Recombinant Treponema denticola 50S ribosomal protein L24 (rplX)

What is Treponema denticola 50S ribosomal protein L24 and its function in bacterial physiology?

T. denticola's complete genome sequence of 2,843,201 bp (ATCC 35405 strain) enables identification and characterization of essential genes like rplX . The protein synthesis machinery is particularly important for T. denticola as it requires specific proteins to establish itself in the subgingival dental plaque and interact with the host immune system .

How can recombinant T. denticola rplX be expressed and purified for research purposes?

Expression and purification of recombinant T. denticola rplX can be accomplished through several methodological approaches:

• Vector Selection and Cloning: The rplX gene can be amplified from T. denticola genomic DNA (ATCC 35405 strain) using PCR with specific primers designed based on the published genome sequence . The amplified gene can then be cloned into an appropriate expression vector such as pET-based systems.

• Expression System: E. coli is typically used as the heterologous host for recombinant expression. Specialized strains like BL21(DE3) are particularly suitable for ribosomal protein expression. For challenging expression, a T. denticola-E. coli shuttle plasmid system might be considered, similar to those developed for other T. denticola proteins .

• Purification Strategy: A His-tag or other affinity tag can be incorporated to facilitate purification. The copy number of the expression plasmid is an important consideration, with studies on T. denticola plasmids suggesting approximately 24-27 copies per cell for optimal expression .

• Expression Verification: qPCR methods, similar to those used to compare expression levels of other T. denticola genes such as flaA, msp, and fhbB, can be employed to verify expression levels of recombinant rplX .

What are the structural characteristics of T. denticola 50S ribosomal protein L24?

The T. denticola 50S ribosomal protein L24 shares structural features with ribosomal L24 proteins from other bacteria, but with specific characteristics related to its adaptation in the oral spirochete environment:

• Primary Structure: The protein typically contains conserved regions that interact with ribosomal RNA and other ribosomal proteins. Analysis of the T. denticola genome reveals that its ribosomal proteins, including L24, have evolved differently compared to those in T. pallidum, reflecting their adaptation to different host environments .

• Secondary and Tertiary Structure: The protein likely adopts a globular fold with alpha-helical and beta-sheet elements, as is common for ribosomal proteins. The specific structural features would be determined by the unique sequence characteristics of T. denticola L24, which has evolved in the context of the oral microbiome.

• Interactions: Within the ribosome, L24 interacts with both 23S rRNA and neighboring ribosomal proteins, forming part of the nascent peptide exit tunnel. These interactions are critical for proper ribosome assembly and function.

What techniques are available for detecting the expression of native rplX in T. denticola samples?

Several techniques can be employed for detecting native rplX expression in T. denticola samples:

• RT-qPCR: Real-time quantitative PCR can be used to measure rplX mRNA levels. This technique has been successfully applied to quantify other T. denticola genes and can be adapted for rplX .

• Western Blotting: Using antibodies specific to the L24 protein can detect the protein in cellular extracts. This requires generation of anti-L24 antibodies or using antibodies against a recombinant tagged version of the protein.

• Mass Spectrometry: Proteomic approaches can identify and quantify the presence of L24 in T. denticola samples. This approach is particularly useful for studying post-translational modifications.

• Species-Specific PCR: Similar to methods used for T. denticola detection in clinical samples, specific primers targeting the rplX gene can be developed for detection purposes .

How does T. denticola rplX differ structurally and functionally from homologous proteins in other oral pathogens?

The structural and functional differences between T. denticola rplX and its homologs in other oral pathogens reflect evolutionary adaptations to specific ecological niches:

• Comparative Genomics Perspective: The T. denticola genome is considerably larger (2.84 Mb) than that of related spirochetes like T. pallidum, suggesting potential functional diversification of essential proteins including rplX . Comparative analysis indicates that differences in gene content, including ribosomal proteins, are attributable to genome reduction, lineage-specific expansions, and horizontal gene transfer among oral microbiome members .

• Sequence Variations: T. denticola rplX likely contains specific amino acid substitutions that differentiate it from homologs in other oral bacteria. These variations may affect RNA binding properties, interactions with other ribosomal proteins, or susceptibility to antimicrobial agents targeting protein synthesis.

• Functional Implications: The specific sequence and structural features of T. denticola rplX may contribute to unique aspects of protein synthesis regulation in this organism, potentially relating to its adaptation to the polymicrobial environment of dental plaque and its pathogenic lifestyle .

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What role might rplX play in T. denticola antibiotic resistance and how can this be studied using recombinant protein?

The potential role of rplX in T. denticola antibiotic resistance represents an important research direction:

• Mechanism of Resistance: Ribosomal proteins, including L24, are potential targets for antibiotics that inhibit protein synthesis. Mutations or structural variations in rplX could contribute to altered antibiotic binding and resistance mechanisms. For example, studies on ErmB (23S rRNA methylase) in T. denticola have shown its role in resistance , and similar mechanisms might involve rplX.

• Methodological Approaches:

  • Site-directed mutagenesis of recombinant rplX to identify critical residues for antibiotic interaction

  • In vitro translation assays using purified ribosomes with incorporated recombinant rplX variants

  • Minimum inhibitory concentration (MIC) testing with T. denticola strains expressing modified rplX

  • Structural studies (X-ray crystallography or cryo-EM) of ribosomes containing variant rplX proteins

• Targeted Antibiotics: Antibiotics that target the 50S ribosomal subunit, such as macrolides, lincosamides, and streptogramins, could be studied in relation to rplX variations. The ErmB methylase in T. denticola already suggests the importance of ribosomal modifications in resistance mechanisms .

How can recombinant T. denticola rplX be used to study host-pathogen interactions in periodontal disease?

Recombinant T. denticola rplX offers several avenues for investigating host-pathogen interactions in periodontal disease:

• Immunological Studies: Purified recombinant rplX can be used to assess its potential as an immunogenic protein:

  • Measuring antibody responses in periodontal disease patients

  • Evaluating its ability to stimulate cytokine production by host immune cells

  • Determining if anti-rplX antibodies enhance phagocytosis of T. denticola by macrophages, similar to studies with other T. denticola proteins like MSP

• Diagnostic Applications: If rplX proves to be immunogenic, recombinant protein could serve as an antigen in immunological assays to detect T. denticola-specific antibodies in patient samples, complementing current PCR-based detection methods .

• Interaction with Host Factors: Studies could explore potential moonlighting functions of rplX beyond its canonical role in protein synthesis:

  • Binding assays with host extracellular matrix components

  • Potential interactions with host complement or immune regulatory factors

  • Role in bacterial adhesion to host cells

• Animal Models: Recombinant rplX could be used in animal models of periodontal disease to assess its role in pathogenesis and potential as a vaccine candidate. Previous studies have shown that immune responses play a major role in controlling T. denticola infections .

What are the technical challenges in expressing and maintaining the native conformation of T. denticola rplX in heterologous systems?

The expression of T. denticola proteins in heterologous systems presents several technical challenges:

• Codon Usage Bias: T. denticola has a different codon usage pattern compared to common expression hosts like E. coli. This can lead to:

  • Poor translation efficiency

  • Premature termination of protein synthesis

  • Formation of inclusion bodies

  Solution: Codon optimization of the rplX gene sequence for the expression host or use of strains enriched in rare tRNAs.

• Protein Folding and Stability: Ribosomal proteins typically exist in complex with rRNA and other proteins, which may affect their folding and stability when expressed recombinantly.

  Approaches:

  • Expression as fusion proteins with solubility-enhancing tags

  • Co-expression with chaperones

  • Optimization of induction conditions (temperature, IPTG concentration)

  • Testing different solubilization and refolding protocols

• Post-translational Modifications: Any native modifications of T. denticola rplX may be absent in heterologous hosts.

  Investigation strategies:

  • Mass spectrometry to identify modifications in native protein

  • Engineering expression systems to incorporate necessary modifications

• Expression Level Control: Studies with other T. denticola proteins have shown that copy number and promoter strength significantly affect expression levels . For rplX, balancing expression levels is crucial to avoid toxicity or inclusion body formation.

  Solutions:

  • Testing various promoter strengths

  • Using inducible systems with tight regulation

  • Optimizing plasmid copy number based on findings that T. denticola plasmids maintain approximately 24-27 copies per cell

How might T. denticola rplX expression be regulated under different environmental conditions relevant to periodontal disease?

The regulation of rplX expression in T. denticola under different environmental conditions provides insights into adaptation mechanisms during infection:

• Oxygen Levels: As an anaerobe, T. denticola encounters varying oxygen levels in periodontal pockets. Studies have shown that macrophage interactions with T. denticola differ under aerobic versus anaerobic conditions , suggesting that protein expression, including ribosomal proteins, may be similarly affected.

  Research approach: Compare rplX expression levels in T. denticola cultured under strictly anaerobic versus microaerobic conditions using RT-qPCR or proteomics.

• Nutrient Availability: Periodontal pockets present a complex nutritional environment with fluctuating resources.

  Investigation methods:

  • Grow T. denticola in media with varying nutrient compositions

  • Monitor rplX expression in response to specific nutritional stress

  • Compare with expression patterns of known virulence factors like dentilisin

• Co-infection with Other Periodontal Pathogens: T. denticola exists in polymicrobial biofilms, and interactions with other species may affect gene expression.

  Experimental design:

  • Co-culture T. denticola with other periodontal pathogens like P. gingivalis

  • Analyze changes in rplX expression compared to monoculture

  • Explore potential regulatory links between rplX and virulence factors

• Host Immune Factors: Exposure to host defense mechanisms may alter ribosomal protein expression.

  Research approach:

  • Expose T. denticola to sublethal concentrations of antimicrobial peptides

  • Analyze changes in rplX expression after interaction with macrophages or neutrophils

  • Investigate potential coordination between rplX expression and stress response genes

A comprehensive understanding of these regulatory patterns could provide insights into T. denticola adaptation during infection and identify potential intervention targets.

How can structural analysis of recombinant T. denticola rplX contribute to antibiotic development strategies?

Structural analysis of T. denticola rplX offers potential for developing targeted antimicrobial strategies:

• Structure Determination Approaches:

  • X-ray crystallography of purified recombinant rplX

  • Cryo-electron microscopy of reconstituted ribosomal complexes containing rplX

  • NMR spectroscopy for dynamic structural information

  • In silico modeling based on homologous structures

• Identification of Unique Structural Features: Comparing the structure of T. denticola rplX with homologs from other bacteria could reveal unique pockets or interfaces that might be targeted by novel antibiotics. The genome comparison between T. denticola and T. pallidum has already revealed significant differences that could extend to ribosomal proteins .

• Rational Drug Design Approach:

  • Virtual screening of compound libraries against identified binding pockets

  • Fragment-based drug design targeting unique structural features

  • Structure-activity relationship studies of compounds binding to rplX

  • Evaluation of species-selectivity based on structural differences between T. denticola rplX and human ribosomal proteins

• Integration with Other Targets: Combined targeting of multiple ribosomal components, including rplX and other factors identified in the T. denticola genome, could provide synergistic antimicrobial effects while reducing the risk of resistance development.

What potential epitopes in T. denticola rplX might be useful for developing diagnostic tools or vaccines?

Identifying immunogenic epitopes in T. denticola rplX could advance both diagnostic and vaccine development efforts:

• Epitope Prediction and Validation:

  • Computational prediction of B-cell and T-cell epitopes based on the rplX sequence

  • Synthesis of predicted epitope peptides for immunological testing

  • ELISA or other immunoassays to validate immunoreactivity with patient sera

  • Epitope mapping using recombinant rplX fragments

• Diagnostic Applications:

  • Development of ELISA or lateral flow assays using recombinant rplX or specific epitope peptides

  • Multiplex assays combining rplX epitopes with other T. denticola antigens

  • Point-of-care diagnostics for periodontal disease risk assessment

• Vaccine Development Considerations:

  • Evaluation of epitope conservation across T. denticola strains

  • Assessment of cross-reactivity with human proteins to avoid autoimmune responses

  • Animal studies to evaluate protective efficacy of rplX-based vaccine candidates

  • Combination with other T. denticola antigens for broader protection

How can recombinant T. denticola rplX be used to study ribosome assembly and function in oral spirochetes?

Recombinant T. denticola rplX provides a valuable tool for investigating ribosome biology in oral spirochetes:

• In vitro Ribosome Assembly Studies:

  • Reconstitution experiments with recombinant rplX and other ribosomal components

  • Analysis of assembly intermediates using density gradient centrifugation

  • Identification of assembly factors specific to T. denticola ribosomes

• Functional Analysis:

  • In vitro translation assays using ribosomes with recombinant rplX

  • Measurement of translation efficiency and fidelity

  • Comparison with ribosomes containing rplX from other bacterial species

• Interaction Studies:

  • Pull-down assays to identify proteins interacting with rplX

  • Characterization of rplX-rRNA interactions using RNA footprinting

  • Investigation of potential moonlighting functions beyond the ribosome

• Evolutionary Perspectives:

  • Comparative analysis of rplX function across different spirochete species

  • Investigation of how T. denticola rplX contributes to the organism's adaptation to its unique ecological niche

  • Study of horizontal gene transfer events that might have influenced rplX evolution, as suggested by genomic analyses of T. denticola

These approaches would provide insights into the specific adaptations of the protein synthesis machinery in T. denticola, potentially revealing unique features that contribute to its pathogenicity.

What is the potential role of T. denticola rplX in biofilm formation and interspecies interactions in the oral microbiome?

The potential role of ribosomal proteins like rplX in biofilm formation represents an emerging area of research:

• Moonlighting Functions: Beyond its canonical role in protein synthesis, rplX might serve additional functions in T. denticola, particularly in the context of biofilm formation:

  • Potential surface exposure of ribosomal proteins under specific conditions

  • Possible interaction with extracellular matrix components

  • Role in cell-cell communication or adhesion

• Interspecies Interactions: T. denticola participates in complex polymicrobial biofilms with other periodontal pathogens:

  • Investigation of rplX expression changes during co-culture with P. gingivalis or F. nucleatum

  • Potential role in synergistic interactions that enhance virulence, similar to T. denticola LrrA protein's role in binding to T. forsythia

  • Contribution to metabolic cooperation within dental plaque biofilms

• Experimental Approaches:

  • Biofilm formation assays comparing wild-type T. denticola with strains expressing modified rplX

  • Localization studies using fluorescently tagged rplX to detect potential surface exposure

  • Protein-protein interaction studies between rplX and components from other oral bacteria

• Regulatory Connections: Analysis of potential regulatory links between rplX expression and known biofilm formation factors in T. denticola, similar to studies that have examined connections between dentilisin protease and iron uptake systems .