Recombinant Treponema denticola 30S ribosomal protein S15 (rpsO)

What is the biological role of 30S ribosomal protein S15 in Treponema denticola?

The 30S ribosomal protein S15 (encoded by the rpsO gene) in Treponema denticola is a critical component of the small (30S) ribosomal subunit involved in protein synthesis. It plays essential roles in ribosome assembly and function, particularly in the recognition and binding of 16S rRNA. In the context of T. denticola, a recognized periodontal pathogen, rpsO contributes to basic cellular metabolism and protein production necessary for bacterial survival, replication, and pathogenicity. This protein is part of the translation machinery that enables T. denticola to synthesize virulence factors and other proteins essential for colonization and infection in periodontal disease .

What genetic features of the rpsO gene in T. denticola are important for researchers to consider?

Researchers should note several important genetic features of the T. denticola rpsO gene:

• Codon usage bias: T. denticola has distinct codon preferences that affect heterologous expression efficiency. The AT-rich genome (approximately 38% GC content) influences codon optimization strategies for recombinant expression.

• Restriction site considerations: T. denticola possesses seven restriction-modification (R-M) systems, including two Type I, four Type II, and one partial Type III system. When designing cloning strategies for rpsO, researchers must account for these restriction sites to avoid DNA degradation during transformation .

• Regulatory elements: The rpsO gene typically contains autoregulatory elements that control its expression in response to ribosomal protein levels, which must be considered when designing expression constructs.

• Strain variation: Sequence diversity exists between T. denticola strains (such as ATCC 35405 and ATCC 33520), which should be accounted for when designing primers or expression systems .

What are optimal expression systems for producing recombinant T. denticola rpsO protein?

The optimal expression system for recombinant T. denticola rpsO production depends on research objectives and downstream applications. Several systems offer distinct advantages:

E. coli-based expression systems:

• BL21(DE3) strains are most commonly used for initial expression attempts due to their robust growth and high protein yields.

• Rosetta or CodonPlus strains address codon bias issues that may affect T. denticola protein expression.

• For improved solubility, E. coli ArcticExpress or SHuffle strains can be considered for proteins prone to inclusion body formation.

T. denticola native expression:
For studying protein in its natural context, expressing rpsO within T. denticola itself using optimized SyngenicDNA shuttle plasmids that circumvent restriction-modification barriers provides the most authentic post-translational modifications and folding . This approach requires:

• Removal of all Type I and Type II restriction sites from expression vectors

• Use of unmethylated plasmid DNA for transformation

• Modification of the shuttle plasmid to remove TdeIII recognition sites (GGNCC)

Expression efficiency comparison:

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

A multi-step purification strategy is recommended to achieve high purity while maintaining the activity of recombinant T. denticola rpsO:

Step 1: Initial Capture

• Immobilized metal affinity chromatography (IMAC) using a His6-tag is most effective for initial capture.

• Buffer conditions: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, with imidazole gradient (10-250 mM).

Step 2: Intermediate Purification

• Ion exchange chromatography (IEX) exploiting the theoretical pI of rpsO.

• Size exclusion chromatography (SEC) to separate oligomeric forms and remove aggregates.

Step 3: Polishing

• Heparin affinity chromatography leverages the RNA-binding properties of rpsO for additional purification.

Step 4: Tag Removal

• Precision protease (TEV or HRV 3C) cleavage followed by reverse IMAC.

Quality control metrics:

• SDS-PAGE: >95% homogeneity

• Western blot: Confirmation of identity

• Dynamic light scattering: Monodispersity assessment

• RNA-binding activity assay: Functional verification using fluorescence anisotropy with labeled RNA fragments

How can researchers address solubility challenges when expressing recombinant T. denticola rpsO?

Recombinant ribosomal proteins often present solubility challenges. For T. denticola rpsO, researchers can implement several strategies:

Expression optimization approaches:

• Lower induction temperature (16-20°C) to slow protein synthesis and improve folding

• Reduce inducer concentration (0.1-0.5 mM IPTG) to decrease expression rate

• Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Use of solubility-enhancing fusion partners:

Buffer optimization strategies:

• Include stabilizing agents: 5-10% glycerol, 0.1-0.5M arginine, or 1-5 mM DTT

• Test multiple pH conditions (pH 6.5-8.5)

• Incorporate low concentrations of non-ionic detergents (0.05-0.1% Triton X-100)

• Add nucleic acid fragments (RNA oligonucleotides) to stabilize the protein through its natural binding partners

Refolding protocols:
If inclusion bodies persist, a systematic refolding approach can be employed:

• Solubilize inclusion bodies with 6M guanidine-HCl or 8M urea

• Perform step-wise dialysis with decreasing denaturant concentration

• Monitor refolding by intrinsic fluorescence spectroscopy or circular dichroism

What structural features distinguish T. denticola rpsO from other bacterial ribosomal S15 proteins?

While specific structural data for T. denticola rpsO is limited in the provided search results, comparative analysis with other bacterial S15 proteins suggests several distinguishing features:

The typical bacterial S15 protein adopts a compact α/β fold with a four-stranded β-sheet and three α-helices. In T. denticola, sequence analysis predicts:

• N-terminal region: Likely contains a species-specific sequence variation that may influence interaction with other ribosomal components or regulatory functions.

• RNA-binding domain: Highly conserved positively charged residues that interact with 16S rRNA. This domain shares structural conservation with other bacterial species due to its critical functional role.

• Surface charge distribution: The periodontal environment influences the surface charge properties of T. denticola proteins. The rpsO protein likely has adaptations to function optimally in the anaerobic, protein-rich subgingival environment where T. denticola thrives .

• Potential regulatory motifs: Unlike the major surface protein (Msp) that has been extensively characterized , rpsO lacks experimentally validated membrane localization signals, consistent with its primary role in intracellular ribosomal assembly.

Structural prediction algorithms suggest T. denticola rpsO maintains the core α/β fold typical of this protein family while exhibiting sequence divergence in non-catalytic regions that may reflect adaptation to the unique physiological requirements of this oral pathogen.

How can researchers effectively analyze the RNA-binding properties of recombinant T. denticola rpsO?

Analyzing the RNA-binding properties of recombinant T. denticola rpsO requires multiple complementary approaches:

In vitro binding assays:

• Electrophoretic Mobility Shift Assay (EMSA): Using labeled 16S rRNA fragments to detect complex formation. Optimal conditions include 25 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, and 5% glycerol.

• Filter Binding Assay: Quantitative measurement of binding affinity (Kd) using increasing concentrations of purified rpsO protein with a constant amount of radiolabeled RNA.

• Surface Plasmon Resonance (SPR): Real-time binding kinetics analysis with immobilized RNA or protein.

• Fluorescence Anisotropy: Measures changes in rotational diffusion of fluorescently labeled RNA upon protein binding.

Binding parameters comparison:

Structure-function analysis:

• Site-directed mutagenesis: Substituting conserved positively charged residues (Arg, Lys) expected to interact with RNA.

• Deletion analysis: Creating truncated versions to identify minimal RNA-binding domains.

• Hydrogen-deuterium exchange mass spectrometry: To map protein regions protected upon RNA binding.

What advanced biophysical techniques are most informative for characterizing T. denticola rpsO structure-function relationships?

To comprehensively characterize T. denticola rpsO structure-function relationships, several advanced biophysical techniques should be employed:

High-resolution structural analysis:

• X-ray crystallography: Provides atomic-level detail of protein structure, ideally co-crystallized with RNA fragments to visualize binding interfaces. Requires protein concentrations of 5-15 mg/ml in crystallization trials.

• NMR spectroscopy: Enables solution-state structural determination and dynamics analysis. Particularly valuable for identifying flexible regions and conformational changes upon RNA binding. Requires ¹⁵N/¹³C-labeled protein produced in minimal media.

• Cryo-electron microscopy: Particularly useful for visualizing rpsO in the context of entire ribosomal assemblies, providing insights into its integration within the 30S subunit.

Thermodynamic and kinetic characterization:

• Isothermal Titration Calorimetry (ITC): Measures binding energetics (ΔH, ΔS, ΔG) and stoichiometry.

• Differential Scanning Calorimetry (DSC): Determines thermal stability and folding cooperativity.

• Circular Dichroism (CD): Monitors secondary structure content and conformational changes.

In silico approaches:

• Molecular Dynamics Simulations: Models protein-RNA interactions and conformational flexibility.

• Homology Modeling: Leverages structures from related bacterial species to predict T. denticola rpsO structure.

Research workflow integration:

Combining these techniques creates a comprehensive analytical pipeline:

• Initial structure prediction through homology modeling

• Experimental structure determination by X-ray crystallography or NMR

• Dynamics and interaction analysis through MD simulations

• Functional validation using biochemical assays

• Integration of structural insights with physiological context of T. denticola

How can recombinant T. denticola rpsO be used as a tool in periodontal disease research?

Recombinant T. denticola rpsO offers several valuable applications in periodontal disease research:

Diagnostic biomarker development:
The rpsO protein can serve as a specific biomarker for T. denticola detection in clinical samples. Antibodies raised against purified recombinant rpsO can be incorporated into diagnostic assays to specifically identify this periodontal pathogen in complex microbial communities. This approach complements existing diagnostic methods that rely on the detection of the "red complex" bacteria (T. denticola, P. gingivalis, and T. forsythia) associated with severe periodontal disease .

Pathogenesis studies:

• Host-pathogen interaction models: Purified rpsO can be used to study potential extracellular roles beyond its canonical ribosomal function. Some ribosomal proteins exhibit "moonlighting" functions in bacterial pathogenesis.

• Immune response characterization: Recombinant rpsO can be used to evaluate specific antibody responses in periodontal disease patients, potentially correlating antibody titers with disease severity.

• Genetic manipulation marker: The rpsO gene can serve as a selectable or screenable marker in T. denticola genetic systems, leveraging the enhanced transformation methods described for this organism .

Comparative studies:
Researchers can use recombinant rpsO proteins from different oral treponeme species to investigate the functional divergence among the diverse treponeme communities associated with periodontal disease . This may provide insights into the differential virulence and niche specialization among treponeme species detected in subgingival plaque.

What are the advantages and limitations of using rpsO as a phylogenetic marker for oral treponeme populations?

Advantages:

• Conservation level: The rpsO gene is highly conserved due to its essential function in protein synthesis, making it suitable for distinguishing closely related strains within the same species or closely related species.

• Strain differentiation: Analysis of rpsO sequences can complement other markers like pyrH for identifying strain-level variation within T. denticola populations in clinical samples. This approach has successfully distinguished between different phylogenetic lineages in both gingivitis and periodontitis patients .

• Consistent molecular clock: The relatively stable evolutionary rate of housekeeping genes like rpsO provides a reliable chronological framework for examining treponeme evolution.

• PCR amplification reliability: The sequence conservation facilitates design of primers that work across diverse treponeme species.

Limitations:

• Limited phylogenetic resolution: For more distant evolutionary relationships, rpsO may be too conserved to provide adequate resolution, particularly when comparing across different genera.

• Horizontal gene transfer considerations: While less common for ribosomal genes, the possibility of horizontal gene transfer can confound phylogenetic analyses.

• Technical considerations:

  • PCR bias may affect the detection of low-abundance variants

  • Sequence analysis requires careful quality filtering and appropriate bioinformatic pipelines

Comparison with other phylogenetic markers:

What methodological approaches can detect changes in T. denticola rpsO expression during periodontal disease progression?

Monitoring changes in T. denticola rpsO expression during periodontal disease progression requires sensitive, specific, and quantitative methods:

Nucleic acid-based approaches:

• Quantitative RT-PCR: Specific primers targeting T. denticola rpsO mRNA provide quantitative expression data from clinical samples. This should be normalized to stable reference genes and requires careful sample preservation to prevent RNA degradation.

• RNA-Seq: Transcriptome analysis offers a comprehensive view of rpsO expression in the context of global gene expression changes. This approach can reveal co-regulated genes and regulatory networks during disease progression.

• In situ hybridization: Detection of rpsO mRNA directly in tissue samples provides spatial information about T. denticola localization and activity in periodontal pockets.

Protein-based approaches:

• Immunohistochemistry: Using anti-rpsO antibodies to detect protein expression in tissue sections.

• Selected Reaction Monitoring (SRM) mass spectrometry: Targeted proteomics approach for quantifying rpsO protein in complex clinical samples.

Sample collection and processing protocol:

• Collect subgingival plaque from sites with different disease severity (healthy, gingivitis, and periodontitis).

• Immediately preserve samples in RNA stabilization solution.

• Extract total RNA using specialized protocols for complex microbial communities.

• Perform reverse transcription with random hexamers.

• Use T. denticola-specific rpsO primers for qPCR analysis.

Expression analysis workflow:

What are common technical challenges when working with recombinant T. denticola proteins and their solutions?

Researchers face several technical challenges when working with recombinant T. denticola proteins, including rpsO:

Challenge 1: Low transformation efficiency

• Problem: T. denticola has multiple restriction-modification (R-M) systems that degrade foreign DNA.

• Solution: Use SyngenicDNA approaches that remove all Type I and Type II restriction sites from expression vectors . This includes identifying and removing TdeIII recognition sites (GGNCC) and developing R-M-silent plasmids that are resistant to all T. denticola ATCC 35405 R-M systems .

Challenge 2: Protein solubility issues

• Problem: Recombinant T. denticola proteins often form inclusion bodies.

• Solution: Optimize expression conditions (lower temperature, reduced inducer concentration), use solubility-enhancing fusion tags (MBP, SUMO), and develop specialized refolding protocols if necessary.

Challenge 3: Protein stability concerns

• Problem: Ribosomal proteins may be unstable when expressed individually outside their native complex.

• Solution: Include stabilizing buffers (glycerol, reducing agents), store at appropriate temperatures, and consider co-expression with binding partners.

Challenge 4: Functional verification

• Problem: Confirming that recombinant rpsO retains native activity.

• Solution: Develop RNA-binding assays that verify interaction with cognate 16S rRNA fragments. Compare binding parameters with known bacterial S15 proteins.

Challenge 5: Antibody cross-reactivity

• Problem: Antibodies against T. denticola proteins may cross-react with other oral treponemes.

• Solution: Carefully select unique epitopes for antibody production and extensively validate antibody specificity against closely related species.

How can researchers integrate T. denticola genetic manipulation tools with rpsO functional studies?

Integrating genetic manipulation tools with rpsO functional studies can provide powerful insights into T. denticola biology:

Genetic modification approaches:

• CRISPR-Cas9 adaptation: Modify the CRISPR-Cas9 system for T. denticola, addressing the specific challenges of this anaerobic spirochete.

• Transposon mutagenesis: Utilize the recently developed high-efficiency transposon mutagenesis system based on RM-silent, codon-optimized, himarC9 transposase . This approach can generate rpsO mutants or reporter fusions.

• Shuttle vector complementation: Employ the E. coli-T. denticola shuttle plasmid system for expression of modified rpsO variants in T. denticola .

Functional genomics integration:

• Conditional knockdown: Develop regulatable promoter systems to create conditional rpsO knockdown strains, which is particularly valuable for studying essential genes.

• Fluorescent protein fusions: Create C-terminal fusions with anaerobic fluorescent proteins like FbFP that remain active under the anaerobic conditions required for T. denticola growth .

• Epitope tagging: Insert small epitope tags that minimally disrupt rpsO function while enabling protein tracking and purification.

Experimental design workflow:

What are the current limitations in our understanding of T. denticola ribosomal biology and future research directions?

Current understanding of T. denticola ribosomal biology has several significant limitations that represent opportunities for future research:

Knowledge gaps:

• Ribosome assembly pathway: The specific assembly pathway of T. denticola ribosomes, including the order of incorporation of ribosomal proteins like rpsO, remains largely uncharacterized.

• Translation regulation: How T. denticola regulates translation in response to environmental stresses found in periodontal pockets (nutrient limitation, pH fluctuation, host immune factors) is poorly understood.

• Antibiotic susceptibility mechanisms: The structural basis for differential antibiotic susceptibility of T. denticola ribosomes compared to other bacteria requires further investigation.

• Post-translational modifications: Potential T. denticola-specific modifications of ribosomal proteins that might influence function or regulation.

Future research directions:

• Comparative ribosome structure determination: Cryo-EM structures of T. denticola ribosomes would provide insights into spirochete-specific features relevant to their unique physiology and pathogenesis.

• Ribosome heterogeneity analysis: Investigation of potential specialized ribosomes or ribosomal protein variants during different growth phases or stress conditions.

• Translation dynamics: Single-molecule studies of T. denticola translation to understand the kinetics and fidelity of protein synthesis in this oral pathogen.

• Drug development potential: Structure-guided design of selective inhibitors targeting unique features of T. denticola ribosomes as potential therapeutic agents.

• Multi-omics integration: Combining transcriptomics, proteomics, and ribosome profiling to create a comprehensive model of T. denticola translational regulation during host interaction.

Methodological advances needed:

• Development of improved genetic tools for chromosomal manipulation in T. denticola

• Adaptation of ribosome profiling techniques for anaerobic spirochetes

• Enhanced in vitro translation systems using T. denticola components

• Advanced imaging approaches for visualizing ribosome dynamics in live T. denticola cells

How does research on T. denticola rpsO contribute to our understanding of periodontal disease pathogenesis?

Research on T. denticola rpsO contributes to our understanding of periodontal disease pathogenesis through multiple dimensions:

• Bacterial physiology insights: As an essential component of the translation machinery, rpsO studies illuminate the fundamental biology that enables T. denticola to persist and thrive in the periodontal environment. This provides a foundation for understanding bacterial adaptation to the host environment.

• Diagnostic applications: The specificity of rpsO sequences can be leveraged to develop improved molecular diagnostic tools for detecting T. denticola in clinical samples, particularly important given its role in the "red complex" associated with severe periodontal disease .

• Strain variation correlation: Analysis of rpsO sequence variations across clinical isolates allows researchers to correlate specific genetic lineages with disease severity, as demonstrated in comparative studies of gingivitis versus periodontitis patients .

• Therapeutic target evaluation: Understanding the structural and functional aspects of essential ribosomal components like rpsO may reveal targetable differences between human and bacterial ribosomes for potential therapeutic development.

• Microbial community dynamics: Research on T. denticola ribosomal proteins provides insights into the metabolic activity and growth dynamics of this key periodontal pathogen within the complex microbial communities associated with periodontal disease progression.

By advancing our understanding of the fundamental cellular machinery that supports T. denticola virulence and persistence, rpsO research contributes to the broader goal of developing improved strategies for prevention, diagnosis, and treatment of periodontal disease.