Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Ribosome maturation factor RimP (rimP)

Protein Expression System Optimization

These hypothetical results suggest that while E. coli SHuffle enhances solubility, trade-offs in yield must be addressed through iterative optimization .

Functional Validation in Ribosome Assembly

Assessing RimP’s role in ribosome maturation necessitates ribosome profiling and co-sedimentation assays. A protocol validated for LIC11505 binding to integrins can be adapted:

• Isolate 30S and 50S ribosomal subunits from L. interrogans cultures under low-Mg²⁺ conditions.

• Incubate recombinant RimP with subunits at varying molar ratios (1:1 to 1:10).

• Analyze binding kinetics via surface plasmon resonance (SPR), using a Biacore T200 system with immobilized ribosomal RNA.

• Validate assembly efficiency by quantifying 70S formation via sucrose density gradients.

Discrepancies between in vitro binding data and in vivo ribosome assembly phenotypes often arise from missing chaperones or post-translational modifications. Parallel experiments in Leptospira knockout strains are essential for resolving such contradictions .

Structural Determinants of Ribosome Interaction

Cryo-EM and X-ray crystallography are pivotal for mapping RimP’s binding interfaces. For LRR-proteins like LIC11505, structural analyses revealed leucine-rich repeats critical for host ligand binding . Applying similar approaches to RimP:

• Generate truncation mutants targeting predicted RNA-binding domains (e.g., KH or OB folds).

• Perform crosslinking-MS to identify proximal ribosomal proteins (e.g., S12, S17).

Preliminary data might show disrupted 30S subunit assembly in ΔrimP strains, with compensatory upregulation of other assembly factors like RbfA or Era.

Interaction With Host Pathways

Pathogenic Leptospira proteins often exhibit dual roles in bacterial physiology and host interaction. For example, LIC11505 binds integrins and glycosaminoglycans (GAGs) to facilitate host cell adhesion . To test if RimP influences host responses:

• Treat human endothelial cells with recombinant RimP (0–10 µg/mL) for 24 hr.

• Quantify inflammatory markers (IL-6, TNF-α) via ELISA.

• Assess ribosomal stress via phosphorylation of RPS6 (Ser235/236).

Unexpected cytokine induction would suggest moonlighting functions unrelated to ribosome biogenesis, requiring orthogonal validation (e.g., RNAi knockdown in host cells).

Discrepancies Between In Vitro and In Vivo Activity

A common issue arises when recombinant RimP shows robust ribosome-binding activity in vitro but fails to complement ΔrimP strains. Potential solutions include:

• Check for protein misfolding via circular dichroism (CD) spectroscopy.

• Test activity in minimal media mimicking Leptospira’s intracellular niche (e.g., low iron, high osmolarity).

• Evaluate post-translational modifications via phosphoproteomics or acetylome analysis.

For instance, LIC11051 exhibits differential binding to laminin under varying pH conditions , highlighting the need for physiologically relevant assay parameters.

Conflicting Ligand Binding Profiles

If RimP shows inconsistent interactions with ribosomal subunits across studies, consider:

• Standardize buffer conditions (e.g., Mg²⁺ concentration, redox state).

• Use crosslinkers like DSS to stabilize transient interactions.

• Compare wild-type and mutant Ribosome profiles via RNA-seq to identify misassembled rRNA regions.

Risk Assessment in Pathogen Handling

The Stanford Laboratory Risk Assessment Tool provides a framework for experiments involving recombinant Leptospira proteins:

• Explore: Review biosafety level (BSL) requirements for L. interrogans (BSL-2).

• Plan: Implement aerosol-containment measures during protein purification.

• Challenge: Simulate accidental exposure scenarios (e.g., centrifuge rotor failure).

• Assess: Validate decontamination protocols using surrogate markers (e.g., phage ΦX174).

Data Reproducibility Strategies

• Pre-register experimental protocols detailing buffer compositions and equipment settings.

• Include internal controls (e.g., commercial ribosome assembly kits) in every assay batch.

• Share raw datasets (e.g., SPR sensorgrams, density gradient profiles) via repositories like Zenodo.

Targeted Mutagenesis for Vaccine Development

Attenuated Leptospira strains with rimP deletions could serve as live vaccines. Key steps:

• Design allelic exchange vectors with rimP flanking regions and antibiotic markers.

• Electroporate into L. interrogans using parameters optimized for high-efficiency transformation.

• Verify attenuation via hamster challenge models, comparing histopathology and bacterial loads.

High-Throughput Screening Assays

Develop a fluorescence-polarization assay to identify RimP inhibitors:

• Label 16S rRNA fragments with Cy3.

• Measure anisotropy changes upon RimP-inhibitor binding.

• Counter-screen against human ribosome factors to ensure specificity.