Ion exchange chromatography or gel filtration

  • Quality control: SDS-PAGE and Western blotting verification

For functional studies, enzymatic activity assays must be developed to confirm that the recombinant protein retains its phenylalanylation activity with appropriate tRNA substrates.

What are the optimal conditions for assessing the enzymatic activity of recombinant PheT?

Determining the enzymatic activity of recombinant Phenylalanine--tRNA ligase beta subunit (PheT) from Leptospira interrogans requires carefully designed assays that measure the aminoacylation of tRNA^Phe with phenylalanine. While specific activity studies for L. interrogans PheT are not detailed in the provided search results, a methodological approach can be extrapolated from standard aminoacyl-tRNA synthetase assays.

Recommended enzymatic activity assay protocol:

• Reaction components:

  • Purified recombinant PheT protein (typically 10-100 nM)

  • Purified PheS protein (alpha subunit) for reconstitution of the functional complex

  • tRNA^Phe substrate (either purified from E. coli or synthetic)

  • L-phenylalanine (0.1-1 mM)

  • ATP (2-5 mM)

  • Magnesium ions (5-10 mM MgCl₂)

  • Buffer system (typically Tris-HCl, pH 7.5-8.0)

• Detection methods:

  • Radioactive assay: Using [³H]-phenylalanine or [¹⁴C]-phenylalanine and measuring trichloroacetic acid-precipitable radioactivity

  • Pyrophosphate release assay: Coupling the PPi released during aminoacylation to enzymatic reactions that generate a colorimetric or fluorescent signal

  • HPLC-based assay: Separation and quantification of charged vs. uncharged tRNA species

• Optimal reaction conditions:

  • Temperature: 30-37°C (based on Leptospira growth optima)

  • pH: 7.5-8.0

  • Incubation time: 5-30 minutes (establish linear range)

• Controls and validation:

  • Negative control: Omission of ATP or enzyme

  • Positive control: Commercially available phenylalanyl-tRNA synthetase

  • Enzyme inactivation: Heat-denatured enzyme

For studying the effects of environmental conditions on PheT activity, the assay can be modified to include varying concentrations of iron, different pH levels, or serum components to mimic conditions used in proteomic studies of L. interrogans .

How do proteomics approaches reveal PheT expression patterns in Leptospira under different environmental conditions?

Proteomics approaches have provided valuable insights into protein expression patterns in Leptospira interrogans under various environmental conditions, although specific data on PheT expression is limited. Comparative proteomic analyses have been particularly informative for understanding how Leptospira adapts to different environments, including conditions that mimic in vivo infection.

A comprehensive study by Malmström et al. (2009) performed comparative global proteome analyses on L. interrogans serovar Copenhageni strain Fiocruz L1-130 under conventional in vitro conditions versus conditions mimicking in vivo infection (iron limitation and serum presence) . The study employed both gel-based and non-gel-based proteomic approaches:

Methodological workflow for proteomic analysis:

• Sample preparation:

  • Cultivation of L. interrogans under different conditions

  • Protein extraction and quantification

  • Pooling of biological replicates (75 independent cultures for primary samples)

• Analytical techniques:

  • iTRAQ labeling for quantitative analysis

  • Two-dimensional gel electrophoresis for comparative analysis

  • LC-MS/MS for protein identification

• Data analysis:

  • Relative quantification of proteins between conditions

  • Identification of differentially expressed proteins

  • Functional categorization of regulated proteins

The study revealed significant proteome alterations in response to environmental conditions that mimic in vivo infection . While specific data on PheT expression was not highlighted in the search results, the study identified numerous proteins that were differentially expressed upon exposure to host components and iron limitation.

For researchers interested in specifically studying PheT expression patterns, similar methodological approaches could be applied with targeted analysis for aminoacyl-tRNA synthetases. This would involve:

• Western blot analysis with specific antibodies against PheT

• Selected reaction monitoring (SRM) mass spectrometry for targeted quantification

• Transcriptome analysis using RT-qPCR or RNA-seq to correlate protein expression with gene expression levels

Can recombinant PheT be used as a diagnostic marker for leptospirosis detection?

While specific studies on using recombinant PheT as a diagnostic marker for leptospirosis are not available in the search results, insights can be gained from studies evaluating other recombinant Leptospira proteins for serodiagnosis. The potential of PheT as a diagnostic marker should be assessed in the context of current diagnostic challenges and existing recombinant protein-based approaches.

Leptospirosis diagnosis faces significant challenges:

• The microscopic agglutination test (MAT), recommended by WHO, has reduced sensitivity at disease onset

• Early diagnosis is critical for effective treatment and disease management

Studies evaluating recombinant Leptospira proteins as diagnostic markers have shown promising results:

For PheT to be evaluated as a diagnostic marker, the following methodological approach would be recommended:

• Antigen preparation:

  • Expression and purification of recombinant PheT with appropriate tags

  • Quality control to ensure proper folding and epitope presentation

• Immunological assessment:

  • ELISA development using purified recombinant PheT

  • Determination of cutoff values using sera from healthy individuals

  • Evaluation of IgM and IgG responses in patient sera

• Diagnostic performance evaluation:

  • Testing paired sera from confirmed leptospirosis cases (acute and convalescent phases)

  • Assessment of sensitivity and specificity

  • Cross-reactivity testing with sera from patients with similar clinical presentations

• Comparative analysis:

  • Comparison with established diagnostic markers like LipL32

  • Evaluation of potential for inclusion in multi-antigen panels

The recombinant chimeric protein approach, as demonstrated with rChi2 , shows particular promise, suggesting that PheT might be more valuable as part of a multi-epitope diagnostic panel rather than as a standalone marker.

What genomic analyses have revealed about the pheT gene and its conservation across Leptospira species?

Genomic analyses have provided significant insights into the Leptospira interrogans genome, including the context and conservation of genes like pheT across different species and serovars. The complete genome sequence of L. interrogans serovar Copenhageni has been particularly informative in understanding the genetic basis of leptospiral physiology and pathogenesis.

The genome of L. interrogans serovar Copenhageni strain Fiocruz L1-130 reveals the presence of a comprehensive set of genes encoding for protein synthesis machinery, including aminoacyl-tRNA synthetases like PheT . These genes are critical for the organism's ability to adapt to diverse environmental conditions.

Comparative genomic features across Leptospira species:

While specific analyses of pheT conservation are not detailed in the search results, studies on orthologous proteins in Leptospira provide a framework for understanding gene conservation patterns. A study by Ramadass et al. (2004) on extracellular proteins found:

• Highest coverage and identity of orthologous proteins (>50% identity) predominantly found among pathogenic Leptospira species

• Intermediate and saprophytic species showing less than 50% identity despite good coverage against query sequences

This pattern suggests that essential genes involved in protein synthesis, like pheT, would likely show high conservation among pathogenic Leptospira species but may have more sequence divergence in non-pathogenic species.

Methodological approaches for analyzing pheT conservation include:

• Multiple sequence alignment:

  • BLAST analysis of pheT sequences across different Leptospira species

  • Calculation of percent identity and similarity scores

  • Identification of conserved domains and critical residues

• Phylogenetic analysis:

  • Construction of phylogenetic trees based on pheT sequences

  • Comparison with species phylogeny based on standard markers like 16S rRNA or ppk genes

  • Identification of potential horizontal gene transfer events

• Synteny analysis:

  • Examination of gene order and organization around the pheT locus

  • Identification of conserved gene clusters or operons

The high conservation of essential genes like pheT across pathogenic species makes them potential targets for broad-spectrum diagnostics and therapeutics against leptospirosis.

What animal models are most appropriate for evaluating recombinant Leptospira proteins like PheT?

Animal models play a crucial role in evaluating the pathogenicity of Leptospira interrogans and the potential of recombinant proteins as vaccine candidates or therapeutic targets. Based on the search results, several animal models have been successfully employed in leptospirosis research, each with specific advantages for different research questions.

Golden Syrian hamsters have emerged as a preferred animal model for leptospirosis studies, particularly for evaluating vaccine candidates:

• Hamsters are highly susceptible to L. interrogans infection and develop a disease that resembles severe human leptospirosis

• The LD₅₀ for L. interrogans serovar Pomona infection in hamsters is approximately 10⁸ leptospires

• Immunization with recombinant proteins followed by challenge with 10⁸ leptospires provides a reliable assessment of protective efficacy

A methodological protocol for evaluating recombinant proteins in the hamster model:

• Immunization schedule:

  • Primary immunization at 3 weeks of age

  • Booster immunization at 6 weeks of age

  • Use of appropriate adjuvants (e.g., aluminum hydroxide)

• Challenge protocol:

  • Intraperitoneal challenge with 10⁸ virulent leptospires 3 weeks after final immunization

  • Monitoring of survival, clinical signs, and pathological changes

  • Collection of serum samples to assess antibody responses

• Assessment parameters:

  • Survival rate and time to death

  • Histopathological examination of tissues, particularly kidneys and liver

  • Serological responses (antibody titers)

  • Bacterial burden in tissues (culture and qPCR)

Other animal models mentioned in the search results include:

• Rats: Natural reservoir hosts for serovars icterohaemorrhagiae and copenhageni

• Mice: Less ideal for general pathogenicity studies but used for specific immunological investigations

• Cattle: Used for studies on serovar Hardjo infections and vaccine evaluation

For evaluating PheT specifically, the hamster model would be most appropriate given its established use in assessing recombinant Leptospira proteins. The search results indicate that recombinant proteins such as LigA have shown promising protective efficacy in this model, with 100% survival in immunized hamsters compared to control groups .

What challenges exist in developing recombinant protein-based vaccines for leptospirosis, and how might these apply to PheT?

The development of recombinant protein-based vaccines for leptospirosis faces several significant challenges, which would also apply to potential PheT-based vaccine candidates. Understanding these challenges is essential for designing effective experimental approaches and interpreting results accurately.

Key challenges in recombinant leptospiral vaccine development:

• Antigenic diversity and serovar specificity:

  • Over 250 pathogenic serovars of Leptospira exist, complicating broad-spectrum protection

  • Immunity is often serovar-specific, requiring multivalent approaches

• Protein expression and purification challenges:

  • Maintaining proper protein folding and epitope presentation is critical

  • Optimization of expression systems may be necessary for each protein target

• Adjuvant selection:

  • Choice of adjuvant significantly impacts immune response quality and duration

  • Aluminum hydroxide has been used successfully in hamster models

• Correlates of protection:

  • Unclear relationship between antibody titers and protective immunity

  • Need to assess both humoral and cell-mediated immune responses

• Target protein selection criteria:

  • Surface exposure and conservation across serovars are critical factors

  • Proteins must be expressed during infection to be effective targets

Studies have shown promising results with certain recombinant proteins as vaccine candidates:

Data compiled from search results

For PheT to be evaluated as a vaccine candidate, several strategic approaches would be recommended:

• Epitope mapping and selection:

  • Identification of surface-exposed, immunogenic epitopes within PheT

  • Creation of chimeric constructs combining selected epitopes with known immunogenic proteins

• Multi-component vaccine strategy:

  • Combination with other protective antigens like LigA or LipL32

  • Development of a chimeric multiepitope protein similar to rChi2

• Immune response characterization:

  • Assessment of both Th1 and Th2 responses

  • Evaluation of memory B-cell and T-cell responses for long-term protection

The study by Forster et al. (2020) demonstrated that recombinant chimeric proteins containing multiple conserved leptospiral antigens can provide broad cross-protection, suggesting a potential path forward for utilizing proteins like PheT in vaccine development .

How can structural biology approaches contribute to understanding PheT function and potentially aid drug discovery?

Structural biology approaches offer powerful tools for understanding the function of PheT from Leptospira interrogans at the molecular level and can significantly accelerate drug discovery efforts targeting this enzyme. While the search results don't contain specific structural studies on L. interrogans PheT, the approaches and methodologies can be extrapolated from related research.

Key structural biology approaches for studying PheT:

• X-ray crystallography:

  • Determination of high-resolution 3D structures

  • Visualization of active site architecture and substrate binding pockets

  • Co-crystallization with substrates, products, or inhibitors to capture different functional states

• Cryo-electron microscopy (cryo-EM):

  • Visualization of larger complexes (e.g., PheT in complex with tRNA or other proteins)

  • Study of conformational changes during the catalytic cycle

  • Analysis of quaternary structure arrangements

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Analysis of protein dynamics in solution

  • Study of protein-ligand interactions

  • Investigation of conformational changes upon substrate binding

• Computational approaches:

  • Homology modeling based on structures of homologous proteins

  • Molecular dynamics simulations to study protein flexibility and ligand binding

  • Virtual screening for potential inhibitors targeting the active site

The potential of PheT as a drug target is supported by recent research on aminoacyl-tRNA synthetases in other bacterial pathogens. A 2025 study demonstrated that PheT is a promising drug target in Mycobacterium abscessus, where genetic disruption of the gene led to growth inhibition both in vitro and in vivo .

Structure-based drug discovery strategy for PheT inhibitors:

• Target validation and preparation:

  • Expression and purification of recombinant PheT to >95% purity

  • Confirmation of enzymatic activity and establishment of high-throughput assays

  • Crystallization condition optimization

• Structure determination and analysis:

  • Identification of catalytic residues and binding pockets

  • Comparison with human homologs to identify selectivity-determining features

  • Analysis of species-specific structural differences

• Virtual screening and rational design:

  • In silico screening of compound libraries against identified binding sites

  • Fragment-based approaches to identify initial chemical matter

  • Structure-based optimization of hit compounds

• Iterative optimization:

  • Co-crystallization with lead compounds

  • Structure-activity relationship studies

  • Improvement of potency, selectivity, and drug-like properties

The fact that PheT plays an essential role in protein synthesis and that bacterial aminoacyl-tRNA synthetases often differ structurally from their human counterparts makes them attractive targets for antimicrobial development. The recent success in targeting PheT in M. abscessus suggests that similar approaches could be productive for Leptospira interrogans.

What detection methods can be optimized for studying the expression of PheT in Leptospira during infection?

Detecting and quantifying PheT expression in Leptospira interrogans during infection requires sensitive and specific methodologies that can overcome the challenges of low abundance proteins and complex biological matrices. Based on the search results, several approaches have been successfully applied to study leptospiral proteins during infection and could be optimized for PheT-specific studies.

Recommended detection methods for PheT expression analysis:

• Quantitative Proteomics:

  • iTRAQ (isobaric tags for relative and absolute quantitation) labeling for comparative proteomics

  • SILAC (stable isotope labeling with amino acids in cell culture) for metabolic labeling

  • Label-free quantitation using LC-MS/MS

• Immunological Detection:

  • Development of specific anti-PheT antibodies

  • Western blotting of bacterial lysates from in vitro and in vivo conditions

  • Immunohistochemistry of infected tissues

• Transcriptional Analysis:

  • RT-qPCR for targeted quantification of pheT mRNA

  • RNA-seq for global transcriptional profiling

  • In situ hybridization to localize pheT expression in tissues

• Reporter Systems:

  • Construction of pheT promoter-reporter fusions

  • Luciferase or fluorescent protein-based reporters

  • Flow cytometry for single-cell analysis of expression

Methodological considerations for optimizing detection in infection models:

• Sample preparation from infection models:

  • Enrichment techniques for leptospiral cells from tissue homogenates

  • Laser capture microdissection for tissue-specific analysis

  • Magnetic bead-based purification using specific antibodies

• Controls and validation:

  • Comparison with housekeeping genes/proteins

  • Spike-in standards for absolute quantification

  • Multiple technical and biological replicates

• Time-course analysis:

  • Sampling at different stages of infection

  • Correlation with disease progression

  • Comparison between acute and chronic infection phases

The global proteome analysis by Malmström et al. demonstrated that L. interrogans undergoes significant proteome alterations under conditions mimicking in vivo infection, with differential expression of various proteins . Similar approaches could be applied specifically to track PheT expression during infection, potentially revealing its role in leptospiral adaptation to the host environment.

How does the function of PheT in Leptospira compare to its homologs in other bacterial pathogens, and what are the implications for broad-spectrum therapeutics?

The function of Phenylalanine--tRNA ligase beta subunit (PheT) is highly conserved across bacterial species as an essential component of the protein synthesis machinery, yet there may be important structural and functional differences that could be exploited for species-specific therapeutic targeting. Comparing Leptospira PheT with homologs in other bacterial pathogens provides valuable insights for broad-spectrum therapeutic development.

Functional conservation and divergence:

• The 2025 study on Mycobacterium abscessus PheT demonstrated that genetic disruption affected not only bacterial growth but also altered host gene expression related to cell cycle, apoptotic signaling, and inflammatory responses

• Some aminoacyl-tRNA synthetases have been found to have moonlighting functions in bacterial pathogenesis, potentially serving as virulence factors

Structural considerations for drug development:

While specific structural data for L. interrogans PheT is not available in the search results, comparative analysis of aminoacyl-tRNA synthetase structures across bacterial pathogens reveals:

• Conserved catalytic domains that could be targeted for broad-spectrum activity

• Species-specific surface loops and binding pockets that might enable selective targeting

• Differences in quaternary structure organization that could affect drug binding

Comparative table of PheT characteristics across bacterial pathogens:

Methodological approach for comparative analysis:

• Sequence-based comparison:

  • Multiple sequence alignment of PheT sequences from diverse pathogens

  • Identification of conserved and variable regions

  • Phylogenetic analysis to understand evolutionary relationships

• Structure-based analysis:

  • Homology modeling of L. interrogans PheT based on crystallized homologs

  • Comparison of active site architecture

  • Analysis of species-specific structural features

• Functional assays:

  • Comparative enzymatic activity under different conditions

  • Cross-species complementation experiments

  • Inhibitor screening against PheT from multiple species