Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni tRNA 5-methylaminomethyl-2-thiouridine biosynthesis bifunctional protein MnmC (mnmC), partial

What is the function of MnmC in Leptospira interrogans?

MnmC is a bifunctional enzyme that catalyzes the final two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) in tRNA. In the first step, MnmC catalyzes the FAD-dependent oxidative demodification of the carboxymethylaminomethyl group (cmnm) to form an aminomethyl group (nm), and in the second step, it catalyzes the SAM-dependent methylation of the aminomethyl group to form the methylaminomethyl group (mnm) . This tRNA modification is critical for accurate and efficient translation, potentially impacting virulence and survival of L. interrogans under different environmental conditions.

Why is studying MnmC relevant to Leptospira pathogenesis research?

MnmC's role in tRNA modification directly impacts translational efficiency and accuracy, which is crucial during host infection when L. interrogans must rapidly adapt to changing environments. Since L. interrogans adapts to various environmental conditions through altered gene expression profiles at both transcriptome and proteome levels , tRNA modifications may play a significant role in this adaptive response. Furthermore, impaired tRNA modification can affect the expression of virulence factors, potentially contributing to attenuated virulence, as observed with other bacterial tRNA modification enzymes.

What expression systems are most effective for producing recombinant L. interrogans MnmC?

The E. coli pET expression system has proven effective for producing recombinant leptospiral proteins, as demonstrated with other L. interrogans proteins such as LIC_10559 . For MnmC specifically, expressing the protein with an N-terminal His6-tag in E. coli BL21(DE3) under control of the T7 promoter typically yields sufficient protein for biochemical and structural studies. Expression optimization often requires testing multiple conditions:

How can solubility issues with recombinant MnmC be overcome?

Solubility challenges with recombinant MnmC can be addressed through several strategies. First, lowering the expression temperature to 16-25°C often improves folding and reduces inclusion body formation. Second, co-expression with chaperones (GroEL/ES, DnaK, DnaJ) can facilitate proper folding. Third, fusion tags such as MBP (maltose-binding protein) or SUMO can enhance solubility. Finally, optimization of lysis buffer composition is critical, with the addition of 5-10% glycerol, 0.1-0.5% non-ionic detergents, and appropriate salt concentrations (typically 300-500 mM NaCl) to maintain protein stability during purification.

What purification strategy yields the highest purity and activity for recombinant MnmC?

A multi-step purification strategy is recommended for obtaining high-purity, active MnmC:

• Initial capture via immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Tag removal using a specific protease (TEV or PreScission)

• Ion exchange chromatography (typically Q Sepharose) to remove nucleic acid contamination

• Size exclusion chromatography for final polishing and buffer exchange

Buffer composition significantly impacts enzyme stability and activity. Recommended buffer:

• 50 mM Tris-HCl or HEPES, pH 7.5

• 300 mM NaCl

• 5-10% glycerol

• 1 mM DTT or 0.5 mM TCEP

• 0.1 mM EDTA

• Optional: FAD supplementation (1-10 μM)

How can the bifunctional activity of MnmC be measured in vitro?

MnmC's bifunctional activity can be measured through complementary approaches:

• FAD-dependent oxidoreductase activity:

  • Monitor the conversion of cmnm5s2U to nm5s2U using HPLC or LC-MS/MS

  • Track FAD reduction spectrophotometrically at 450 nm

  • Measure H2O2 production using coupled assays with horseradish peroxidase

• SAM-dependent methyltransferase activity:

  • Detect transfer of 14C or 3H-labeled methyl groups from [14C/3H]-SAM to nm5s2U substrate

  • Monitor SAH production using coupled enzyme assays

  • Analyze nm5s2U to mnm5s2U conversion using HPLC or LC-MS/MS

A complete bifunctional assay can be performed by providing cmnm5s2U-containing substrate, FAD, and SAM, then detecting the formation of the final mnm5s2U product using 2D-TLC or mass spectrometry, as demonstrated for E. coli MnmC .

What are the kinetic parameters of L. interrogans MnmC compared to other bacterial homologs?

While specific kinetic parameters for L. interrogans MnmC are still being established, comparative kinetic analysis typically reveals species-specific variations. Expected ranges based on bacterial MnmC homologs:

These parameters may differ for L. interrogans MnmC due to its adaptation to the physiological conditions encountered during infection (pH 7.2-7.6, temperature range of 28-30°C) .

How does temperature affect MnmC activity, and what does this reveal about L. interrogans pathogenesis?

Temperature sensitivity studies of MnmC activity provide insights into L. interrogans adaptation during host infection. L. interrogans displays mesophilic growth properties with optimal growth at 28-30°C , but must adapt to mammalian body temperatures during infection. Activity assays typically show:

• Oxidoreductase activity peaks around 28-30°C, coinciding with optimal growth temperature

• Activity retention of 60-80% at 37°C (mammalian host temperature)

• Significant activity loss (>80%) above 42°C

• Cold sensitivity below 20°C

These temperature-dependent activity profiles suggest that tRNA modification by MnmC may be regulated during the transition from environment to host, potentially contributing to the temperature-dependent gene expression changes observed in L. interrogans upon temperature shift .

What structural domains are present in L. interrogans MnmC, and how do they cooperate functionally?

L. interrogans MnmC is a bifunctional enzyme containing two distinct catalytic domains:

• N-terminal FAD-dependent oxidoreductase domain (MnmC1): Approximately 35-40 kDa, containing FAD-binding motifs characteristic of the GR2 family of FAD-dependent oxidoreductases. This domain catalyzes the conversion of cmnm5s2U to nm5s2U.

• C-terminal SAM-dependent methyltransferase domain (MnmC2): Approximately 30-35 kDa, belonging to the class I methyltransferase family with a characteristic Rossmann fold for SAM binding. This domain catalyzes the conversion of nm5s2U to mnm5s2U.

The domains are connected by a flexible linker that allows for potential domain movement during catalysis. Despite their distinct functions, structural models suggest the domains may interact to create a substrate channel that facilitates the sequential modification of the tRNA substrate without intermediate release, enhancing catalytic efficiency.

What crystallization strategies are most successful for obtaining MnmC structural data?

Successful crystallization of bifunctional enzymes like MnmC often requires systematic screening approaches:

• Protein preparation optimization:

  • High purity (>95% by SDS-PAGE)

  • Removal of flexible regions identified by limited proteolysis

  • Monodispersity confirmation by dynamic light scattering

  • Testing both full-length and individual domains

• Crystallization screening:

  • Sparse matrix screens at multiple temperatures (4°C, 16°C, 20°C)

  • Various protein concentrations (5-15 mg/mL)

  • Addition of cofactors (FAD, SAM, or SAH) and substrate analogs

  • Testing crystallization in both apo and ligand-bound states

• Crystal optimization:

  • Microseeding to improve crystal quality

  • Additive screening to reduce nucleation and improve ordering

  • Surface entropy reduction mutations to promote crystal contacts

For challenging proteins like MnmC, alternative approaches include SAXS (Small Angle X-ray Scattering) for low-resolution solution structures or cryo-EM for structure determination without crystallization.

How can molecular dynamics simulations complement experimental structural data for MnmC?

Molecular dynamics (MD) simulations offer valuable insights that complement experimental structural data for enzymes like MnmC:

• Conformational dynamics: MD simulations can reveal domain movements and conformational changes during substrate binding and catalysis that may not be captured in static crystal structures.

• Substrate binding pathway analysis: Simulations can elucidate how substrates navigate from solvent to active sites, particularly important for bifunctional enzymes where intermediates may transit between active sites.

• Water and ion networks: MD reveals functionally important water molecules and ion coordination sites that mediate substrate recognition or catalysis.

• Allostery and communication between domains: Simulations can identify allosteric pathways between the oxidoreductase and methyltransferase domains that regulate activity.

• Impact of mutations: In silico mutagenesis combined with MD can predict the effects of mutations on structure, stability, and function before experimental validation.

For optimal results, simulations should be performed with appropriate force fields for nucleic acid-protein complexes and include explicit consideration of cofactors using carefully parameterized force field extensions.

How does MnmC activity affect L. interrogans virulence gene expression?

MnmC's tRNA modification activity likely influences virulence gene expression through several mechanisms:

• Translational efficiency: mnm5s2U modification in the wobble position of tRNAs for Lys, Glu, and Gln affects codon recognition efficiency, potentially impacting the translation of virulence factors enriched in these codons.

• Stress response regulation: During host infection, L. interrogans faces various stressors that require rapid adaptation . tRNA modifications could modulate the translation of stress response proteins, including known virulence factors like LigA, LigB, and Loa22 .

• Environmental sensing: tRNA modification status may serve as a sensor for environmental conditions, similar to other bacteria where tRNA modification enzymes respond to nutrient availability and stress conditions.

Analysis of L. interrogans proteome during infection-mimicking conditions (iron limitation and serum exposure) has shown upregulation of several virulence factors , and the translational regulation of these factors may be influenced by MnmC activity. Codon usage analysis of upregulated virulence genes often reveals enrichment in codons that rely on mnm5s2U-modified tRNAs for optimal translation.

Could MnmC serve as a target for developing novel anti-Leptospira therapeutics?

MnmC presents several characteristics that make it a potentially attractive therapeutic target:

• Essential function: tRNA modifications are often critical for bacterial fitness, particularly under stress conditions encountered during infection.

• Structural distinctiveness: Bacterial MnmC enzymes have structural features distinct from human tRNA modification enzymes, potentially allowing for selective targeting.

• Bifunctional nature: The presence of two enzymatic activities in one protein provides multiple sites for inhibitor development.

Therapeutic development strategies might include:

While MnmC inhibition might not be directly bactericidal, it could attenuate virulence and enhance host clearance, similar to other "anti-virulence" approaches in development for bacterial pathogens.

How does MnmC contribute to L. interrogans survival under different host environmental conditions?

L. interrogans encounters diverse microenvironments during infection, and MnmC likely contributes to adaptation through fine-tuning translation:

• pH adaptation: L. interrogans displays neutralophilic properties, growing optimally at pH 7.4 . MnmC activity may be pH-sensitive, affecting translation of pH response genes during transit through different host tissues.

• Temperature response: As L. interrogans transitions from environmental temperatures to mammalian body temperature, temperature-dependent changes in MnmC activity could modulate the translation of temperature-responsive genes.

• Oxidative stress resistance: During macrophage encounters, L. interrogans must withstand oxidative attack . MnmC-mediated tRNA modifications may enhance translation of oxidative stress response proteins containing codons dependent on modified tRNAs.

• Iron limitation adaptation: In iron-limiting conditions, L. interrogans upregulates various proteins , and optimal translation of these proteins may depend on MnmC activity.

Experimental approaches combining tRNA modification profiling under different stress conditions with ribosome profiling can reveal how MnmC-dependent modifications influence the translatome in response to host environments.

How do you identify MnmC homologs across different Leptospira species and serovars?

Comprehensive identification of MnmC homologs requires a multi-faceted bioinformatic approach:

• Sequence-based identification:

  • PSI-BLAST searches using known MnmC sequences (including E. coli YfcK) as queries

  • HMMER searches with custom profiles built from validated MnmC sequences

  • Analysis of syntenic regions across Leptospira genomes

• Domain architecture verification:

  • Confirmation of both oxidoreductase and methyltransferase domains using InterProScan

  • Verification of key catalytic residues in both domains

  • Analysis of domain organization and linker regions

• Phylogenetic analysis:

  • Construction of maximum likelihood trees to distinguish true orthologs from paralogs

  • Comparison with 16S rRNA phylogeny to identify potential horizontal gene transfer events

  • Evaluation of evolutionary rates within pathogenic versus saprophytic Leptospira

This approach typically reveals MnmC conservation across pathogenic Leptospira species, with greater sequence divergence in saprophytic species, suggesting potential adaptation to pathogenic lifestyles.

What computational approaches can predict the impact of MnmC mutations on tRNA modification and bacterial fitness?

Advanced computational approaches can predict mutation impacts on MnmC function:

• Structural impact prediction:

  • Homology modeling combined with energy minimization

  • Molecular dynamics simulations to assess stability changes

  • Binding pocket analysis to identify critical substrate interaction residues

• Functional impact prediction:

  • Conservation analysis across homologs

  • Coevolutionary analysis to identify functionally coupled residues

  • Machine learning approaches integrating structural and evolutionary features

• Systems-level impact prediction:

  • Codon usage analysis to identify genes dependent on MnmC-modified tRNAs

  • Integration with transcriptome data to predict translational effects

  • Flux balance analysis to model metabolic consequences

These approaches can guide experimental mutagenesis by prioritizing residues likely to affect only one domain of the bifunctional protein or residues that might decouple the two activities, allowing for more precise determination of each activity's contribution to pathogenesis.

How can codon usage analysis integrate with tRNA modification data to predict genes affected by MnmC deficiency?

Integrative analysis of codon usage and tRNA modification can predict genes most affected by MnmC deficiency:

• Codon usage profiling:

  • Calculate codon adaptation index for all genes

  • Identify genes enriched in codons read by tRNAs modified by MnmC (particularly AAA, GAA, and CAA)

  • Cluster genes by codon usage patterns

• tRNA modification mapping:

  • Quantify mnm5s2U levels in tRNAs using LC-MS/MS

  • Compare modification levels across growth conditions

  • Correlate modification changes with stress responses

• Integrative analysis:

  • Identify genes with high dependency on MnmC-modified tRNAs

  • Correlate with proteomics data from wild-type vs. MnmC-deficient strains

  • Predict translational efficiency changes using ribosome profiling data

This approach typically identifies virulence factors, stress response proteins, and metabolic enzymes as particularly sensitive to MnmC activity, providing targets for experimental validation and insights into pathogenesis mechanisms.

How can CRISPR-Cas9 genome editing be optimized for studying MnmC function in L. interrogans?

CRISPR-Cas9 genome editing in L. interrogans requires careful optimization due to the challenging nature of leptospiral genetic manipulation:

• Delivery system optimization:

  • Electroporation parameters: 1.8-2.5 kV, 200-400 Ω, 25 μF

  • Temperature recovery: 30°C for 18-24 hours

  • Selection timing: gradual introduction of antibiotics after recovery period

• Guide RNA design considerations:

  • Target PAM-rich regions unique to mnmC

  • Validate specificity using whole-genome off-target prediction

  • Design guides targeting either the oxidoreductase or methyltransferase domain separately

  • Include controls targeting non-essential genes

• Repair template strategies:

  • Homology arms of 750-1000 bp for efficient recombination

  • Silent mutations in PAM or seed region to prevent re-cutting

  • Strategic placement of selection markers

  • Consider domain-specific knockouts versus full gene deletion

• Verification approaches:

  • PCR and sequencing of modification site

  • RT-qPCR for transcript analysis

  • Western blotting with domain-specific antibodies

  • tRNA modification analysis by mass spectrometry

This approach has been successfully used for gene inactivation in L. interrogans as demonstrated by previous studies using transposon-based approaches for other genes .

What proteomics approaches can reveal the global impact of MnmC deficiency on L. interrogans?

Comprehensive proteomics approaches can elucidate the global impact of MnmC deficiency:

• Quantitative proteomics:

  • iTRAQ or TMT labeling for multiplexed comparison

  • SILAC labeling for in vivo incorporation

  • Label-free quantification with high-resolution mass spectrometry

• Targeted analysis of translation fidelity:

  • Ribosome profiling to identify translation pausing

  • Pulse labeling with azide-containing amino acids

  • Mass spectrometry detection of mistranslation events

• Post-translational modification analysis:

  • Phosphoproteomics to detect stress response activation

  • Glycoproteomics to assess cell surface modifications

  • Redox proteomics to evaluate oxidative stress effects

• Protein-protein interaction changes:

  • Proximity labeling (BioID, APEX) of MnmC interactors

  • Crosslinking mass spectrometry

  • Co-immunoprecipitation combined with quantitative proteomics

This multi-faceted approach would reveal not only which proteins change in abundance but also provide mechanistic insights into how MnmC deficiency affects translational fidelity, stress responses, and protein function regulation.

How can single-molecule techniques advance our understanding of MnmC catalytic mechanism?

Single-molecule techniques offer unique insights into MnmC's catalytic mechanisms:

• Single-molecule FRET (smFRET):

  • Label MnmC domains with appropriate FRET pairs

  • Monitor domain movements during substrate binding and catalysis

  • Detect potential conformational changes between oxidation and methylation steps

  • Observe real-time kinetics without population averaging

• Optical tweezers combined with fluorescence:

  • Manipulate tRNA substrates while monitoring MnmC binding

  • Measure force-dependent binding and dissociation

  • Detect conformational changes in both enzyme and substrate

• Single-molecule enzymology:

  • Zero-mode waveguides to observe sequential catalytic events

  • Microfluidic approaches to detect product formation

  • Correlation of conformational dynamics with catalytic events

• High-speed AFM:

  • Visualize MnmC-tRNA complexes in real-time

  • Observe structural changes during catalysis

  • Map interaction sites through topographical analysis

Does MnmC elicit an immune response during Leptospira infection, and could it serve as a diagnostic marker?

Similar to other leptospiral proteins like LIC_10559 , MnmC may elicit an immune response during infection. To evaluate its immunoreactivity:

• Serological testing:

  • Western blot analysis using recombinant MnmC against sera from infected animals

  • ELISA assays with purified MnmC to quantify antibody responses

  • Comparison with established serodiagnostic antigens like LipL32

• Epitope mapping:

  • Peptide arrays to identify immunodominant regions

  • Phage display to isolate antibody-binding peptides

  • Computational prediction of B-cell epitopes

• Diagnostic potential assessment:

  • Sensitivity and specificity determination across different infection stages

  • Cross-reactivity testing with sera from other infectious diseases

  • Comparison with microscopic agglutination test (MAT) results

If MnmC proves immunogenic, it could be incorporated into multi-epitope diagnostic proteins similar to the recombinant leptospirosis multiepitope protein (r-LMP) , which has shown promise in detecting anti-leptospire antibodies without cross-reactions with other patient sera.

What is the most effective protocol for producing MnmC-specific antibodies for research applications?

Production of high-quality MnmC-specific antibodies requires careful antigen design and immunization protocols:

• Antigen design strategies:

  • Full-length MnmC for comprehensive antibody repertoire

  • Domain-specific constructs for distinguishing activities

  • Synthetic peptides from predicted surface-exposed regions

  • Consideration of species-specific vs. conserved epitopes

• Immunization protocol optimization:

  Parameter	Recommendation
  Animal model	Rabbits for polyclonal; mice for monoclonal
  Adjuvant	Freund's complete for primary; incomplete for boosters
  Dose	50-200 μg per immunization
  Schedule	Primary + 3 boosters at 2-week intervals
  Route	Subcutaneous for polyclonal; intraperitoneal for monoclonal

• Antibody purification and validation:

  • Affinity purification against immobilized antigen

  • Specificity testing via Western blot against recombinant protein and cellular extracts

  • Cross-reactivity assessment with related bacterial proteins

  • Functional validation through immunoprecipitation of active enzyme

Domain-specific antibodies are particularly valuable for studying MnmC, as they allow independent tracking of the oxidoreductase and methyltransferase domains and can be used to assess potential conformational changes during catalysis.

How can immunoproteomics approaches identify MnmC interactions with the host immune system during infection?

Immunoproteomics approaches provide comprehensive insights into how MnmC interacts with the host immune system:

• Serological proteome analysis (SERPA):

  • 2D gel separation of leptospiral proteins

  • Western blotting with sera from infected animals

  • Mass spectrometry identification of immunoreactive spots

  • Comparison across infection stages and host species

• Immunopeptidome analysis:

  • Isolation of MHC-bound peptides from infected host cells

  • LC-MS/MS identification of presented peptides

  • Mapping peptides to MnmC sequence

  • Correlation with predicted T-cell epitopes

• B-cell receptor (BCR) repertoire sequencing:

  • Next-generation sequencing of BCR from infected hosts

  • Identification of clonally expanded B-cell populations

  • Recombinant expression of disease-specific antibodies

  • Testing specificity against MnmC epitopes

• Cytokine profiling in response to MnmC:

  • Stimulation of host immune cells with recombinant MnmC

  • Multiplex cytokine assays to determine response profile

  • Comparison with other leptospiral immunogens

  • Correlation with pathology markers