Recombinant Chlamydophila caviae Leucine--tRNA ligase (leuS), partial

What is Leucine--tRNA ligase (leuS) and what is its biological role in Chlamydophila caviae?

Leucine--tRNA ligase (LeuRS), encoded by the leuS gene, is a critical aminoacyl-tRNA synthetase that catalyzes the attachment of leucine to its cognate tRNA molecule (tRNA^Leu) in a two-step reaction. First, it activates leucine using ATP to form leucyl-adenylate, then transfers the activated leucine to tRNA^Leu, producing charged leucyl-tRNA essential for protein synthesis .

In Chlamydophila caviae (strain GPIC), leuS is designated as CCA_00612 and encodes an enzyme crucial for bacterial protein synthesis and survival . As a member of class I aminoacyl-tRNA synthetases, LeuRS contains a characteristic Rossmann fold catalytic domain and plays a fundamental role in translating the genetic code by ensuring accurate amino acid incorporation during protein synthesis .

How does recombinant Chlamydophila caviae leuS differ from native leuS protein?

Recombinant Chlamydophila caviae leuS differs from its native counterpart in several key aspects:

• Expression system influence: The recombinant protein can be expressed in various systems including yeast, E. coli, baculovirus, and mammalian cells, each imparting different post-translational modifications and folding characteristics to the final protein .

• Structural modifications: Recombinant leuS typically contains additional tag sequences (such as Avi-tag for biotinylation) that facilitate purification, detection, and experimental manipulation .

• Partial vs. complete sequence: The designated "partial" status indicates that the recombinant protein represents a fragment of the full-length native leuS, likely including the catalytic domain but possibly missing regulatory regions present in the complete protein .

• Purity and homogeneity: Recombinant preparations typically achieve >85% purity (as measured by SDS-PAGE), whereas native leuS exists in complex with other cellular components .

What are the primary experimental applications of recombinant leuS in Chlamydia research?

Recombinant Chlamydophila caviae leuS serves multiple critical research functions:

• Study of persistence mechanisms: Since tRNA synthetases are targets for inducing persistence in Chlamydia, recombinant leuS enables investigation of amino acid starvation responses and developmental cycle regulation .

• Phylogenetic and evolutionary studies: The leuS gene sequence and protein structure provide valuable markers for studying evolutionary relationships within Chlamydiaceae, enabling researchers to determine species boundaries and genetic relationships .

• Drug development platforms: As bacterial tRNA synthetases represent attractive antimicrobial targets, recombinant leuS facilitates high-throughput screening of inhibitors like AN3365 that specifically target bacterial leucyl-tRNA synthetases .

• Recombination and lateral gene transfer research: Purified leuS can be used to investigate the mechanisms of interspecies genetic exchange, particularly important given that lateral gene transfer is observed in laboratory experiments with Chlamydia species .

What expression systems provide optimal yields and functionality for recombinant Chlamydophila caviae leuS?

The choice of expression system significantly impacts both yield and functionality of recombinant leuS:

How should researchers optimize storage and handling of recombinant leuS to maintain enzymatic activity?

To preserve functionality of recombinant Chlamydophila caviae leuS:

• Initial reconstitution: Centrifuge the lyophilized powder briefly before opening to ensure contents settle at the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Stabilization: Add glycerol to a final concentration of 5-50% to prevent freeze-thaw damage .

• Aliquoting strategy: Prepare small single-use aliquots to avoid repeated freeze-thaw cycles that progressively reduce enzymatic activity .

• Temperature considerations: Store aliquots at -80°C for long-term preservation. For experiments requiring multiple sampling, maintain working aliquots at -20°C for up to one month .

• Activity preservation: During experimental procedures, keep the enzyme on ice and minimize exposure to room temperature. Consider adding reducing agents (1-5 mM DTT) to prevent oxidation of critical cysteine residues in the catalytic domain.

• Activity verification: Periodically confirm enzymatic activity through aminoacylation assays measuring the attachment of leucine to tRNA^Leu substrates.

What are the critical parameters for designing functional assays to measure leuS activity?

When designing assays to evaluate recombinant leuS activity, researchers should consider:

• Two-step reaction monitoring: Implement assays that can monitor both ATP-dependent activation of leucine (forming leucyl-adenylate) and the transfer to tRNA, such as:

  • ATP-PPi exchange assays for the first step

  • Filter-binding assays with radioisotope-labeled leucine for complete reaction

• Buffer optimization:

  • Maintain pH between 7.0-7.5

  • Include 5-10 mM MgCl₂ as a critical cofactor

  • Add 1-5 mM ATP as substrate

  • Consider 50-100 mM KCl or NaCl for ionic strength

  • Include 1-5 mM DTT to maintain reduced state of critical cysteines

• Substrate considerations:

  • Use purified tRNA^Leu specific to Chlamydophila caviae for highest activity

  • Ensure L-leucine purity (>99%)

  • Consider temperature (optimal around 30-37°C for chlamydial enzymes)

• Inhibition studies: When testing potential inhibitors, establish appropriate controls:

  • Include known inhibitors like AN3365 as positive controls

  • Use non-cognate amino acids to demonstrate specificity

• Data analysis: Calculate kinetic parameters (KM, kcat) for both leucine and tRNA^Leu substrates to completely characterize enzymatic efficiency.

How can recombinant leuS be used to investigate chlamydial persistence mechanisms?

Recombinant leuS provides a valuable tool for understanding the fundamental persistence mechanisms in Chlamydia:

• Amino acid starvation models: Use purified leuS along with specific inhibitors like AN3365 to create controlled leucine starvation conditions, enabling the study of persistence entry, maintenance, and exit without the confounding variables introduced by IFN-γ treatment .

• Comparative persistence analysis: Engineer systems where various amino acid-tRNA synthetases are individually inhibited to determine if persistence mechanisms are universal or amino acid-specific. Research indicates that tRNA synthetase inhibition drops chlamydial growth to basal levels (near detection limits) and induces persistence-like states .

• Transcriptional response mapping: Employ recombinant leuS in cell-free systems to study how tRNA charging status influences codon-dependent transcriptional changes observed during persistence. Research demonstrates that chlamydial persistence is characterized by Trp codon-dependent changes in transcription, suggesting potential mechanisms for other amino acids .

• Structure-function relationship studies: Utilize site-directed mutagenesis of recombinant leuS to identify critical residues involved in sensing amino acid availability and mediating persistence responses, potentially revealing novel regulatory mechanisms specific to Chlamydiaceae.

• Therapeutic intervention development: Screen compound libraries against recombinant leuS to identify potential antimicrobials that can either prevent persistence (maintaining chlamydial susceptibility to existing treatments) or force exit from the persistent state.

What role does leuS play in recombination and lateral gene transfer among Chlamydia species?

The leuS gene serves as an important element in understanding genetic exchange mechanisms in Chlamydia:

• Recombination hotspot analysis: Research indicates that interspecies genetic exchange in Chlamydia can involve tRNA synthetase genes. Utilizing recombinant leuS can help identify whether this gene resides in recombination hotspots observed between species like C. trachomatis and C. muridarum .

• Sequence conservation implications: Comparative analysis reveals that despite high conservation of enzymatic function, leuS sequences can vary between Chlamydia species, potentially influencing species-specific preferences in recombination events .

• Secondary recombination events: Studies demonstrate that additional recombined sequences ranging from 7 to 8,005 bp can be introduced during interspecies genetic exchange, suggesting potential mechanisms for introduction of novel leuS variants .

• Experimental recombination systems: Recombinant leuS, combined with antibiotic resistance markers, provides a selectable system to study recombination mechanics between Chlamydia species, revealing that "mosaic sequences" often result at recombination junctions .

• Evolutionary implications: The presence of mosaic gene structures in leuS and other tRNA synthetases suggests these genes may contribute to chlamydial adaptation to different host environments and stresses, potentially explaining the evolutionary success of these obligate intracellular parasites .

How do inhibitors targeting leuS compare with other tRNA synthetase inhibitors in Chlamydia research?

Comparative analysis of tRNA synthetase inhibitors reveals important differences:

• Competitive vs. non-competitive inhibition: Unlike indolmycin (a competitive inhibitor of tryptophanyl-tRNA synthetase requiring Trp-depleted media), AN3365 acts as a non-competitive inhibitor of leucyl-tRNA synthetase and remains effective even in the presence of normal medium levels of leucine (105 mg/liter) .

• Inhibition potency variation: AN3365 demonstrates effective inhibition at concentrations exceeding 250 ng/ml, while indolmycin requires higher concentrations (120 μM) combined with tryptophan depletion to achieve similar inhibitory effects .

• Timing of inhibitor efficacy: AN3365 effectively blocks chlamydial growth when added at various time points up to 12 hours post-infection during the developmental cycle, providing greater flexibility in experimental design .

• Host cell impact: Research confirms that neither AN3365 nor indolmycin significantly impacts host cell activity, in contrast to cycloheximide (a eukaryotic protein synthesis inhibitor) or IFN-γ, which do affect host cells .

• Recovery dynamics: Both inhibitors demonstrate reversible inhibition, allowing researchers to model not only entry into persistence but also recovery dynamics when inhibitors are removed – an important consideration for therapeutic development .

How does leuS sequence analysis contribute to understanding Chlamydophila caviae's evolutionary position?

Leucine-tRNA ligase sequences provide valuable phylogenetic information about Chlamydophila caviae:

• Molecular clock applications: The leuS gene evolves at a measurable rate, making it useful for determining evolutionary relationships within the Chlamydiaceae family when analyzed alongside other genetic markers such as 16S-23S rRNA genes .

• Sequence conservation patterns: Comparative analysis of leuS sequences across Chlamydia species reveals conserved catalytic domains alongside variable regions that reflect species-specific adaptations to different hosts and environmental niches .

• Phylogenetic tree construction: Analysis using programs like MEGA (Molecular Evolutionary Genetics Analysis) with the Neighbour-Joining method can place Chlamydophila caviae in relation to other chlamydial species based on leuS sequence similarities and differences .

• Evidence of horizontal gene transfer: The presence or absence of mosaic sequences in leuS can indicate historical recombination events, potentially revealing cross-species interactions that contributed to Chlamydophila caviae's current genetic makeup .

• Host adaptation signatures: Specific sequence motifs within leuS may correlate with adaptation to particular hosts, potentially explaining C. caviae's specific ecological niche (primarily observed in guinea pigs) compared to more widespread chlamydial pathogens .

What are the major sequence differences between Chlamydophila caviae leuS and homologous proteins in related Chlamydia species?

Key sequence variations in leuS across Chlamydia species include:

• Catalytic domain conservation: The core enzymatic domains responsible for ATP binding, leucine activation, and tRNA charging show high conservation (typically >80% sequence identity) across Chlamydia species due to the essential nature of these functions .

• Variable regions: Regions outside the catalytic core, particularly in the N-terminal and C-terminal domains, show greater sequence divergence, potentially reflecting species-specific regulatory mechanisms or protein-protein interactions .

• Codon usage patterns: Analysis of leuS genes across Chlamydia species reveals distinct codon usage patterns that may influence translation efficiency under different host conditions, particularly relevant during persistent infections when amino acid availability fluctuates .

• Amino acid substitutions at key positions: Specific substitutions near the active site may influence substrate specificity or inhibitor sensitivity without compromising core enzymatic function, potentially explaining differential responses to antibiotics targeting tRNA synthetases across Chlamydia species .

• Recombination junction points: Interspecies genomic analyses reveal potential recombination "hotspots" near or within the leuS gene, suggesting these regions play a role in facilitating genetic exchange between Chlamydia species .

What is the significance of leuS in Chlamydophila caviae pathogenicity and persistence?

The leuS gene product plays crucial roles in chlamydial pathogenicity through several mechanisms:

• Persistence regulation: As a target for inhibition during amino acid limitation, leuS mediates entry into the persistent state, which is characterized by a halt in the division cycle, aberrant morphology, and transcriptional changes that allow long-term survival within the host .

• Adaptation to host environments: During infection, the host immune system may release gamma interferon (IFN-γ), which can deplete amino acid pools. LeuS functionality under these stressed conditions is essential for chlamydial survival and continued infection .

• Response to antimicrobial pressure: The ability of leuS to function under various stress conditions, including exposure to antibiotics targeting protein synthesis, contributes to the resilience of Chlamydophila caviae infections .

• Developmental cycle progression: Proper leuS function is required for the normal developmental cycle, including transitions between elementary bodies (infectious form) and reticulate bodies (replicative form), processes fundamental to establishing and maintaining infection .

• Host immune evasion: The persistent state mediated by leuS response to stress conditions helps Chlamydophila caviae evade host immune clearance by entering a low-metabolic, non-replicating state until conditions improve .

How might targeting leuS contribute to novel therapeutic approaches for chlamydial infections?

Targeting leucyl-tRNA synthetase opens several therapeutic avenues:

• Selective inhibition strategies: The structural differences between bacterial and mammalian leucyl-tRNA synthetases enable the development of selective inhibitors like AN3365 that can target chlamydial leuS without affecting host cell protein synthesis .

• Anti-persistence compounds: Developing compounds that specifically prevent leuS-mediated entry into persistence could render Chlamydia more susceptible to existing antibiotics and prevent recurrent infections associated with persistent forms .

• Combination therapy approaches: Using leuS inhibitors alongside conventional antibiotics may increase efficacy by preventing adaptation to stress conditions, potentially reducing treatment duration and preventing development of chronic infections .

• Species-specific targeting: Exploiting sequence variations in leuS between different Chlamydia species could enable development of species-targeted therapeutics, reducing broad-spectrum antibiotic use and associated complications .

• Vaccination strategies: Recombinant leuS or specific epitopes derived from it might serve as candidate antigens for vaccine development, potentially stimulating protective immunity against chlamydial infection while avoiding cross-reactivity with human proteins .

What are the current limitations in studying recombinant Chlamydophila caviae leuS?

Several significant challenges remain in this research area:

• Structural characterization difficulties: The large size and complex domain organization of leuS make high-resolution structural studies challenging, limiting understanding of species-specific structural features.

• In vivo relevance of in vitro findings: While recombinant leuS provides valuable insights in controlled experiments, translating these findings to the complex environment of an actual chlamydial infection presents significant challenges .

• Expression system limitations: Current expression systems may not perfectly replicate the native folding, post-translational modifications, or interaction partners present in Chlamydophila caviae, potentially affecting functional studies .

• Difficulties in studying persistence mechanisms: The complex nature of persistence and the multifactorial signals involved make it challenging to isolate the specific contribution of leuS to this process .

• Limited comparative data: Incomplete genomic and proteomic data for many Chlamydia species limits comprehensive comparative analyses that could reveal important evolutionary patterns in leuS function and regulation .

What emerging technologies might enhance future research on Chlamydophila caviae leuS?

Several innovative approaches promise to advance this research field:

• Cryo-electron microscopy advancements: Emerging cryo-EM techniques may enable high-resolution structural determination of leuS in complex with tRNA and inhibitors, revealing critical interaction details.

• CRISPR-based genetic manipulation: As genetic manipulation systems for Chlamydia continue to improve, direct editing of leuS in its native context will enable more precise functional studies .

• Single-cell analysis techniques: Advanced microfluidic and imaging technologies may allow real-time monitoring of leuS activity and persistence entry/exit within individual infected cells.

• Computational modeling advancements: Improved molecular dynamics simulations and machine learning approaches may help predict leuS structural changes during catalysis and inhibitor interactions.

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data sets will provide comprehensive understanding of how leuS functions within the broader metabolic and regulatory networks of Chlamydophila caviae during different growth phases and stress conditions .

The continued study of recombinant Chlamydophila caviae leucine-tRNA ligase holds promise for advancing both fundamental understanding of prokaryotic translation systems and applied research toward novel anti-chlamydial therapeutics, particularly those targeting the clinically challenging persistent state .