Recombinant Tropheryma whipplei 50S ribosomal protein L5 (rplE)

What is Tropheryma whipplei and why is its ribosomal protein L5 of research interest?

Tropheryma whipplei is the causative agent of Whipple's disease, a rare systemic infection. It is a fastidious gram-positive bacterium measuring approximately 0.25μm, with a distinctive trilaminar cell membrane visible under electron microscopy . The organism is found in environmental samples including soil and sewerage, suggesting it is ubiquitous in nature . The 50S ribosomal protein L5 (rplE) is of particular interest because ribosomal proteins play critical roles in protein synthesis and can have moonlighting functions in bacterial pathogenesis. Additionally, understanding T. whipplei rplE may provide insights into the organism's unique survival mechanisms, as this bacterium employs sophisticated methods to evade host immune responses by inhibiting phago-lysosome biogenesis in macrophages .

How does T. whipplei rplE structure compare to ribosomal L5 proteins in other bacterial species?

While specific structural information about T. whipplei rplE is limited in current literature, comparative genomic analyses suggest it shares homology with other bacterial L5 proteins. In eukaryotes like Xenopus laevis, Rpl5 forms part of the 60S ribosomal subunit and, together with Rpl11 and 5S rRNA, creates the 5S-ribonucleoprotein (RNP) complex . By analogy, T. whipplei rplE likely participates in similar structural arrangements within the bacterial ribosome.

To conduct comparative analysis of T. whipplei rplE with other bacterial species, researchers should:

• Perform multiple sequence alignments using CLUSTAL W or similar tools

• Generate phylogenetic trees to visualize evolutionary relationships

• Employ homology modeling using crystallographic data from related bacterial species as templates

• Analyze conserved domains and binding motifs through tools like PROSITE or InterPro

What genes are adjacent to rplE in the T. whipplei genome, and what does this synteny reveal?

To conduct a thorough synteny analysis of T. whipplei rplE:

• Retrieve the complete genome sequence from databases like NCBI

• Identify the position of rplE and flanking genes

• Compare the syntenic organization with other bacterial species using tools like SyntTax or MicrobesOnline

• Assess whether gene order is conserved or rearranged compared to related bacterial species

What expression systems are most effective for producing recombinant T. whipplei rplE?

Based on approaches used for similar bacterial ribosomal proteins, several expression systems can be considered for T. whipplei rplE:

When designing expression constructs, researchers should consider:

• Adding affinity tags (His6, GST, MBP) to facilitate purification

• Including protease cleavage sites for tag removal

• Optimizing codon usage for the expression host

• Incorporating solubility-enhancing fusion partners if aggregation occurs

What are the primary challenges in purifying recombinant T. whipplei rplE and how can they be addressed?

Purification of recombinant ribosomal proteins presents several challenges:

• Solubility issues: Ribosomal proteins often form inclusion bodies due to improper folding or aggregation. This can be addressed by:

  • Expressing the protein at lower temperatures (16-20°C)

  • Using solubility-enhancing fusion tags like MBP or SUMO

  • Adding detergents or stabilizing agents to buffers

  • Employing refolding protocols if inclusion bodies form

• RNA contamination: Ribosomal proteins naturally bind RNA, which can co-purify with the protein. Solutions include:

  • Including high salt concentrations (0.5-1M NaCl) in buffers

  • Adding RNase treatment steps

  • Incorporating ion exchange chromatography to separate nucleic acids

• Proteolytic degradation: Prevent by:

  • Adding protease inhibitors to all buffers

  • Maintaining samples at 4°C throughout purification

  • Minimizing purification time with optimized protocols

• Maintaining native conformation: Preserve by:

  • Carefully selecting buffer conditions (pH, ionic strength)

  • Including stabilizing agents like glycerol or specific divalent cations

  • Using mild elution conditions in affinity chromatography

How can researchers validate the structural integrity of purified recombinant T. whipplei rplE?

To ensure the recombinant T. whipplei rplE maintains its structural integrity after purification, researchers should employ multiple complementary techniques:

• Circular Dichroism (CD) Spectroscopy: Measures secondary structure content (α-helices, β-sheets) to confirm proper folding

• Size Exclusion Chromatography (SEC): Assesses whether the protein exists in monomeric form or forms aggregates

• Thermal Shift Assays: Evaluates protein stability under different buffer conditions

• Limited Proteolysis: Probes the accessibility of cleavage sites to verify proper folding

• Functional Assays: Tests the ability to bind known interacting partners (e.g., 5S rRNA, other ribosomal proteins)

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For detailed structural analysis of properly folded protein

• Mass Spectrometry: Confirms protein identity and detects any post-translational modifications

What methods are most suitable for studying interactions between T. whipplei rplE and other ribosomal components?

Several complementary approaches can be used to characterize the interactions of T. whipplei rplE with other ribosomal components:

• Electrophoretic Mobility Shift Assays (EMSA): To detect binding with 5S rRNA and determine binding affinity

• Surface Plasmon Resonance (SPR): For real-time, label-free measurement of binding kinetics with other ribosomal proteins or RNA

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of binding interactions

• Fluorescence Anisotropy: For measuring binding to fluorescently labeled RNA or protein partners

• Chemical Cross-linking coupled with Mass Spectrometry (XL-MS): To map interaction interfaces within the ribosomal complex

• Cryo-Electron Microscopy: For structural visualization of rplE within the assembled ribosome

• Pull-down Assays: Using tagged recombinant rplE to identify interacting partners from T. whipplei lysates

A systematic approach combining these methods would provide comprehensive insights into how T. whipplei rplE contributes to ribosome assembly and function.

Could T. whipplei rplE have moonlighting functions beyond protein synthesis?

While the primary function of ribosomal protein L5 is in protein synthesis, numerous ribosomal proteins have been found to have secondary "moonlighting" functions, particularly in pathogens. Based on information from related systems, T. whipplei rplE might potentially:

• Participate in host-pathogen interactions: Similar to how T. whipplei inhibits phago-lysosome biogenesis through its GAPDH homolog , rplE could potentially interact with host factors when released from damaged bacterial cells.

• Regulate gene expression: In eukaryotes, Rpl5 regulates gene expression through interactions with transcription factors like c-Myc . Bacterial rplE might have similar regulatory functions.

• Contribute to stress responses: Under stress conditions, ribosomal proteins can dissociate from ribosomes and participate in alternative cellular processes.

To investigate potential moonlighting functions, researchers should:

• Perform yeast two-hybrid or bacterial two-hybrid screens to identify non-ribosomal interaction partners

• Conduct pull-down experiments using recombinant rplE as bait with host cell lysates

• Test rplE's effect on cellular processes in cell culture models

• Examine phenotypic effects of rplE overexpression or deletion mutants

How might T. whipplei rplE contribute to the organism's unique phagosome maturation block?

T. whipplei is notable for creating a "chimeric" phagosome that stably expresses both Rab5 and Rab7, representing a novel mechanism for subverting phagosome maturation . While the search results indicate that a GAPDH homolog may be responsible for blocking Rab5 activity, it's possible that ribosomal proteins like rplE could also play roles in this process.

To investigate potential contributions of rplE to phagosome maturation block:

• Localization studies: Perform immunofluorescence microscopy to determine if rplE localizes to the phagosomal membrane during infection

• Protein-protein interaction studies: Investigate whether rplE interacts with host factors involved in phagosome maturation (particularly Rab5, Rab7, or their effectors)

• Complementation assays: Express recombinant T. whipplei rplE in macrophages and assess effects on phagosome maturation

• Comparative studies: Compare the effects of rplE from T. whipplei with rplE from bacteria that do not block phagosome maturation

• Mutational analysis: Identify critical domains in rplE that might mediate interactions with host factors

How can recombinant T. whipplei rplE be used to develop improved diagnostic tools for Whipple's disease?

Whipple's disease is challenging to diagnose due to its rarity and diverse clinical manifestations. Recombinant T. whipplei rplE could potentially improve diagnostic approaches through:

• Serological assays: Developing ELISA or Western blot assays using recombinant rplE to detect anti-T. whipplei antibodies in patient sera

• Antigen detection: Creating immunoassays to detect rplE in patient samples

• PCR enhancement: Using knowledge of rplE sequence conservation to design improved PCR primers for detection

• Imaging probes: Developing labeled anti-rplE antibodies for immunohistochemistry

To evaluate the diagnostic utility of recombinant rplE, researchers should:

• Determine the conservation of rplE sequence across T. whipplei isolates

• Assess cross-reactivity with related bacterial species

• Compare sensitivity and specificity with existing diagnostic methods

• Evaluate performance in different sample types (blood, tissue, cerebrospinal fluid)

What research approaches could determine if T. whipplei rplE is a viable target for antimicrobial development?

Evaluating T. whipplei rplE as a potential antimicrobial target would involve:

• Essentiality assessment: Determine if rplE is essential for T. whipplei survival, possibly through:

  • Antisense RNA approaches

  • CRISPR interference if applicable to T. whipplei

  • Conditional expression systems

• Structural uniqueness analysis: Compare T. whipplei rplE structure with human ribosomal proteins to identify unique features that could be targeted

• Small molecule screening:

  • Develop in vitro binding assays for high-throughput screening

  • Test compounds that disrupt rplE interactions with rRNA or other ribosomal proteins

  • Evaluate effects on T. whipplei growth in cell culture models

• Peptide inhibitor design:

  • Identify interface regions in protein-protein or protein-RNA interactions

  • Design peptides that mimic these interfaces to disrupt essential interactions

  • Test inhibitory effects on T. whipplei ribosome assembly

• Drug repurposing approaches:

  • Test known antibiotics that target bacterial protein synthesis for specific activity against T. whipplei

  • Investigate whether structural differences in T. whipplei rplE could be exploited for selective targeting

How does the role of T. whipplei rplE in bacterial survival compare to the role of Rpl5 in ribosomopathies?

This question bridges bacterial and eukaryotic ribosomal biology, comparing T. whipplei rplE with eukaryotic Rpl5, which is implicated in Diamond-Blackfan anemia (DBA) when mutated .

In eukaryotes, Rpl5 functions extend beyond protein synthesis:

• Forms part of the 5S RNP complex with Rpl11 and 5S rRNA

• Regulates Tp53 by binding to MDM2, preventing Tp53 degradation and promoting apoptosis

• Interacts with c-Myc in signaling pathways

• Mutations lead to tissue-specific effects despite ribosomes' ubiquitous necessity

Comparative analysis could reveal:

• Whether T. whipplei rplE has similar regulatory functions in bacterial stress responses

• If rplE mutations affect bacterial fitness or virulence

• Potential convergent or divergent evolution of ribosomal protein functions

Research approaches should include:

• Comparative structural analysis of bacterial rplE and eukaryotic Rpl5

• Functional complementation studies to test cross-kingdom conservation

• Analysis of T. whipplei rplE effects on growth, stress responses, and virulence

• Investigation of whether T. whipplei rplE interacts with bacterial transcription factors

What are the most effective methods for studying T. whipplei rplE's role in bacterial ribosomes using cryo-EM?

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of ribosome structure. To study T. whipplei rplE using this technique:

• Ribosome isolation protocol:

  • Culture T. whipplei in specialized media or infected cell lines

  • Lyse cells under gentle conditions to preserve ribosome integrity

  • Purify ribosomes through sucrose gradient ultracentrifugation

  • Remove contaminants while maintaining ribosomal subunit associations

• Sample preparation optimization:

  • Test different grid types (Quantifoil, C-flat) for optimal particle distribution

  • Optimize freezing conditions to prevent preferred orientation

  • Consider using detergents or additives to improve particle contrast

• Data collection strategy:

  • Collect multiple tilt series to address preferred orientation issues

  • Use energy filters to improve signal-to-noise ratio

  • Implement beam-tilt correction for high-resolution data

• Processing workflow:

  • Classify particles to identify different conformational states

  • Use focused refinement on the L5 region for highest resolution

  • Employ Bayesian polishing and CTF refinement for resolution improvement

• Validation approaches:

  • Generate split dataset reconstructions to confirm structural features

  • Use model-map FSC curves to validate atomic models

  • Perform cross-validation with biochemical data

How can researchers design a targeted mutagenesis study of T. whipplei rplE to identify critical functional domains?

A systematic mutagenesis approach to elucidate rplE function would include:

• Sequence and structure-based mutation design:

  • Align T. whipplei rplE with homologs to identify conserved residues

  • Use homology models to predict functional domains involved in:

    • 5S rRNA binding

    • Interactions with adjacent ribosomal proteins

    • Potential contacts with translation factors

  • Design alanine scanning mutants across predicted functional surfaces

  • Create chimeric proteins with rplE domains from other bacteria

• Expression and functional validation:

  • Express wild-type and mutant proteins in parallel

  • Confirm proper folding using circular dichroism and thermal shift assays

  • Test RNA binding capabilities using EMSAs or filter binding assays

  • Evaluate protein-protein interactions with pull-down experiments

• In vitro translation assays:

  • Reconstitute ribosomes with mutant rplE proteins

  • Measure translation efficiency and fidelity

  • Assess effects on specific steps (initiation, elongation, termination)

• Structural validation:

  • Determine structures of key mutants by X-ray crystallography or cryo-EM

  • Compare with wild-type structures to identify conformational changes

What computational approaches can predict interactions between T. whipplei rplE and host immunity factors?

Several computational methods can be used to predict potential interactions between T. whipplei rplE and host immunity components:

• Protein-protein docking:

  • Generate homology models of T. whipplei rplE

  • Collect structures of candidate host immunity factors (TLRs, NLRs, etc.)

  • Perform rigid and flexible docking using software like HADDOCK, ClusPro, or Rosetta

  • Score and rank potential binding modes

• Molecular dynamics simulations:

  • Validate stability of predicted complexes in explicit solvent

  • Calculate binding free energies using methods like MM/PBSA

  • Identify key residues through contact analysis and free energy decomposition

• Epitope prediction:

  • Use algorithms like BepiPred, DiscoTope, or EPSVR to predict B-cell epitopes

  • Employ NetMHC or similar tools to predict T-cell epitopes

  • Create epitope maps to guide experimental validation

• Network analysis:

  • Construct protein-protein interaction networks

  • Identify potential immune signaling pathways affected by rplE

  • Predict functional consequences using pathway enrichment analysis

These predictions should be validated experimentally through:

• Pull-down assays with recombinant rplE and host cell lysates

• Surface plasmon resonance with predicted interacting partners

• Cell-based reporter assays to detect immune activation

• Immunoprecipitation studies from infected cell models

How might T. whipplei rplE contribute to the bacteria's unusual survival mechanisms in host cells?

T. whipplei employs sophisticated mechanisms to survive within host cells, including the creation of a "chimeric" phagosome that expresses both Rab5 and Rab7 . While specific involvement of rplE in these processes is not established in the search results, potential roles could be explored based on known bacterial adaptation strategies:

• Moonlighting outside the ribosome: rplE might be released or presented on the bacterial surface during infection, similar to how T. whipplei GAPDH appears to block Rab5 activity

• Modulation of host translation: If released into the host cytosol, bacterial ribosomal proteins might interfere with host translation machinery

• Immune evasion: rplE could potentially bind to host immune receptors or signaling molecules, modulating inflammatory responses

Research strategies to investigate these possibilities include:

• Immunofluorescence microscopy to track rplE localization during infection

• Proteomics analysis of T. whipplei phagosomes to detect bacterial proteins

• Yeast two-hybrid or pull-down assays to identify host interaction partners

• Expression of recombinant rplE in host cells to assess phenotypic effects

What role might post-translational modifications of T. whipplei rplE play in bacterial adaptation to host environments?

Post-translational modifications (PTMs) of bacterial ribosomal proteins are increasingly recognized as important regulatory mechanisms. For T. whipplei rplE, potential PTMs could include:

• Phosphorylation: May regulate binding to rRNA or other ribosomal proteins

• Methylation: Could affect RNA binding specificity

• Acetylation: Might regulate protein-protein interactions

• ADP-ribosylation: Potentially modulates translation efficiency

To investigate PTMs of T. whipplei rplE:

• Mass spectrometry approaches:

  • Purify ribosomes from T. whipplei grown under different conditions

  • Perform bottom-up proteomics with enrichment for specific PTMs

  • Use top-down MS to obtain intact protein mass profiles

  • Apply targeted MS/MS for detailed PTM mapping

• Functional studies:

  • Generate site-directed mutants that mimic or prevent specific PTMs

  • Compare activity of modified and unmodified forms

  • Assess PTM patterns in different growth conditions or infection stages

• Enzyme identification:

  • Search for PTM-adding enzymes in the T. whipplei genome

  • Test their activity on recombinant rplE

  • Investigate regulation of these enzymes during infection

How can systems biology approaches integrate T. whipplei rplE research into broader understanding of host-pathogen interactions?

Systems biology offers powerful frameworks to contextualize the role of specific proteins like rplE within the broader host-pathogen interaction network:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from T. whipplei infection models

  • Map changes in rplE expression/modification to specific infection stages

  • Correlate rplE dynamics with global bacterial and host responses

• Network analysis:

  • Construct protein-protein interaction networks centered on rplE

  • Identify potential regulatory connections to virulence pathways

  • Map rplE connections to host defense mechanisms

• Mathematical modeling:

  • Develop ordinary differential equation models of T. whipplei growth and survival

  • Incorporate rplE functions in translation efficiency

  • Simulate effects of targeting rplE on bacterial fitness

• Comparative genomics and evolution:

  • Compare rplE sequences across T. whipplei isolates from different clinical sources

  • Analyze selection pressure on rplE compared to other ribosomal proteins

  • Identify potential host-adaptation signatures in the rplE sequence

• Machine learning applications:

  • Use sequence-based predictions to identify functional domains in rplE

  • Apply unsupervised learning to cluster T. whipplei proteins by expression pattern

  • Develop predictive models for bacterial survival based on ribosomal protein dynamics