Recombinant Treponema denticola 30S ribosomal protein S6 (rpsF)

What is the structure and function of 30S ribosomal protein S6 (rpsF) in Treponema denticola?

The 30S ribosomal protein S6 (rpsF) is a component of the small ribosomal subunit in Treponema denticola, encoded by the rpsF gene. The full-length protein consists of 93 amino acids with the sequence "MRNYELMTVF PVEEDLYKPG IDALHSILAD FGVQIKSEEP FGDRDLAYEI KKKTKGRYVL FNIEADPAKM IELDKRFKLI TQMLTYLFVR LED" as identified in the strain ATCC 35405 . As a ribosomal protein, rpsF plays a crucial role in protein synthesis by helping maintain the structural integrity of the 30S ribosomal subunit and facilitating the translation process. In T. denticola, the proper functioning of ribosomes is essential for bacterial survival and virulence factor production, which contributes to its pathogenicity in periodontal disease contexts.

How is Treponema denticola classified among oral treponemes?

Treponema denticola belongs to phylogroup 2 of oral treponemes, which is one of at least seven phylogroups of oral treponemes identified in human subgingival plaque samples . Phylogenetic analyses based on highly conserved genes such as pyrH have shown that T. denticola forms a distinct cluster within phylogroup 2. Studies have demonstrated that phylogroup 2 treponemes, particularly T. denticola, show significantly higher diversity and abundance in subjects with periodontitis compared to those with gingivitis . The OTU (Operational Taxonomic Unit) 8P47, which corresponds to the type strain of Treponema denticola, has shown the strongest association with periodontitis (p < 0.01) .

How is recombinant T. denticola rpsF typically produced for research applications?

Recombinant T. denticola 30S ribosomal protein S6 (rpsF) is typically produced in yeast expression systems to ensure proper folding and post-translational modifications . The production process involves:

• Cloning the full-length rpsF gene (expression region 1-93) from T. denticola (strain ATCC 35405)

• Inserting the gene into appropriate yeast expression vectors

• Transforming yeast cells with the recombinant vector

• Inducing protein expression under optimized conditions

• Purifying the expressed protein using chromatographic techniques

• Quality control testing to ensure >85% purity as verified by SDS-PAGE

The recombinant protein may include specific tags to facilitate purification and detection, though the exact tag type is often determined during the manufacturing process based on experimental requirements .

How can genetic variation in rpsF be used for strain typing of T. denticola clinical isolates?

While rpsF itself has not been widely used as a strain typing marker for T. denticola, the approach of using highly conserved housekeeping genes for molecular epidemiology is well-established. Research on T. denticola populations has demonstrated that individuals with periodontal disease often harbor multiple genetic lineages of the same treponeme species within their oral cavities . For example, subjects with periodontitis commonly contain at least 3 distinct genotypes corresponding to T. denticola .

For strain typing, researchers could:

• Sequence the rpsF gene from multiple clinical isolates

• Analyze sequence variations to establish genotype clusters

• Compare these with established typing methods like pyrH gene analysis

• Correlate genetic variations with clinical parameters

pyrH genotyping has already revealed that the most prevalent genotype (pyrH001) corresponds to a large cluster of previously identified T. denticola isolates including the reference strains ATCC 33520 and ATCC 35405 T . Similar approaches could be applied to rpsF.

What immunological applications exist for recombinant T. denticola rpsF?

Recombinant T. denticola rpsF has potential applications in immunological research related to periodontal disease:

• Antibody development: The purified recombinant protein can be used as an immunogen to develop polyclonal or monoclonal antibodies specific to T. denticola rpsF . These antibodies can serve as valuable tools for:

  • Immunohistochemistry to detect T. denticola in clinical samples

  • Western blotting to quantify rpsF expression levels

  • Immunoprecipitation to study protein interactions

• Biomarker research: As T. denticola shows strong association with periodontitis , rpsF-specific antibodies could be evaluated as potential diagnostic biomarkers.

• Vaccine development: The recombinant protein could be investigated as a potential antigen for vaccines targeting periodontal pathogens.

• Host-pathogen interaction studies: Purified rpsF could be used to investigate its interactions with host immune receptors and subsequent immune response pathways.

How does T. denticola rpsF compare to rpsF proteins from other oral pathogens?

A comprehensive comparative analysis of rpsF proteins across oral pathogens would involve:

• Sequence alignment: Multiple sequence alignment of rpsF from T. denticola (Q73M32) with rpsF from other oral pathogens to identify conserved domains and variable regions.

• Structural comparison: Prediction or determination of three-dimensional structures to identify structural variations that might influence function.

• Phylogenetic analysis: Construction of phylogenetic trees based on rpsF sequences to understand evolutionary relationships among oral pathogens.

• Functional conservation: Analysis of conserved binding sites and functional domains to predict shared and distinct functional properties.

This type of comparative analysis could provide insights into the evolutionary adaptation of oral treponemes and potentially identify unique features of T. denticola rpsF that could be targeted for diagnostic or therapeutic purposes.

What are the optimal storage and handling conditions for recombinant T. denticola rpsF?

The stability and shelf life of recombinant T. denticola rpsF depends on several factors including storage state, buffer composition, and storage temperature. Based on manufacturer recommendations:

Storage recommendations:

• Lyophilized form: 12 months stability at -20°C/-80°C

• Liquid form: 6 months stability at -20°C/-80°C

• Working aliquots: Store at 4°C for up to one week

Handling recommendations:

• Avoid repeated freeze-thaw cycles as this can lead to protein degradation and loss of activity

• Briefly centrifuge vials before opening to bring contents to the bottom

• For reconstitution of lyophilized protein:

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration (50% is often recommended)

  • Aliquot for long-term storage at -20°C/-80°C

These conditions minimize protein degradation and ensure maximum stability for experimental applications.

What controls should be included when using recombinant T. denticola rpsF in binding studies?

When designing binding studies with recombinant T. denticola rpsF, researchers should include several types of controls to ensure experimental validity:

• Negative controls:

  • Buffer-only control (no protein) to establish baseline

  • Irrelevant protein control (similar size/structure but unrelated function)

  • Denatured rpsF to confirm structure-dependent interactions

• Positive controls:

  • Known binding partners of ribosomal proteins if available

  • Antibodies specific to any tags present on the recombinant protein

• Specificity controls:

  • Competitive binding with excess unlabeled rpsF

  • Dose-response curves to demonstrate specific binding

  • Cross-reactivity tests with similar ribosomal proteins

• Technical controls:

  • Reproducibility controls (multiple technical and biological replicates)

  • Temperature controls (binding at different temperatures)

  • pH and salt concentration gradients to optimize binding conditions

These controls help validate experimental results and ensure that observed interactions are specific to the native conformation of T. denticola rpsF.

How can researchers validate the functional activity of recombinant T. denticola rpsF?

Validating the functional activity of recombinant T. denticola rpsF requires several approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper folding

  • Size exclusion chromatography to verify monomeric state

  • Dynamic light scattering to check for aggregation

• Ribosomal assembly assays:

  • In vitro reconstitution of 30S ribosomal subunits using recombinant rpsF

  • Monitoring incorporation of rpsF into partial ribosomal complexes

  • Electron microscopy to visualize ribosome assembly intermediates

• Functional assays:

  • RNA binding assays to confirm interaction with ribosomal RNA

  • Translation efficiency assays using in vitro translation systems

  • Complementation assays in rpsF-deficient bacterial strains

• Comparative analysis:

  • Side-by-side testing with native rpsF (if available)

  • Activity comparison with rpsF from related bacterial species

These validation approaches ensure that the recombinant protein maintains its native structural and functional properties, which is essential for meaningful research applications.

How should researchers analyze genetic diversity data of T. denticola strains based on rpsF sequences?

Analysis of genetic diversity using rpsF sequences requires a systematic approach:

• Sequence alignment and processing:

  • Multiple sequence alignment of rpsF sequences from different isolates

  • Quality filtering to remove erroneous sequences

  • Determination of appropriate sequence identity cutoffs (97% is commonly used for genotype designation)

• Diversity metrics calculation:

  • Genotype richness (number of distinct genotypes)

  • Shannon diversity index to account for both richness and evenness

  • Phylogenetic diversity metrics that incorporate evolutionary distances

• Statistical comparisons:

  • Mann-Whitney U test for comparing diversity between different clinical groups

  • UniFrac analysis (both weighted and unweighted) to compare community structures

  • Parsimony p-tests to determine statistical significance of population differences

• Visualization techniques:

  • Principal Coordinate Analysis (PCoA) to visualize differences between samples

  • Phylogenetic trees to show relationships between different genotypes

  • Heatmaps to display genotype distribution across samples

This analytical framework would be similar to the approaches used for analyzing pyrH genotypes in T. denticola populations, where significant differences in diversity have been observed between periodontitis and gingivitis subjects .

How can researchers correlate T. denticola rpsF genetic variants with clinical parameters of periodontal disease?

To establish correlations between rpsF genetic variants and clinical periodontal parameters, researchers should:

• Clinical data collection:

  • Comprehensive periodontal examination including probing depths, attachment loss, bleeding on probing

  • Categorization of subjects (e.g., healthy, gingivitis, chronic periodontitis, aggressive periodontitis)

  • Inflammatory marker measurements from gingival crevicular fluid

• Genotyping approach:

  • PCR amplification and sequencing of rpsF from clinical samples

  • Assignment of sequences to genotypes based on sequence identity cutoffs

  • Determination of genotype prevalence in each subject and clinical group

• Statistical analysis:

  • Correlation analysis between specific genotypes and disease severity

  • Multivariate regression to account for confounding factors

  • Odds ratio calculations to quantify associations

• Validation strategies:

  • Cohort validation in different populations

  • Longitudinal studies to track genotype changes with disease progression

  • In vitro functional studies to investigate mechanistic relationships

Similar approaches have demonstrated that certain T. denticola genetic lineages show disease-selective distributions, with one lineage (OTU 8P47) showing the strongest association with periodontitis (p < 0.01) .

What methods are recommended for analyzing proteomic data involving T. denticola rpsF?

Proteomic analysis involving T. denticola rpsF should consider:

• Sample preparation optimization:

  • Efficient extraction protocols for membrane-associated ribosomal proteins

  • Enrichment strategies for low-abundance proteins

  • Appropriate controls for normalization

• Data acquisition approaches:

  • Selected/Multiple Reaction Monitoring (SRM/MRM) for targeted quantification

  • Data-Independent Acquisition (DIA) for comprehensive profiling

  • Post-translational modification mapping using specialized techniques

• Bioinformatic analysis pipeline:

  • Database selection including appropriate T. denticola strain references

  • False discovery rate control for reliable identification

  • Quantification methods (label-free vs. labeled approaches)

• Interpretation frameworks:

  • Pathway analysis incorporating ribosomal assembly and protein synthesis

  • Protein-protein interaction networks to identify functional associations

  • Comparative analysis across different clinical conditions

These approaches allow researchers to understand the expression dynamics of rpsF in different contexts and its potential role in T. denticola virulence and adaptation.

How can recombinant T. denticola rpsF contribute to understanding periodontal disease pathogenesis?

Recombinant T. denticola rpsF can be utilized in several ways to advance our understanding of periodontal disease pathogenesis:

• Host-pathogen interaction studies:

  • Investigation of rpsF interactions with host immune receptors

  • Analysis of pro-inflammatory responses triggered by rpsF

  • Evaluation of rpsF as a potential pathogen-associated molecular pattern (PAMP)

• Biofilm research:

  • Examination of rpsF's role in biofilm formation and maintenance

  • Study of protein expression dynamics during biofilm development

  • Investigation of interspecies interactions in polymicrobial biofilms

• Virulence mechanism exploration:

  • Analysis of rpsF's potential moonlighting functions beyond protein synthesis

  • Evaluation of its contribution to stress response and antibiotic resistance

  • Investigation of its potential role in host cell invasion or adhesion

• Translational research applications:

  • Development of diagnostic tools based on rpsF detection

  • Evaluation as a potential vaccine candidate

  • Target identification for novel therapeutic approaches

These research directions leverage the availability of pure recombinant protein to dissect specific molecular mechanisms underlying T. denticola's role in periodontal disease.

What is the relationship between T. denticola genetic diversity and periodontal disease progression?

Studies of T. denticola genetic diversity have revealed important relationships with periodontal disease:

• Strain diversity correlations:

  • Subjects with periodontitis harbor a greater diversity of treponeme genotypes than subjects with gingivitis

  • The diversity of phylogroup 2 oral treponemes (including T. denticola) is significantly higher in periodontitis subjects

  • Individuals with periodontitis commonly harbor multiple distinct genetic lineages of the same treponeme species within their oral cavities

• Disease-selective distribution patterns:

  • Certain phylogenetic lineages of T. denticola show disease-selective distributions

  • One T. denticola lineage (OTU 8P47) has demonstrated the strongest statistical association with periodontitis (p < 0.01)

• Community structure distinctions:

  • Phylogeny-based analyses (∫-libshuff, UniFrac, Parsimony p-tests) confirm that treponeme communities in periodontitis and health/gingivitis are significantly different

  • These differences are particularly pronounced for phylogroups 1, 2, 5, and 6

This evidence suggests that specific genetic variants of T. denticola may contribute differentially to periodontal disease pathogenesis, with certain strains potentially possessing enhanced virulence properties.

How can researchers troubleshoot protein degradation issues when working with recombinant T. denticola rpsF?

Protein degradation is a common challenge when working with recombinant proteins. For T. denticola rpsF, consider the following troubleshooting approaches:

• Storage optimization:

  • Add protease inhibitors to storage buffers

  • Maintain consistent cold chain during handling

  • Aliquot stock solutions to avoid repeated freeze-thaw cycles

  • Add glycerol (5-50%) to prevent freeze-damage

• Buffer optimization:

  • Test different pH conditions to identify optimal stability range

  • Adjust salt concentration to enhance protein stability

  • Consider adding stabilizing agents (e.g., glycerol, sucrose, BSA)

  • Evaluate reducing agents to maintain disulfide bonds

• Analytical monitoring:

  • Use SDS-PAGE to regularly check protein integrity

  • Consider size exclusion chromatography to detect aggregation

  • Implement activity assays to monitor functional degradation

  • Use mass spectrometry to identify specific degradation sites

• Procedural modifications:

  • Minimize exposure to room temperature

  • Reduce physical agitation during handling

  • Consider lyophilization for long-term storage

  • Use low-protein-binding tubes and pipette tips

These approaches help maintain the structural and functional integrity of recombinant T. denticola rpsF throughout experimental procedures.

What strategies can address cross-reactivity issues in immunological studies involving T. denticola rpsF?

Cross-reactivity can complicate immunological studies with T. denticola rpsF. Researchers can implement these strategies to address such issues:

• Antibody validation:

  • Extensive pre-adsorption against related bacterial lysates

  • Epitope mapping to identify unique regions for targeting

  • Competitive ELISA to confirm specificity

  • Western blot analysis against multiple bacterial species

• Assay optimization:

  • Titration of antibody concentrations to minimize non-specific binding

  • Optimization of blocking reagents and conditions

  • Inclusion of detergents to reduce hydrophobic interactions

  • Addition of competing antigens to block cross-reactive antibodies

• Advanced approaches:

  • Development of monoclonal antibodies against unique epitopes

  • Peptide-based approaches targeting highly specific regions

  • Affinity purification of antibodies against the specific target

  • Development of aptamers as alternative recognition molecules

• Control implementations:

  • rpsF knockout strains as negative controls

  • Closely related species as specificity controls

  • Pre-immune sera for background determination

  • Isotype controls for non-specific binding assessment

These strategies help ensure that experimental results reflect true T. denticola rpsF-specific interactions rather than cross-reactivity with related proteins.