Recombinant Treponema denticola 50S ribosomal protein L5 (rplE)

What is the role of ribosomal protein L5 in Treponema denticola?

Ribosomal protein L5 in T. denticola, like in other bacteria such as E. coli, plays a crucial role in the assembly of the large 50S ribosomal subunit. It is specifically involved in the formation of the central protuberance (CP) of the ribosome. The absence of L5 leads to the accumulation of defective 45S particles that lack several CP components and are unable to associate with the small ribosomal subunit, ultimately impairing protein synthesis . In the context of T. denticola, an oral spirochete associated with periodontal disease, proper ribosomal assembly is essential for bacterial survival and virulence expression.

What is the relationship between rplE expression and T. denticola virulence?

While the search results do not directly address the relationship between rplE and T. denticola virulence, we can infer connections based on the fundamental role of ribosomes in protein synthesis. As a component essential for ribosomal assembly and function, rplE indirectly affects the expression of virulence factors such as the major surface protein (Msp) and dentilisin protease complex, which are known contributors to T. denticola pathogenicity . The proper expression of these virulence factors depends on functional ribosomes for their translation. Additionally, like other bacteria, T. denticola likely regulates ribosomal protein expression in response to environmental conditions encountered during infection.

What are the optimal expression systems for producing recombinant T. denticola rplE?

For recombinant expression of T. denticola proteins, including rplE, several expression systems can be employed based on the research requirements:

• E. coli expression systems: These represent the most commonly used approach for bacterial protein expression. For T. denticola rplE, vectors such as pET series with T7 promoters in E. coli BL21(DE3) strains often yield good results for ribosomal proteins. The methodology similar to that used for Msp protein fragments could be adapted .

• Cell-free expression systems: These can be particularly useful for proteins that may affect host cell ribosomal function.

• Native expression: For certain studies requiring authentic post-translational modifications, expression in T. denticola itself may be necessary, though technically more challenging.

The choice depends on research goals, required protein yields, and downstream applications. Optimization of expression conditions (temperature, induction time, media composition) is essential for obtaining soluble, correctly folded protein.

What purification strategy yields the highest purity and activity for recombinant T. denticola rplE?

A multi-step purification strategy is recommended for obtaining high-purity, functionally active recombinant T. denticola rplE:

• Affinity chromatography: Using His-tag or GST-tag systems for initial capture.

• Ion exchange chromatography: To separate based on charge differences.

• Size exclusion chromatography: For final polishing and buffer exchange.

For ribosomal proteins like L5 that interact with RNA, care must be taken to remove nucleic acid contamination, potentially using high-salt washes or nuclease treatments. Based on methods used for other T. denticola proteins, the purification protocol might include sequential extraction steps, ultracentrifugation, and extensive washing . Protein activity should be verified through RNA binding assays and/or ribosome assembly assays.

How can the solubility of recombinant T. denticola rplE be improved during expression?

Improving solubility of recombinant T. denticola rplE may require several strategies:

• Expression temperature optimization: Lower temperatures (16-25°C) often increase solubility by slowing protein folding.

• Codon optimization: Adapting the rplE sequence to the codon usage bias of the expression host.

• Fusion tags: Adding solubility-enhancing tags such as MBP, SUMO, or Thioredoxin.

• Buffer optimization: Including stabilizing additives like glycerol, specific salts, or mild detergents.

• Co-expression with chaperones: GroEL/GroES or DnaK systems can assist proper folding.

For ribosomal proteins specifically, co-expression with their binding partners (e.g., 5S rRNA) or expression as defined fragments may enhance solubility by providing natural stabilization.

What techniques are most effective for studying T. denticola rplE interactions with 5S rRNA?

Several complementary techniques are recommended for studying T. denticola rplE interactions with 5S rRNA:

• Electrophoretic Mobility Shift Assays (EMSA): To detect rplE-5S rRNA complex formation.

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics and affinity measurements.

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

• Fluorescence techniques: Including FRET to monitor conformational changes upon binding.

• Structural biology approaches: X-ray crystallography, NMR, or cryo-EM for high-resolution structural characterization.

Data from E. coli suggest that L5 forms a specific complex with 5S rRNA in the cytoplasm before incorporation into the ribosome , and similar studies in T. denticola would help understand species-specific aspects of this interaction.

How does the T. denticola ribosomal assembly process differ from that of model organisms like E. coli?

While specific differences between T. denticola and E. coli ribosomal assembly are not directly addressed in the search results, several potential differences may exist:

• Assembly factors: T. denticola may utilize different auxiliary factors or chaperones during ribosome assembly.

• Environmental adaptations: As an oral spirochete, T. denticola may have evolved assembly processes adapted to its unique ecological niche.

• Temporal regulation: The order and timing of component incorporation could differ.

• Response to stress: Assembly pathways under stress conditions relevant to periodontal disease may be specialized.

Research in E. coli has shown that L5 plays a key role in CP formation, with its absence leading to accumulation of defective 45S particles lacking multiple CP components . Comparative studies would be needed to determine if this critical role is conserved in T. denticola and if there are species-specific nuances in the assembly process.

How can researchers generate conditional knockdown models of rplE in T. denticola to study its function?

Creating conditional knockdown models of essential genes like rplE in T. denticola requires sophisticated genetic approaches:

• Inducible antisense RNA systems: Expressing antisense RNA complementary to rplE mRNA under an inducible promoter to regulate expression levels.

• CRISPR interference (CRISPRi): Using catalytically inactive Cas9 (dCas9) to block transcription of rplE without cleaving DNA.

• Riboswitch-based regulation: Incorporating engineered riboswitches in the 5' UTR of rplE to control translation in response to specific ligands.

• Tetracycline-inducible systems: Adapting Tet-On/Tet-Off systems for T. denticola.

Methods for genetic manipulation in T. denticola have been established for other genes, including targeted mutagenesis approaches using selectable markers like ermB or aphA2 . Similar methodologies could be adapted for rplE, though careful design is needed given its likely essential nature.

What is the impact of rplE mutations on antibiotic resistance in T. denticola?

Mutations in ribosomal proteins, including L5, can potentially affect antibiotic susceptibility in bacteria:

• Macrolide resistance: L5 mutations might alter binding sites for macrolides that target the 50S subunit.

• Pleuromutilin resistance: Changes in L5 could affect the binding pocket for these antibiotics.

• Cross-resistance patterns: Mutations may confer resistance to multiple antibiotic classes that target overlapping ribosomal regions.

While specific data on T. denticola rplE mutations and antibiotic resistance are not provided in the search results, understanding such relationships would be valuable for both basic science and clinical considerations in treating periodontal infections.

How does rplE contribute to stress responses in T. denticola during infection?

Ribosomal proteins, including L5, likely play important roles in T. denticola's adaptation to stress conditions encountered during infection:

• Translational regulation: rplE may contribute to selective translation of stress-response genes.

• Extraribosomal functions: Beyond its structural role in ribosomes, L5 might participate in stress signaling networks.

• Ribosome heterogeneity: Stress conditions might alter the composition of ribosomes, including L5 incorporation.

• Coordination with virulence expression: Stress responses and virulence factor expression appear to be interlinked in T. denticola, as suggested by studies on dentilisin and Msp .

Research approaches to study these aspects could include transcriptomics and proteomics under various stress conditions, examination of protein-protein interactions outside the ribosome, and functional studies of rplE under conditions mimicking the periodontal pocket environment.

How can recombinant T. denticola rplE be used to study ribosomal assembly in vitro?

Recombinant T. denticola rplE provides valuable opportunities for in vitro studies of ribosomal assembly:

• Reconstitution experiments: Using purified components to reconstruct the central protuberance and study assembly hierarchies.

• Time-resolved structural studies: Analyzing intermediates in CP formation using cryo-EM or other structural approaches.

• Single-molecule techniques: Real-time observation of rplE incorporation using fluorescently labeled components.

• Assembly defect complementation: Testing whether recombinant rplE can rescue assembly defects in depleted systems.

Based on E. coli studies, L5 appears to first form a complex with 5S rRNA and certain proteins (like L18 and L25) in the cytoplasm before incorporation into the nascent large subunit . Similar experiments with T. denticola components would reveal conservation or divergence in assembly pathways.

What techniques can be used to study the interplay between rplE and virulence factor expression in T. denticola?

Several sophisticated techniques can elucidate connections between rplE and virulence factor expression:

• Ribosome profiling: To examine translation efficiency of virulence genes under various conditions affecting rplE.

• RIP-Seq (RNA immunoprecipitation sequencing): To identify mRNAs associated with ribosomes containing particular rplE variants.

• Protein-protein interaction networks: Using proximity labeling approaches to identify non-ribosomal interaction partners of rplE.

• Conditional expression systems: To analyze effects of rplE depletion on virulence factor expression.

The search results indicate that expression of T. denticola virulence factors like Msp and dentilisin is regulated by complex networks involving transcriptional regulators such as TDE_0127 . Understanding how ribosomal proteins integrate into these regulatory networks would provide novel insights into pathogen biology.

How does post-translational modification of rplE affect its function in T. denticola?

Post-translational modifications (PTMs) of ribosomal proteins can significantly impact their function:

• Identification of PTMs: Mass spectrometry-based approaches to map modifications like methylation, acetylation, or phosphorylation on T. denticola rplE.

• Functional significance: Site-directed mutagenesis of modified residues to assess impact on ribosome assembly and function.

• Regulatory aspects: Examining how environmental conditions affect the PTM profile of rplE.

• Comparative analysis: Contrasting PTM patterns between T. denticola and other bacteria to identify unique features.

While specific information on T. denticola rplE modifications is not provided in the search results, research on other bacterial species suggests that ribosomal protein modifications can fine-tune translation and may represent adaptations to specific environmental niches.

How has rplE evolved across Treponema species and what implications does this have for pathogenicity?

Evolutionary analysis of rplE across Treponema species can provide insights into adaptation and pathogenicity:

• Sequence conservation analysis: Identifying highly conserved regions likely essential for core functions versus variable regions potentially related to species-specific adaptations.

• Selection pressure analysis: Detecting signatures of positive or purifying selection that might indicate functional constraints or adaptive evolution.

• Structural comparison: Examining how sequence differences translate to structural variation that might affect interactions with other ribosomal components.

• Horizontal gene transfer assessment: Evaluating evidence for lateral transfer events that might have contributed to pathogenic potential.

While T. denticola is associated with periodontal disease , other Treponema species cause different human diseases or are non-pathogenic. Comparative analysis of rplE across these species may reveal correlations between specific sequence features and pathogenic potential.

What experimental approaches can determine if T. denticola rplE has moonlighting functions beyond ribosome assembly?

Several experimental approaches can uncover potential moonlighting functions of T. denticola rplE:

• Protein-protein interaction screening: Using yeast two-hybrid, pull-down assays, or proximity labeling to identify non-ribosomal interaction partners.

• Subcellular localization studies: Immunofluorescence or fractionation to detect rplE in unexpected cellular compartments.

• Phenotypic analysis of overexpression: Examining effects of increased rplE levels on various cellular processes.

• In vitro activity assays: Testing purified rplE for enzymatic or binding activities unrelated to ribosome function.

Ribosomal proteins in other organisms have been found to perform secondary functions in DNA repair, transcriptional regulation, and stress responses. Similar multifunctionality in T. denticola rplE could provide insights into this pathogen's adaptability and virulence mechanisms.

Table 1. Comparison of Key Properties of T. denticola Ribosomal Components and Their Role in Assembly

Table 2. Recommended Expression and Purification Conditions for Recombinant T. denticola rplE

Table 3. Potential Regulatory Connections Between rplE and Virulence Factors in T. denticola

What are promising approaches for targeting T. denticola rplE in periodontal disease therapeutics?

Innovative approaches for targeting T. denticola rplE in therapeutic contexts include:

• Structure-based drug design: Using atomic-level structural information to develop small molecules that specifically disrupt L5-5S rRNA interactions or L5 incorporation into ribosomes.

• Antisense oligonucleotides: Designing nucleic acid-based therapeutics that selectively inhibit rplE expression.

• Peptide mimetics: Developing peptides that mimic interaction interfaces and compete with natural binding partners.

• CRISPR-based antimicrobials: Targeted nucleases designed to cleave the rplE gene specifically in T. denticola.

Since ribosomal proteins are essential for bacterial survival, targeting rplE could potentially inhibit T. denticola growth and virulence in periodontal pockets, representing a novel approach to periodontal disease management.

How might rplE contribute to T. denticola's ability to form biofilms in the oral cavity?

Ribosomal protein L5 might influence T. denticola biofilm formation through several mechanisms:

• Translational regulation: Selective translation of biofilm-associated genes under specific conditions.

• Stress adaptation: Contribution to survival in the competitive polymicrobial environment of oral biofilms.

• Indirect effects via virulence factors: Proper expression of surface proteins like Msp that mediate adhesion and intercellular interactions .

• Coordination with environmental sensing: Potential links between ribosome function and signaling pathways that detect conditions favorable for biofilm formation.

T. denticola is known to participate in complex polymicrobial oral biofilms associated with periodontal disease . Understanding how fundamental cellular components like L5 contribute to this behavior would provide new insights into pathogenesis and potential intervention strategies.