Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Glycine--tRNA ligase (glyQS)

What is the basic function of Glycine--tRNA ligase (glyQS) in Leptospira interrogans?

Glycine--tRNA ligase (glyQS) in Leptospira interrogans catalyzes the attachment of glycine to tRNA(Gly), which is a critical step in protein synthesis. This enzyme belongs to the class-II aminoacyl-tRNA synthetase family and facilitates the charging of tRNAGly with its cognate amino acid glycine through an ATP-dependent reaction: ATP + glycine + tRNAGly → AMP + diphosphate + glycyl-tRNAGly. The enzyme plays an essential role in translation accuracy by ensuring the correct amino acid is incorporated into growing polypeptide chains according to the genetic code .

How does Leptospira interrogans glyQS differ from human glycyl-tRNA synthetase?

The Leptospira interrogans glyQS differs from human glycyl-tRNA synthetase (encoded by GARS1) in several significant ways:

These differences make the bacterial enzyme a potential antimicrobial target as selective inhibition could theoretically be achieved without affecting the human ortholog. Additionally, the bacterial enzyme lacks domains associated with non-canonical functions that have been described for the human enzyme .

What are the optimal expression systems for producing recombinant Leptospira interrogans glyQS?

For the expression of recombinant Leptospira interrogans glyQS, several expression systems can be employed, with E. coli being the most commonly used due to its simplicity and high yield. The methodology should include:

• Gene synthesis or amplification: The glyQS gene can be PCR-amplified from Leptospira interrogans serogroup Icterohaemorrhagiae genomic DNA using primers designed based on the available sequence.

• Vector selection: pET series vectors (particularly pET28a with an N-terminal His-tag) are highly recommended due to their strong T7 promoter system and compatibility with E. coli BL21(DE3) or Rosetta(DE3) strains.

• Expression conditions:

  • Induction with 0.5-1.0 mM IPTG when culture reaches OD600 of 0.6-0.8

  • Post-induction temperature: 16-18°C for 16-18 hours (to enhance solubility)

  • Media supplementation with 2% glucose to repress basal expression

• Alternative systems: For studies requiring post-translational modifications or when facing solubility issues, consider:

  • Cell-free expression systems

  • Yeast expression systems (P. pastoris)

  • Insect cell expression systems (baculovirus)

Each system has advantages and limitations regarding yield, post-translational modifications, and scalability. The choice should be guided by the specific research requirements and downstream applications .

What purification strategies yield the highest enzymatic activity for recombinant glyQS?

A multi-step purification strategy is recommended to obtain high-purity, enzymatically active recombinant glyQS:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

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

  • Elution with 250-300 mM imidazole

• Intermediate purification: Ion exchange chromatography

  • Q-Sepharose column (anion exchange) with a linear gradient of 0-500 mM NaCl

• Polishing step: Size exclusion chromatography

  • Superdex 200 column in 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT

• Critical factors affecting enzyme activity:

  • Buffer optimization: 20-50 mM HEPES or Tris buffer (pH 7.5-8.0)

  • Salt concentration: 100-200 mM NaCl or KCl

  • Addition of stabilizing agents: 5-10% glycerol, 1-2 mM DTT or β-mercaptoethanol

  • Enzyme concentration: 0.5-1.0 mg/ml for optimal stability

• Activity preservation:

  • Storage at -80°C in small aliquots with 20% glycerol

  • Avoid repeated freeze-thaw cycles

  • Addition of 0.1 mM ATP and 1 mM MgCl2 to storage buffer can enhance stability

Typical yield from a 1L bacterial culture is approximately 5-10 mg of purified protein with >95% purity and specific activity of 1000-1500 nmol/min/mg under optimal conditions .

How can I verify the proper folding and functionality of purified recombinant glyQS?

To verify proper folding and functionality of purified recombinant Leptospira interrogans glyQS, employ multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure composition (expected profile: significant α-helical and β-sheet content)

  • Thermal shift assay (TSA) to determine protein stability (Tm typically between 45-55°C)

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to confirm oligomeric state (expected to be dimeric)

• Enzymatic activity assays:

  • Aminoacylation assay: Measure the formation of glycyl-tRNAGly using [14C]-glycine or [3H]-glycine

  • ATP-PPi exchange assay: Quantify the reverse reaction through isotope exchange between PPi and ATP

  • Colorimetric pyrophosphate release assay: Monitor the release of PPi using commercially available kits

• Substrate binding assessments:

  • Isothermal titration calorimetry (ITC) to determine binding affinities for glycine, ATP, and tRNAGly

  • Microscale thermophoresis (MST) as an alternative approach for binding studies

• Expected kinetic parameters for properly folded and functional enzyme:

  • Km for glycine: 10-50 μM

  • Km for ATP: 100-500 μM

  • Km for tRNAGly: 0.5-2 μM

  • kcat: 1-5 s-1

• Controls to include:

  • Known aminoacyl-tRNA synthetase inhibitors as negative controls

  • Denatured enzyme preparation as negative control

  • Commercial E. coli GlyRS as positive control for comparative analysis

What approaches can be used to study the role of glyQS in Leptospira interrogans pathogenesis?

To investigate the role of glyQS in Leptospira interrogans pathogenesis, researchers should implement a multi-faceted approach:

• Gene expression analysis during infection:

  • Real-time RT-PCR to quantify glyQS expression levels under various conditions (different hosts, temperatures, pH, osmolarity)

  • RNA-Seq for comprehensive transcriptome analysis, comparing pathogenic versus saprophytic Leptospira strains

  • In vivo expression technology (IVET) to identify glyQS expression during specific stages of infection

• Genetic manipulation strategies:

  • Conditional knockdown using antisense RNA or CRISPRi (complete knockout may be lethal)

  • Site-directed mutagenesis of key catalytic residues to create attenuated strains

  • Complementation studies to verify phenotypes

  • Overexpression analysis to assess potential toxicity or phenotypic changes

• Protein-protein interaction studies:

  • Pull-down assays coupled with mass spectrometry to identify interaction partners

  • Bacterial two-hybrid screening

  • Co-immunoprecipitation with tagged glyQS

  • Proximity labeling approaches (BioID or APEX) to identify neighborhood proteins

• Animal model studies:

  • Hamster or guinea pig models for acute leptospirosis

  • Comparative virulence studies between wild-type and glyQS-attenuated strains

  • Tissue distribution analysis using immunohistochemistry with anti-glyQS antibodies

  • Immune response assessment against glyQS using ELISA and ELISpot

• Ex vivo cellular studies:

  • Infection of relevant host cells (macrophages, kidney epithelial cells) with wild-type and glyQS-modified strains

  • Assessment of bacterial adhesion, invasion, and intracellular survival

  • Host cell transcriptomics and proteomics to identify differential responses

These methodologies should be employed in a systematic manner to establish potential connections between glyQS function and virulence, similar to approaches used for studying leucine-rich repeat proteins in Leptospira pathogenesis .

How does physiological osmolarity affect glyQS expression and function in Leptospira interrogans?

The effect of physiological osmolarity on glyQS expression and function in Leptospira interrogans can be studied using comprehensive methodological approaches:

• Transcriptional regulation analysis:

  • Real-time RT-PCR quantification of glyQS mRNA levels under various osmotic conditions (physiological osmolarity ~300 mOsm/L versus environmental conditions ~30 mOsm/L)

  • Promoter analysis using reporter gene fusions (glyQS promoter::luciferase or GFP constructs)

  • Identification of osmolarity-responsive transcription factors through ChIP-seq or DNA-protein interaction analyses

• Experimental design for osmolarity studies:

  • Culture bacteria in EMJH media adjusted to different osmolarities using NaCl, sucrose, or other osmolytes

  • Time-course analysis to capture both immediate and adaptive responses

  • Parallel analysis of known osmolarity-responsive genes as positive controls

• Protein expression and functional assessment:

  • Western blot analysis of glyQS protein levels using specific antibodies

  • Measurement of enzymatic activity in cell lysates from bacteria grown at different osmolarities

  • Analysis of protein localization using immunofluorescence microscopy

  • Post-translational modification analysis through mass spectrometry

• Physiological impact determination:

  • Global protein synthesis rate measurement using puromycin incorporation assays

  • Ribosome profiling to assess translational efficiency

  • tRNAGly charging levels analysis through acid-urea PAGE and Northern blotting

  • Cellular glycine pools quantification using HPLC or LC-MS

• Data integration and interpretation:

  • Correlate glyQS expression/activity with growth rates and viability

  • Examine effects on virulence factor expression

  • Establish connections between osmotic stress response and tRNA aminoacylation

  • Compare responses with other aminoacyl-tRNA synthetases to identify specific versus general effects

Previous studies have shown that many Leptospira genes respond to physiologic osmolarity, which may represent an important signal for adaptation during host infection. Research designs should include appropriate controls and multiple technical and biological replicates to ensure statistical robustness .

What methods are effective for studying potential tRNAGly sequestration by mutant forms of glyQS?

Investigating tRNAGly sequestration by mutant forms of glyQS requires specialized techniques that can detect abnormal tRNA-protein interactions and their downstream effects:

• Generation of relevant mutants:

  • Site-directed mutagenesis to create glyQS variants based on:

    • Sequence homology with known human pathogenic GlyRS variants (e.g., equivalents to S211F, H418R, K456Q)

    • Conservation analysis to identify key residues in tRNA binding domains

    • In silico molecular docking predictions

• In vitro tRNA binding analysis:

  • Electrophoretic mobility shift assays (EMSA) with radiolabeled or fluorescently labeled tRNAGly

  • Filter binding assays to quantify binding affinities (Kd determination)

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) for real-time binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Competitive binding assays to assess relative binding strengths

• Cellular assays for tRNA sequestration:

  • Fluorescence in situ hybridization (FISH) to visualize tRNAGly cellular distribution

  • Subcellular fractionation followed by Northern blot analysis

  • RNA immunoprecipitation (RIP) to isolate glyQS-bound tRNAs

  • Proximity ligation assays to detect glyQS-tRNAGly interactions in situ

• Functional consequences assessment:

  • Global protein synthesis measurement using puromycin incorporation or [35S]-methionine labeling

  • Polysome profiling to assess translation efficiency

  • Ribosome footprinting to identify translational pausing at glycine codons

  • Glycine-rich protein expression analysis through targeted proteomics

• Heterologous expression systems:

  • Expression of mutant Leptospira glyQS in model organisms (E. coli, yeast, Drosophila)

  • Complementation assays in temperature-sensitive GlyRS mutant strains

  • Transgenic Drosophila models for in vivo phenotypic analysis

These methodologies have successfully been applied in studies of human GlyRS mutations, where tRNAGly sequestration has been identified as a unifying mechanism underlying peripheral neuropathy. Similar approaches would be valuable for understanding potential pathogenic mechanisms of mutant Leptospira glyQS variants .

What crystallization and structural determination methods are recommended for Leptospira interrogans glyQS?

For successful crystallization and structural determination of Leptospira interrogans glyQS, researchers should follow this comprehensive methodology:

• Pre-crystallization sample preparation:

  • Achieve protein concentration of 5-15 mg/ml in a buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT

  • Verify sample homogeneity via dynamic light scattering (DLS)

  • Perform limited proteolysis to identify stable domains if full-length protein proves challenging

  • Consider removal of the His-tag with TEV protease to improve crystal quality

  • Prepare co-crystallization samples with substrates (glycine, ATP, tRNAGly, or non-hydrolyzable analogs)

• Initial crystallization screening:

  • Employ sitting-drop vapor diffusion method with commercial sparse matrix screens

  • Utilize automated crystallization platforms for higher throughput

  • Incubate at both 4°C and 18°C to identify temperature-dependent conditions

  • Test 1:1, 1:2, and 2:1 protein:reservoir ratios

  • Incorporate microseeding from initial crystalline material

• Optimization strategies:

  • Fine grid screens around promising conditions

  • Additive screens to improve crystal quality

  • Utilize counter-diffusion methods for larger, better-diffracting crystals

  • Apply surface entropy reduction approach if necessary

  • For co-crystal structures, verify ligand occupancy through pre-crystallization binding assays

• X-ray diffraction data collection:

  • Cryoprotection optimization (typically 20-25% glycerol, ethylene glycol, or PEG 400)

  • Initial diffraction testing at home source

  • High-resolution data collection at synchrotron facilities

  • Collection of multiple datasets for molecular replacement or experimental phasing

• Structure determination workflow:

  • Molecular replacement using existing aminoacyl-tRNA synthetase structures

  • If molecular replacement fails, prepare selenomethionine-labeled protein for SAD/MAD phasing

  • Model building with Coot, refinement with PHENIX or REFMAC5

  • Validation using MolProbity and wwPDB validation tools

• Alternative approaches if crystallization proves challenging:

  • Cryo-electron microscopy (cryo-EM) for structural determination

  • Small-angle X-ray scattering (SAXS) for low-resolution envelope

  • NMR spectroscopy for structural information of domains <25 kDa

Expected resolution for good quality crystals should be in the range of 1.8-2.5 Å, which would allow detailed analysis of active site architecture and substrate-binding modes .

What are the recommended assays for measuring glyQS enzymatic activity and inhibition?

To accurately measure Leptospira interrogans glyQS enzymatic activity and inhibition, researchers should employ these methodologically rigorous assays:

• Standard aminoacylation assay:

  • Reaction components: 100 mM HEPES pH 7.5, 10 mM KCl, 10 mM MgCl2, 4 mM ATP, 0.1 mg/mL BSA, 2 mM DTT

  • Substrate concentrations: 50-100 μM [14C]-glycine or [3H]-glycine, 2-4 μM total tRNAGly or total Leptospira tRNA

  • Enzyme concentration: 10-50 nM purified glyQS

  • Temperature: 30-37°C

  • Time course: 0-10 minutes with sampling at 1-minute intervals

  • Quantification: TCA precipitation on filter discs, washing, and scintillation counting

• High-throughput adaptations:

  • Pyrophosphate-release coupled assay with EnzChek Pyrophosphate Assay Kit

  • malachite green assay to detect orthophosphate after pyrophosphatase treatment

  • Luminescent ATP consumption assay (e.g., Kinase-Glo)

  • Time-resolved FRET using fluorescently labeled tRNAGly

• Kinetic parameters determination:

  • Vary glycine concentration (1-1000 μM) at fixed ATP and tRNAGly

  • Vary ATP concentration (10-1000 μM) at fixed glycine and tRNAGly

  • Vary tRNAGly concentration (0.1-10 μM) at fixed glycine and ATP

  • Use Michaelis-Menten, Lineweaver-Burk, or non-linear regression analysis

• Inhibition studies methodology:

  • IC50 determination: Test compounds at 6-8 concentrations in semi-log dilutions

  • Mechanism of inhibition: Vary substrate concentrations in presence of fixed inhibitor

  • Ki determination: Global fitting of data to competitive, uncompetitive, or non-competitive models

  • Time-dependence assessment: Pre-incubation of enzyme with inhibitor for various times

• Data presentation and analysis:

  Parameter	Expected Range	Units
  Km (glycine)	10-50	μM
  Km (ATP)	100-500	μM
  Km (tRNAGly)	0.5-2	μM
  kcat	1-5	s-1
  kcat/Km (glycine)	105-106	M-1s-1
  Assay Z' factor	>0.7	dimensionless
  Coefficient of variation	<10%	%

• Controls and validation:

  • Positive control: Commercial E. coli GlyRS

  • Negative controls: Heat-inactivated enzyme, no-enzyme reaction

  • Validation compounds: Known aminoacyl-tRNA synthetase inhibitors (e.g., mupirocin)

  • Orthogonal assay confirmation for potential inhibitors

These methodologies provide a comprehensive framework for rigorous enzymatic characterization and inhibitor identification for Leptospira interrogans glyQS .

How can I investigate the potential non-canonical functions of glyQS in Leptospira interrogans?

Investigating potential non-canonical functions of glyQS in Leptospira interrogans requires multidisciplinary approaches that extend beyond the traditional aminoacylation role:

• Interaction partner identification:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Bacterial two-hybrid or split-luciferase complementation assays

  • Protein microarray screening with purified glyQS as a probe

  • Crosslinking mass spectrometry to capture transient interactions

  • Co-immunoprecipitation with anti-glyQS antibodies from Leptospira lysates

• Subcellular localization studies:

  • Immunofluorescence microscopy with anti-glyQS antibodies

  • Creation of GFP-glyQS fusion proteins for live-cell imaging

  • Subcellular fractionation followed by Western blot analysis

  • Proteinase K accessibility assays to determine membrane association

  • Immunogold electron microscopy for high-resolution localization

• Post-translational modification analysis:

  • Phosphoproteomics to identify potential phosphorylation sites

  • Analysis of other modifications (acetylation, methylation) by mass spectrometry

  • In vitro modification assays with purified kinases or other modifying enzymes

  • Creation of phosphomimetic or phospho-null mutants to assess functional impact

• Secretion and extracellular function assessment:

  • Analysis of secretome for presence of glyQS

  • Investigation of potential moonlighting functions in extracellular space

  • Binding assays with host extracellular matrix components

  • Host cell interaction studies with purified glyQS

• Experimental design for functional validation:

  • Domain deletion constructs to map non-canonical functions

  • Separation-of-function mutations that affect non-canonical roles without impacting aminoacylation

  • Complementation studies with domain-specific mutants

  • Heterologous expression in systems lacking endogenous glyQS

  • Competitive inhibition assays using peptides derived from interaction interfaces

By analogy with other aminoacyl-tRNA synthetases that have evolved secondary functions, glyQS may participate in unexpected cellular processes beyond translation, such as bacterial adhesion to host tissues, stress responses, or signaling pathways. The methodology outlined above would help uncover such non-canonical functions, similar to approaches used for leucine-rich repeat proteins in Leptospira pathogenesis .

How can recombinant glyQS be used as a target for anti-leptospiral drug discovery?

Recombinant Leptospira interrogans glyQS offers significant potential as a target for anti-leptospiral drug discovery, which can be approached through the following methodological framework:

• Target validation strategies:

  • Essentiality assessment through conditional knockdown systems

  • Demonstration of growth inhibition upon glyQS activity reduction

  • In silico comparative analysis between bacterial and human orthologues to identify selective targeting potential

  • Establishment of minimal inhibitory activity threshold required for antimicrobial effect

• High-throughput screening (HTS) methodology:

  • Primary biochemical screen using aminoacylation assays adapted to 384-well format

  • Screening library composition: natural product extracts, synthetic compound libraries, repurposing libraries

  • Hit confirmation through dose-response curves (8-point, 3-fold dilutions)

  • Counter-screening against human GARS1 to establish selectivity window

  • Secondary whole-cell assays against Leptospira cultures

• Structure-based drug design approach:

  • Virtual screening against glyQS crystal structure or homology model

  • Focus on unique pockets absent in the human orthologue

  • Molecular docking of compound libraries (e.g., ZINC, ChEMBL)

  • Fragment-based screening using thermal shift assays, STD-NMR, or X-ray crystallography

  • Structure-activity relationship (SAR) studies of promising scaffolds

• Compound optimization workflow:

  Property	Primary Screen	Lead Optimization	Candidate Selection
  glyQS IC50	<10 μM	<100 nM	<50 nM
  Selectivity (human/bacterial)	>10×	>100×	>500×
  MIC against Leptospira	<50 μM	<5 μM	<1 μM
  Cytotoxicity (CC50)	Not required	>100 μM	>200 μM
  Solubility	Not required	>100 μM	>200 μM
  Metabolic stability	Not required	t1/2 >30 min	t1/2 >2 h

• Lead validation methodologies:

  • Mechanism of action confirmation (binding site verification through resistance mutations)

  • Efficacy evaluation in cellular infection models

  • PK studies in rodent models

  • Efficacy evaluation in hamster or guinea pig leptospirosis models

  • Combination studies with standard antibiotics (e.g., doxycycline, penicillin)

• Target engagement verification:

  • Cellular thermal shift assay (CETSA) in bacterial cultures

  • Metabolomic profiling to confirm pathway disruption

  • Transcriptomic response analysis to confirm stress signature

  • Time-kill studies to characterize bactericidal vs. bacteriostatic action

This systematic approach leverages the essential nature of aminoacyl-tRNA synthetases while focusing on exploiting structural and functional differences between bacterial and human enzymes to develop selective inhibitors as potential therapeutics against leptospirosis .

What are the challenges and methodologies for using recombinant glyQS in developing serodiagnostic tests for leptospirosis?

Developing serodiagnostic tests for leptospirosis using recombinant Leptospira interrogans glyQS involves specific challenges and methodological considerations:

• Antigenicity assessment methodology:

  • Human sera panel screening:

    • Acute leptospirosis patients (MAT-positive)

    • Convalescent leptospirosis patients

    • Healthy controls from endemic and non-endemic regions

    • Patients with other infectious diseases (cross-reactivity panel)

  • Western blot and ELISA analysis to determine immunoreactivity

  • Epitope mapping using peptide arrays or phage display

  • Assessment of correlation between antibody titers and disease severity

• Immunoassay development workflow:

  • Protein preparation considerations:

    • Full-length versus immunodominant fragments

    • Native versus denatured conformation

    • Expression system selection for optimal antigenicity

    • Quality control parameters (purity >95%, consistent lot-to-lot performance)

  • Assay formats:

    • ELISA (indirect, sandwich, competitive)

    • Lateral flow immunoassay

    • Multiplex bead-based assays

    • Chemiluminescent immunoassay

• Performance optimization strategy:

  • Surface chemistry optimization for protein immobilization

  • Buffer composition adjustments to minimize background

  • Blocking agent selection to prevent non-specific binding

  • Signal amplification methods for increased sensitivity

  • Calibrator and control preparation for standardization

• Diagnostic performance targets:

  Parameter	Minimal Target	Optimal Target
  Clinical sensitivity	>85%	>95%
  Clinical specificity	>90%	>98%
  Cross-reactivity	<10%	<5%
  Limit of detection	100 ng/mL IgG/IgM	10 ng/mL IgG/IgM
  Precision (CV)	<15%	<10%
  Sample types	Serum	Serum, whole blood, urine

• Validation methodology:

  • Analytical validation:

    • Precision (intra-assay, inter-assay, inter-lot)

    • Linearity and reportable range

    • Interference studies (hemolysis, lipemia, icterus)

    • Stability (on-board, freeze-thaw, shipping conditions)

  • Clinical validation:

    • Sample size calculation based on prevalence

    • Multi-center evaluation

    • Comparison with reference methods (MAT, PCR, culture)

    • ROC analysis for cut-off determination

• Key challenges and mitigation strategies:

  • Challenge: Cross-reactivity with other spirochetes

    • Solution: Identify unique epitopes through comparative analysis

  • Challenge: Variable antibody response timing and magnitude

    • Solution: Combined IgM/IgG detection with time-course studies

  • Challenge: Strain variation in antigen sequence

    • Solution: Conservation analysis across serovars, use of conserved epitopes

  • Challenge: Low sensitivity in early disease phase

    • Solution: Combination with other biomarkers or direct detection methods

This methodological framework addresses the specific challenges in developing serodiagnostic tests using recombinant proteins, with potential for improved sensitivity and specificity compared to traditional whole-cell based assays .

How does the comparative analysis of glyQS across Leptospira species inform evolutionary and pathogenesis studies?

Comparative analysis of glyQS across Leptospira species provides valuable insights into evolutionary patterns and pathogenesis mechanisms, requiring specific methodological approaches:

• Phylogenetic analysis methodology:

  • Sequence data collection:

    • Retrieval of glyQS sequences from all available Leptospira genomes (pathogenic, intermediate, saprophytic)

    • Inclusion of other spirochete and bacterial glyQS sequences as outgroups

    • Multiple sequence alignment using MUSCLE, MAFFT, or T-Coffee with manual curation

  • Phylogenetic reconstruction:

    • Maximum likelihood analysis using RAxML or IQ-TREE

    • Bayesian inference using MrBayes or BEAST

    • Selection of appropriate evolutionary models through ModelTest or similar tools

    • Bootstrap analysis (>1000 replicates) or posterior probability assessment

• Evolutionary pressure analysis:

  • Calculation of dN/dS ratios to identify selection patterns

  • Codon-based Z-test of selection

  • Branch-site models to detect episodic selection

  • FUBAR or MEME analysis to identify sites under positive selection

  • Evolutionary rate analysis using relative rate tests

• Structural and functional domain analysis:

  • Domain conservation mapping on 3D structures

  • Identification of conserved catalytic residues versus variable surface regions

  • Substrate specificity determinant analysis

  • Correlation of sequence variations with biochemical properties

  • Detection of potential lateral gene transfer events

• Experimental validation of evolutionary hypotheses:

  • Site-directed mutagenesis of variant residues

  • Chimeric protein construction between pathogenic and saprophytic variants

  • Complementation studies in heterologous systems

  • Comparative kinetic analysis of representative enzymes

  • Host adaptation assays with variant proteins

• Correlation with pathogenesis data:

  • Comparative expression analysis across species during infection

  • Integration with host-pathogen interaction datasets

  • Analysis of immune recognition patterns between variants

  • Assessment of antigenic drift in relation to immune evasion

  • Correlation with virulence in animal models

• Key insights table from comparative analysis:

  Aspect	Pathogenic Leptospira	Intermediate Leptospira	Saprophytic Leptospira
  Sequence conservation	Highest within P1 subclade	Intermediate, between P1 and S	Highest within S subclade
  Catalytic efficiency	Potentially optimized for host conditions	Moderately adapted	Adapted to environmental conditions
  Expression regulation	Response to host signals	Variable response	Limited response to host signals
  Structural adaptations	Potential surface-exposed unique regions	Intermediate features	Environmental adaptation features
  Evolutionary rate	Potentially accelerated in host-interaction regions	Intermediate	More constrained evolution

This comprehensive methodology enables researchers to understand how glyQS has evolved across the Leptospira genus and how these evolutionary patterns might correlate with the spectrum of pathogenicity observed across species, from highly virulent to saprophytic lifestyles. Similar approaches have been applied to other Leptospira proteins, revealing that genes may be found primarily in pathogenic strains (subclade P1) or more broadly distributed across pathogenic, intermediate, and saprophytic groups .