Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni tRNA modification GTPase MnmE (mnmE)

What is Leptospira interrogans serovar Copenhageni and why is it significant for research?

Leptospira interrogans serovar Copenhageni is one of the most pathogenic serovars within the Icterohaemorrhagiae serogroup and represents more than half of the leptospires encountered in severe human infections . It is a zoonotic pathogen that causes leptospirosis, a disease of significant human and veterinary concern. The strain most commonly used in research is Fiocruz L1-130, which has been extensively characterized for studying leptospiral pathogenesis .

This serovar is particularly significant because:

• It shows high pathogenicity compared to other Leptospira serovars

• It has been isolated from multiple mammalian hosts, including humans and rodents

• It demonstrates specific patterns of tissue colonization, particularly in kidneys

• It elicits distinct immune responses depending on the route of infection

When designing experiments with this organism, researchers should consider that Copenhageni is the predominant serovar in the Icterohaemorrhagiae serogroup isolated in the British Isles, with 16 out of 19 isolates identified as Copenhageni in a representative study .

What is MnmE and what is its role in tRNA modification processes?

MnmE is a multi-domain GTPase that is evolutionarily conserved from bacteria to humans. It functions in conjunction with its partner protein MnmG to catalyze a specific tRNA wobble uridine modification . This modification is critical for accurate and efficient protein translation.

Key characteristics of MnmE include:

• Unlike classical small GTP-binding proteins that are regulated by GEFs and GAPs, MnmE's GTPase activity is activated through potassium-dependent homodimerization of its G domains

• The protein undergoes large-scale conformational changes throughout its GTPase cycle

• These conformational changes are believed to drive and fine-tune the complex tRNA modification reaction

• In eukaryotes, MnmE orthologues are targeted to mitochondria, and mutations in the encoding genes are associated with severe mitochondrial diseases

Understanding MnmE's mechanism is particularly valuable for researchers investigating fundamental aspects of bacterial physiology as well as for those exploring the molecular basis of certain mitochondrial disorders.

How should researchers approach experimental design when investigating Leptospira interrogans pathogenesis?

When designing experiments to study L. interrogans pathogenesis, several methodological considerations are essential:

• Infection Route Selection: Different routes of infection significantly impact experimental outcomes. Research has shown that transdermal and nasal mucosa infections lead to weight loss, renal colonization, and inflammation, while oral mucosa inoculation does not produce these effects . Consider the following comparative data:

• Animal Model Selection: C3H/HeJ mice have been validated for studying sublethal infection with L. interrogans, allowing examination of kidney colonization and immune responses .

• Bacterial Burden Quantification Methods:

  • qPCR of Leptospira 16S rRNA from tissues

  • Culture of tissues to confirm viability

  • Blood sampling at multiple timepoints to track dissemination kinetics

• Immune Response Assessment:

  • Transcriptional analysis of pro-inflammatory mediators (CxCL1/KC, CxCL2/MIP-2, CCL5/RANTES, TNF-α)

  • Th1 cytokine (IFN-γ) measurement

  • Fibrosis markers (collagen A1, iNOS)

  • Immunoglobulin isotyping in serum

What methodological approaches are most effective for studying recombinant proteins from Leptospira interrogans?

Based on established research protocols, the following methodological approach is recommended for studying recombinant proteins from L. interrogans:

• Gene Selection and Verification:

  • Identify target genes through bioinformatic analysis

  • Confirm gene presence across relevant serovars using PCR

  • Verify gene expression via RT-PCR under different environmental conditions to account for regulation (especially important as LIC10258 and LIC12880 show differential expression patterns across serovars)

• Cloning and Expression Strategy:

  • Amplify the complete open reading frame without signal peptide sequence

  • Clone into expression vectors with appropriate tags (His-tag commonly used)

  • Express in E. coli BL21 (SI) strain, which has been successfully employed for leptospiral proteins

  • Optimize induction conditions (temperature, IPTG concentration, duration)

• Protein Purification Protocol:

  • Perform metal affinity chromatography for His-tagged proteins

  • Consider additional purification steps (ion exchange, size exclusion) to achieve high purity

  • Verify protein identity by western blot with anti-His antibodies and mass spectrometry

• Functional Characterization Assays:

  • Binding studies with potential interaction partners (e.g., ECM components, plasminogen)

  • Determine binding kinetics using methods like surface plasmon resonance

  • Calculate binding constants (KD values) for quantitative comparisons

For example, in characterizing novel OmpA-like proteins from L. interrogans, researchers successfully employed Escherichia coli BL21 (SI) as the host expression system, followed by functional characterization revealing plasminogen binding with KD values of 68.8±25.2 nM and 167.39±60.1 nM for rLIC10258 and rLIC12880, respectively .

How can researchers effectively distinguish between Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni?

Distinguishing between serovars Icterohaemorrhagiae and Copenhageni presents significant challenges due to their close genetic relationship. Based on research findings, the following methodological approach is recommended:

• Monoclonal Antibody Typing:

  • Use of specific monoclonal antibodies (particularly F12 C3, F70 C14, and F70 C24)

  • Monoclonal antibody F12 C3 has been shown to be highly specific for both Icterohaemorrhagiae and Copenhageni serovars

  • Additional antibodies can then differentiate between these two closely related serovars

• Genetic Typing Methods:

  • Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA): This method has identified three loci with differences in repeat numbers, indicating genetic diversity between isolates

  • lic12008 Gene Sequence Analysis: All isolates identified as Icterohaemorrhagiae serotype have been shown to have a single base insertion compared to Copenhageni serotype sequences

  • Restriction Endonuclease Analysis (REA): While commonly used, research has shown this method is not able to discriminate between Icterohaemorrhagiae and Copenhageni serovars

Comparative effectiveness of different typing methods:

Research has demonstrated that using a combination of these approaches provides the most reliable differentiation, with molecular methods supporting traditional serological typing.

What are the conformational dynamics of MnmE and how do they drive tRNA modification?

MnmE exhibits complex conformational dynamics that are integral to its function in tRNA modification. The following mechanisms have been elucidated:

• GTPase Cycle and Conformational Changes:

  • MnmE's GTPase activity is uniquely activated through potassium-dependent homodimerization of its G domains

  • This differs from classical GTPases that require auxiliary GEFs and GAPs for regulation

  • The protein undergoes large-scale conformational changes throughout the GTPase cycle

• Functional Mechanism:

  • The conformational changes in MnmE are believed to:

    • Position the catalytic residues optimally for the tRNA modification reaction

    • Facilitate interaction with partner protein MnmG

    • Enable proper binding and orientation of the tRNA substrate

    • Coordinate the chemistry of the modification reaction

• Structural Features Supporting Function:

  • Multi-domain architecture allows for complex movements during catalysis

  • G domains undergo dimerization in a potassium-dependent manner

  • Conformational states are tightly coupled to the nucleotide state (GTP, GDP, or nucleotide-free)

• Methodological Approaches to Study Dynamics:

  • X-ray crystallography of different nucleotide-bound states

  • FRET-based assays to track domain movements in real-time

  • Mutational analysis of key residues involved in dimerization and catalysis

  • Computational molecular dynamics simulations to predict motion trajectories

These conformational changes are believed to drive and tune the complex tRNA modification reaction by controlling the sequential steps of substrate binding, chemistry, and product release .

What is the role of Leptospira surface proteins in host-pathogen interactions and how can they be experimentally characterized?

Leptospira surface proteins play crucial roles in host-pathogen interactions, particularly in bacterial adhesion, invasion, and immune evasion. Their experimental characterization requires a multi-faceted approach:

• Identification and Classification:

  • Bioinformatic analysis to identify potential surface proteins

  • Classification based on structural features (e.g., OmpA-like proteins like Lsa66)

  • Expression analysis under different conditions to determine regulation patterns

• Functional Characterization Methodologies:

  • Binding Assays: Test interaction with host components (ECM molecules, plasminogen)

  • Kinetic Analysis: Determine binding constants (KD) through dose-dependent assays

  • For example, Lsa66 (LIC10258) showed specific binding to:

    • Laminin (KD = 55.4±15.9 nM)

    • Plasma fibronectin (KD = 290.8±11.8 nM)

    • Plasminogen (KD = 68.8±25.2 nM)

• Inhibition Studies:

  • Competitive inhibition assays to assess functional relevance

  • Lsa66 demonstrated inhibitory effects on leptospiral adherence to:

    • Laminin (P<0.05)

    • Plasma fibronectin (P<0.05)

    • Plasminogen (P<0.05)

• Immunological Analysis:

  • Assessment of protein recognition by convalescent sera

  • Antibody production for detection and functional studies

  • Immunofluorescence assays to confirm surface localization

• Expression Analysis Under Host-Relevant Conditions:

  • Osmolarity changes that mimic physiological conditions (≈300 mosmol per liter) encountered upon host entry

  • Temperature shifts that simulate fever or environmental-to-host transition

  • Nutrient availability changes

This methodological approach has successfully identified novel proteins like Lsa66, an OmpA-like protein with dual binding activity to ECM components and plasminogen, which may contribute to leptospiral attachment to host tissues and subsequent invasion .

How do variations in experimental infection routes impact research outcomes when studying Leptospira interrogans pathogenesis?

The choice of infection route significantly impacts research outcomes when studying L. interrogans pathogenesis. Researchers should consider the following methodological implications:

• Differential Tissue Colonization Patterns:

  • Transdermal (TD) and nasal mucosa (NM) routes lead to significant kidney colonization, similar to intraperitoneal (IP) infection

  • Oral mucosa (OM) route shows minimal to no kidney colonization

  • The timing of bacterial dissemination varies significantly between routes

• Distinct Immune Response Profiles:

• Methodological Recommendations:

  • Study Design: Include multiple infection routes when assessing pathogenesis mechanisms

  • Timeline Considerations: Adjust sampling schedules based on route-specific dissemination kinetics

  • Immune Analysis: Incorporate both innate and adaptive immune markers

  • Controls: Always include standard IP route as reference for comparison

• Relevance to Natural Infection:

  • TD and NM routes more closely mimic natural infection scenarios

  • IP route, while convenient, may not accurately represent the natural course of infection

  • Different routes may be more appropriate depending on the specific research question

This comparative understanding is essential for designing studies that accurately model natural infection while maintaining experimental control and reproducibility.

What are the critical parameters for successful expression and purification of recombinant MnmE from Leptospira interrogans?

Based on established methodologies for similar proteins, the following protocol recommendations are critical for successful expression and purification of recombinant MnmE:

• Vector Selection and Construct Design:

  • Use pAE or similar vectors that provide N-terminal His-tags for purification

  • Exclude signal peptide sequences when designing constructs

  • Consider codon optimization for E. coli expression if initial attempts yield low expression

• Expression System Optimization:

  • E. coli BL21 (SI) strain has proven effective for leptospiral proteins

  • Test multiple induction conditions:

    • Temperature: Compare 30°C vs. 37°C

    • Inducer concentration: Typically 0.5-1.0 mM IPTG

    • Duration: 3-6 hours for standard protocol, overnight for difficult proteins

  • Consider auto-induction media for proteins with toxicity issues

• Purification Strategy:

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

  • Include additional purification steps for functional studies:

    • Size exclusion chromatography to ensure monomeric protein

    • Ion exchange chromatography for removal of nucleic acid contamination

• Buffer Optimization for GTPase Activity:

  • Include potassium (typically 50-100 mM KCl) in buffers to support MnmE's potassium-dependent GTPase activity

  • Test protein stability in different pH conditions (typically pH 7.5-8.0)

  • Add glycerol (5-10%) to prevent aggregation and improve stability

  • Consider adding reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

• Activity Assays and Verification:

  • GTPase activity using malachite green phosphate assay

  • Confirm conformational changes using intrinsic tryptophan fluorescence

  • Verify function through complementation assays in mnmE-deficient strains

• Troubleshooting Common Issues:

  • Low expression: Test different E. coli strains, optimize codon usage

  • Insoluble protein: Lower induction temperature, use solubility enhancers

  • Loss of activity: Ensure potassium is present in all buffers, verify protein folding

  • Aggregation: Add detergents (0.01-0.05% Triton X-100) or stabilizers (arginine, sucrose)

This methodological approach builds upon successful strategies used for other leptospiral proteins while incorporating specific considerations for GTPases like MnmE.

How can researchers address challenges in assessing the role of specific proteins in Leptospira pathogenesis?

Researchers face several challenges when assessing protein roles in Leptospira pathogenesis. The following methodological approaches can address these challenges:

This comprehensive approach has been validated in studies of proteins like Lsa66, which demonstrated specific, dose-dependent, and saturable binding to multiple host components with functional inhibition of bacterial attachment .

What are the emerging techniques for studying the structure-function relationships of bacterial tRNA modification enzymes like MnmE?

Several cutting-edge methodologies are emerging for investigating structure-function relationships in bacterial tRNA modification enzymes:

• Cryo-Electron Microscopy (Cryo-EM):

  • Allows visualization of different conformational states without crystallization

  • Particularly valuable for capturing the MnmE-MnmG complex during various stages of the catalytic cycle

  • Can reveal dynamic interactions between the enzyme complex and tRNA substrates

• Time-Resolved X-ray Crystallography:

  • Captures transient conformational states during the GTPase cycle

  • Provides insights into how potassium-dependent dimerization drives structural changes

  • Can be combined with substrate analogs to trap reaction intermediates

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

  • Maps protein dynamics and conformational changes under various conditions

  • Particularly useful for identifying regions that undergo structural rearrangements during substrate binding

  • Can probe effects of mutations or ligands on protein dynamics

• Single-Molecule FRET Techniques:

  • Monitors real-time conformational changes during enzyme function

  • Can track the timing and sequence of structural rearrangements during the catalytic cycle

  • Allows correlation of structural dynamics with catalytic steps

• Integrative Computational Approaches:

  • Molecular dynamics simulations to predict conformational changes

  • Quantum mechanics/molecular mechanics (QM/MM) calculations to model the reaction mechanism

  • Machine learning algorithms to predict effects of mutations on enzyme function

• CRISPR-Based Approaches for In Vivo Functional Analysis:

  • Base editing to introduce specific mutations in the native genomic context

  • CRISPRi for conditional knockdown to assess physiological consequences

  • CRISPR screens to identify genetic interactions with other cellular components

These emerging techniques, when applied to MnmE from Leptospira interrogans, could provide unprecedented insights into how this enzyme's conformational dynamics drive tRNA modification and potentially reveal species-specific features that might be exploited for therapeutic targeting.

How might comparative analysis of MnmE across different bacterial species inform our understanding of pathogenesis?

Comparative analysis of MnmE across bacterial species offers valuable insights into both evolutionary conservation and pathogen-specific adaptations:

• Structural and Functional Conservation Analysis:

  • Identify core structural elements conserved across species

  • Map species-specific variations that might relate to pathogenesis

  • Determine if pathogenic species show distinctive features in MnmE

• Methodological Approach for Comparative Studies:

  • Sequence Analysis:

    • Multiple sequence alignment of MnmE from diverse bacterial species

    • Phylogenetic analysis to correlate MnmE variations with pathogenicity

    • Identification of signature sequences in pathogenic vs. non-pathogenic species

  • Structural Comparison:

    • Homology modeling based on existing MnmE structures

    • Structure-based alignment to identify functional differences

    • Analysis of surface properties and interaction interfaces

  • Functional Comparison:

    • Heterologous expression and biochemical characterization

    • Cross-complementation studies in model organisms

    • Comparative analysis of GTPase activity and regulation

• Relevance to Translational Research:

  • Identification of pathogen-specific features that could be targeted therapeutically

  • Understanding of evolutionary constraints on MnmE function

  • Insight into how tRNA modification impacts bacterial physiology and virulence

• Specific Research Questions to Address:

  • Does MnmE from pathogenic Leptospira show unique structural or functional adaptations?

  • Are there correlations between MnmE variations and bacterial host range or virulence?

  • How do environmental adaptations in different bacterial species affect MnmE function?

  • Does the potassium-dependent activation mechanism vary across species in ways that might impact pathogenesis?

This comparative approach could reveal whether MnmE functions as a basic cellular maintenance factor or if it has acquired specialized roles in pathogenic species like Leptospira interrogans that contribute to their virulence.

What are the implications of tRNA modification for bacterial adaptation to different host environments?

tRNA modification systems, including those involving MnmE, play critical roles in bacterial adaptation to diverse host environments:

• Translational Efficiency and Accuracy:

  • tRNA modifications optimize codon-anticodon interactions

  • This may be particularly important during host infection when rapid protein synthesis is required

  • Different modifications may be preferentially required for expression of virulence factors

• Stress Response Regulation:

  • tRNA modification patterns can change in response to environmental stressors

  • This dynamic regulation may help bacteria adapt to changing host conditions

  • For pathogenic bacteria like Leptospira interrogans that encounter diverse environments (environment → host → different tissues), this adaptability is particularly relevant

• Experimental Approaches to Investigate These Connections:

  • Transcriptomics Under Host-Relevant Conditions:

    • RNA-seq analysis in response to temperature shifts, osmolarity changes, and pH variations

    • Quantification of tRNA modification levels under different conditions

    • Correlation of modification patterns with expression of virulence factors

  • Infection Models With Variable Routes:

    • Analysis of bacterial gene expression after infection via different routes (TD, NM, IP)

    • Comparison of tRNA modification patterns in bacteria recovered from different tissues

    • Assessment of mnmE expression levels during different infection stages

• Methodological Approaches for tRNA Modification Analysis:

  • Mass spectrometry-based methods to quantify modification levels

  • Next-generation sequencing approaches for tRNA modification mapping

  • Bacterial genetics to create modification-deficient strains for in vivo testing

• Research Questions at the Intersection of tRNA Biology and Pathogenesis:

  • Does Leptospira interrogans modulate its tRNA modification pattern during infection?

  • Are specific modifications required for expression of virulence factors?

  • Does the bacterial response to host immune attack involve changes in tRNA modification?

  • Can tRNA modification systems be targeted for antimicrobial development?

This research direction connects fundamental aspects of bacterial physiology with pathogenesis mechanisms and may reveal new targets for therapeutic intervention.

What resources and protocols are available for researchers studying recombinant proteins from Leptospira interrogans?

Researchers studying recombinant proteins from Leptospira interrogans can access several methodological resources:

• Expression System Selection:

  • E. coli BL21 (SI) strain has been validated for successful expression of leptospiral proteins

  • Vector systems with N-terminal His-tags (e.g., pAE) facilitate purification

  • Optimization protocols for difficult-to-express leptospiral proteins

• Standardized Protocols for Functional Characterization:

  • Binding Assays:

    • ELISA-based protocols for measuring interaction with host components

    • Surface plasmon resonance methods for kinetic analysis

    • Protocols for calculating binding constants (KD) as demonstrated with Lsa66

  • Inhibition Studies:

    • ELISA-like assays for quantifying inhibition of leptospiral adherence

    • Standardized methods for assessing statistical significance (e.g., Student's t-test)

• Transcriptional Analysis Methods:

  • RT-PCR protocols for assessing gene expression under various conditions

  • Controls for RNA integrity assessment using 16S ribosomal cDNA

  • Methodologies for testing environmental factors (osmolarity, temperature)

• Animal Model Resources:

  • C3H/HeJ mouse model protocols for sublethal infection studies

  • Standardized infection procedures for different routes (TD, NM, OM, IP)

  • Methods for assessing bacterial dissemination, colonization, and immune response

• Data Analysis Resources:

  • Statistical approaches for comparing binding affinities

  • Methods for analyzing inhibition studies

  • Protocols for quantifying bacterial loads in tissues

• Strain Typing Resources:

  • Monoclonal antibody panels for serological typing

  • MLVA protocols for genetic characterization

  • lic12008 gene sequence analysis methods for differentiating closely related serovars

These methodological resources provide researchers with validated approaches for investigating recombinant proteins from Leptospira interrogans, enabling reproducible and comparable results across different laboratories.

What are the recommended controls and validation methods for studying bacterial GTPases like MnmE?

Robust experimental design for studying bacterial GTPases like MnmE requires comprehensive controls and validation methods:

• Biochemical Characterization Controls:

  • Negative Controls:

    • Heat-inactivated enzyme preparations

    • Catalytically inactive mutants (e.g., mutations in the G domain)

    • Assays performed in the absence of potassium for potassium-dependent GTPases like MnmE

  • Positive Controls:

    • Well-characterized GTPases with known activity profiles

    • Previously validated recombinant MnmE from model organisms

    • Commercial GTPase standards for activity calibration

• Structural and Functional Validation Methods:

  • Circular Dichroism (CD) Spectroscopy:

    • Verification of proper protein folding

    • Monitoring structural changes upon nucleotide binding

  • Thermal Shift Assays:

    • Assessment of protein stability under various conditions

    • Evaluation of ligand effects on protein stability

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

    • Determination of oligomeric state

    • Monitoring of potassium-dependent dimerization

• Activity Assay Validation:

  • Multiple Independent Methods:

    • Malachite green phosphate assay for Pi release

    • HPLC-based methods for nucleotide conversion

    • Coupled enzyme assays for real-time monitoring

  • Kinetic Parameter Determination:

    • Km and Vmax calculations under varying conditions

    • Effects of potassium concentration on activity

    • Temperature and pH optima determination

• Functional Complementation:

  • In vivo Validation:

    • Complementation of mnmE-deficient strains

    • Phenotypic rescue assessment

    • tRNA modification analysis in complemented strains

• Interaction Studies Validation:

  • Reciprocal Co-IP Controls:

    • Verification of interactions from both directions

    • Non-specific binding controls using unrelated proteins

  • Microscale Thermophoresis Controls:

    • Titration with non-interacting proteins as negative controls

    • Competition assays with unlabeled proteins

• Conformational Dynamics Validation:

  • Multiple Structural Methods:

    • Correlation between X-ray crystallography and solution studies

    • Validation of FRET-based conformational measurements with alternative approaches