Recombinant Treponema denticola Ribosome maturation factor RimM (rimM)

What is Treponema denticola and why is its RimM factor significant for research?

Treponema denticola is an oral spirochete strongly implicated in the etiology of chronic periodontitis and other periodontal diseases. It forms part of the pathogenic "Red Complex" bacterial consortium alongside Porphyromonas gingivalis and Tannerella forsythia, which is strongly associated with clinical progression of chronic periodontitis . The ribosome maturation factor RimM in T. denticola is significant because ribosomal assembly and function are crucial for bacterial protein synthesis, survival, and virulence. Understanding RimM's role provides insights into fundamental bacterial processes and potential targets for intervention in periodontal disease.

How does RimM function in ribosome maturation?

RimM functions as a critical accessory factor in 16S rRNA maturation and 30S ribosomal subunit assembly. While specific T. denticola RimM mechanisms aren't fully characterized in the provided research, studies of RimM in other organisms show it typically associates with the 30S ribosomal subunit during assembly . RimM binds to specific regions of the 16S rRNA to facilitate proper folding and processing, ensuring the formation of functional ribosomes. This process is essential for efficient translation and protein synthesis, which underpin bacterial growth, adaptation, and virulence factor expression.

What structural features characterize T. denticola RimM compared to RimM in other bacterial species?

While the specific structural details of T. denticola RimM aren't explicitly described in the available research, RimM proteins typically contain conserved domains that facilitate RNA binding and protein-protein interactions. Researchers investigating T. denticola RimM should consider comparative structural analysis with better-characterized RimM proteins from other bacterial species. Important structural analyses would include identification of RNA-binding motifs, interaction domains with ribosomal proteins, and any spirochete-specific domains that might contribute to specialized functions in T. denticola.

What are the optimal conditions for expressing recombinant T. denticola RimM in heterologous systems?

For efficient expression of recombinant T. denticola RimM, researchers should consider the following methodological approach:

Expression System Selection:

• E. coli-based systems: BL21(DE3) or Rosetta strains are recommended for addressing potential codon bias issues, as T. denticola has different codon usage patterns than E. coli.

• Vector design: Incorporate a fusion tag (His6, GST, or MBP) to facilitate purification and potentially enhance solubility.

Expression Conditions:

The expression should be validated using Western blot analysis with antibodies against the fusion tag or RimM protein. Optimization may be necessary through systematic variation of these parameters to determine ideal conditions for your specific construct.

What are the most effective methods for studying RimM interactions with ribosomal components?

To investigate RimM interactions with ribosomal components, researchers should employ a multi-method approach:

• Co-immunoprecipitation (Co-IP) using antibodies against RimM to pull down associated ribosomal proteins and RNA, followed by mass spectrometry for protein identification and RT-PCR for RNA detection.

• Surface Plasmon Resonance (SPR) or Microscale Thermophoresis (MST) to determine binding kinetics (kon, koff) and affinity (KD) between purified RimM and isolated ribosomal components.

• Bacterial Two-Hybrid Systems to confirm protein-protein interactions in vivo, particularly useful for identifying other maturation factors that may cooperate with RimM .

• RNA-protein binding assays such as Electrophoretic Mobility Shift Assays (EMSA) to characterize interactions between RimM and specific 16S rRNA regions.

• Cryo-electron microscopy (Cryo-EM) for structural visualization of RimM associated with ribosomal subunits, providing insights into binding interfaces and conformational changes.

These methods can be complemented by comparing wild-type and RimM-deficient strains to assess functional impacts on ribosome assembly and maturation.

How can researchers design experiments to assess the impact of RimM mutations on T. denticola virulence?

A comprehensive experimental approach to assess the impact of RimM mutations on T. denticola virulence should include:

In Vitro Studies:

• Generate site-directed mutants targeting conserved RimM domains

• Assess growth rates and stress responses of mutants versus wild-type

• Quantify biofilm formation capabilities on relevant surfaces

• Measure expression levels of known virulence factors, such as dentilisin protease and leucine-rich repeat proteins

• Conduct co-culture experiments with other Red Complex bacteria to assess interspecies interactions

Cell Culture Models:

• Evaluate adhesion and invasion of oral epithelial cells

• Measure cytokine responses in human gingival fibroblasts

• Assess immunomodulatory effects on macrophages and neutrophils

Animal Models:

• Use established rodent periodontitis models with wild-type and RimM mutant strains

• Measure alveolar bone loss as a primary outcome

• Analyze bacterial load and host inflammatory responses

• Consider polymicrobial infections with other Red Complex members to model natural disease state

Data from these experiments should be integrated to develop a comprehensive model of how RimM mutations affect T. denticola pathogenicity through altered ribosome function and subsequent impacts on virulence factor expression.

How does T. denticola RimM contribute to bacterial adaptation in the periodontal pocket environment?

T. denticola RimM likely plays a crucial role in bacterial adaptation to the periodontal pocket environment through multiple mechanisms:

• Stress Response Modulation: RimM's function in ribosome maturation may influence the translation efficiency of stress response proteins. In the periodontal pocket, where conditions include oxygen gradients, pH fluctuations, and nutrient limitations, proper ribosomal function is essential for rapid adaptation.

• Virulence Factor Regulation: RimM may indirectly regulate the expression of virulence determinants such as the leucine-rich repeat proteins (like LrrA) that mediate interactions with other bacteria in the Red Complex and with host cells . By ensuring efficient translation, RimM enables the bacterium to produce appropriate levels of virulence factors.

• Metabolic Flexibility: T. denticola relies on complex anaerobic fermentation of amino acids, producing toxic metabolites that contribute to tissue damage . Efficient ribosome assembly mediated by RimM ensures appropriate expression of metabolic enzymes needed for this nutritional strategy.

• Biofilm Integration: Within polymicrobial biofilms, T. denticola must coordinate its gene expression to interact with other species. RimM-mediated translational efficiency may be critical for producing the adhesins and other factors that facilitate incorporation into the polymicrobial community associated with periodontitis .

Research methodology to investigate these contributions would involve comparative transcriptomic and proteomic analyses of wild-type and RimM-deficient strains under conditions mimicking the periodontal pocket.

What are the potential interactions between RimM and other ribosome assembly factors in T. denticola?

RimM likely functions within a network of ribosome assembly factors in T. denticola. While specific interactions in T. denticola haven't been fully characterized, research in other bacteria suggests several potential interacting partners:

Potential RimM Interaction Network:

• RimP and RbfA: These factors likely cooperate with RimM during early 30S subunit assembly, with each binding to specific regions of the 16S rRNA.

• RsmD methyltransferase: Based on findings in other systems, RimM may interact with RsmD, which methylates specific residues in 16S rRNA. This interaction could coordinate rRNA modification with assembly steps .

• Era and RsgA GTPases: These assembly factors may coordinate with RimM to ensure proper 30S subunit maturation through GTP-dependent conformational changes.

• S19 and S13 ribosomal proteins: RimM likely interacts with these proteins during assembly of the 30S subunit head domain.

Research methods to investigate these interactions should include:

• Bacterial two-hybrid screens to identify novel interacting partners

• Co-immunoprecipitation followed by mass spectrometry

• Genetic suppressor screens to identify functional relationships

• Fluorescence resonance energy transfer (FRET) experiments to detect direct interactions in vivo

Understanding this interaction network would provide insights into spirochete-specific aspects of ribosome assembly and potential vulnerabilities that could be targeted for therapeutic intervention.

How might inhibition of RimM function impact antibiotic susceptibility in T. denticola?

Inhibition of RimM function could significantly alter antibiotic susceptibility in T. denticola through several mechanisms:

• Ribosome-targeting antibiotics: Many antibiotics (macrolides, aminoglycosides, tetracyclines) target bacterial ribosomes. RimM inhibition would disrupt proper ribosome assembly, potentially creating abnormal ribosomal structures with altered binding sites for these antibiotics. This could lead to:

  • Increased sensitivity to some antibiotics due to compromised ribosome function

  • Potential resistance to others if binding sites are malformed

• Stress response modulation: RimM inhibition would create translational stress, potentially triggering upregulation of stress response mechanisms that might cross-protect against antibiotic action.

• Growth rate effects: Compromised RimM function would likely reduce growth rate, which can affect susceptibility to many antibiotics that target actively dividing cells.

Experimental approach to investigate this question:

This research direction is particularly relevant given the emergence of antibiotic resistance in oral pathogens and could identify RimM as a potential target for adjunctive therapy alongside conventional antibiotics.

What are the key challenges in purifying active recombinant T. denticola RimM?

Purifying active recombinant T. denticola RimM presents several technical challenges:

• Protein solubility: RimM proteins often have hydrophobic regions that can cause aggregation. Researchers should:

  • Use solubility-enhancing fusion tags (MBP, SUMO)

  • Express at lower temperatures (16-20°C)

  • Include stabilizing agents (glycerol, arginine) in purification buffers

  • Consider detergent screening if membrane association is suspected

• Maintaining RNA-binding activity: As an RNA-binding protein, RimM requires specific conditions to maintain its functional conformation:

  • Include low concentrations of reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Maintain physiological salt concentrations (100-150 mM NaCl)

  • Avoid excessive exposure to room temperature

  • Consider including RNA stabilizers such as magnesium ions

• Preventing proteolytic degradation:

  • Include protease inhibitor cocktails throughout purification

  • Minimize purification time with streamlined protocols

  • Consider using protease-deficient expression hosts

• Verifying activity:

  • Develop functional assays based on RNA binding (EMSA)

  • Assess interaction with 30S ribosomal subunits using sedimentation assays

  • Consider thermal shift assays to confirm proper folding

A systematic approach to optimization, coupled with rigorous activity testing, is essential for successful purification of functional T. denticola RimM.

How can researchers effectively create and validate RimM knockout or knockdown models in T. denticola?

Creating and validating RimM knockout or knockdown models in T. denticola requires specialized approaches due to the genetic manipulation challenges in spirochetes:

Knockout Strategies:

• Homologous recombination:

  • Design constructs with antibiotic resistance cassettes flanked by 1-2 kb homology arms

  • Optimize electroporation conditions (field strength: 1.8-2.5 kV/cm)

  • Include glycine (0.5-2%) in pre-electroporation culture to weaken the cell wall

  • Extend recovery time (16-24 hours) before antibiotic selection

• CRISPR-Cas9 approaches:

  • Use codon-optimized Cas9 for T. denticola

  • Design guide RNAs targeting non-essential regions of rimM

  • Consider inducible Cas9 systems to minimize toxicity

Knockdown Alternatives:

• Antisense RNA expression:

  • Design antisense constructs targeting the 5' region of rimM mRNA

  • Use inducible promoters to control expression level

  • Monitor knockdown efficiency by qRT-PCR

• CRISPRi (CRISPR interference):

  • Express catalytically inactive Cas9 (dCas9) with guide RNAs targeting rimM

  • Optimize guide RNA design to target the promoter region or early coding sequence

Validation Methods:

These genetic manipulation strategies must be adapted to the specific challenges of T. denticola, including its slow growth rate and unusual cell envelope structure.

What analytical methods can best determine the impact of RimM on global protein synthesis in T. denticola?

To comprehensively assess the impact of RimM on global protein synthesis in T. denticola, researchers should employ multiple complementary analytical approaches:

• Ribosome Profiling:

  • This next-generation sequencing approach provides genome-wide information on ribosome positioning and translation efficiency

  • Compare translation efficiency metrics between wild-type and RimM-depleted strains

  • Identify specific mRNAs most affected by RimM deficiency

  • Protocol adaptation: Include longer flash-freezing in liquid nitrogen and rapid lysis steps to capture the translation status in T. denticola

• Polysome Profiling:

  • Analyze polysome distribution using sucrose gradient centrifugation

  • Quantify monosome and polysome peaks to assess global translation efficiency

  • Examine specific mRNA distribution across polysome fractions using qRT-PCR

  • Technical consideration: Use gentler lysis conditions to preserve intact polysomes from T. denticola

• Metabolic Labeling:

  • Measure incorporation rates of radioactive (35S-methionine) or non-radioactive (BONCAT with azidohomoalanine) amino acids

  • Perform pulse-chase experiments to assess protein synthesis kinetics and stability

  • Combine with 2D gel electrophoresis or mass spectrometry for protein-specific analysis

  • Note: Optimize labeling conditions for the slower growth rate of T. denticola

• Quantitative Proteomics:

  • Use SILAC, TMT or label-free quantification with LC-MS/MS

  • Compare proteome composition between wild-type and RimM-depleted strains

  • Conduct time-course experiments to identify early vs. late effects

  • Bioinformatic analysis: Group affected proteins by function to identify pathways most impacted

• Targeted Analysis of Key Virulence Factors:

  • Develop selected reaction monitoring (SRM) mass spectrometry assays for known T. denticola virulence factors

  • Monitor synthesis rates of specific proteins involved in pathogenesis, including components of the motility apparatus and proteases

Integration of these methods provides a comprehensive view of how RimM affects translation globally and for specific mRNAs, connecting ribosome maturation defects to consequences for bacterial physiology and virulence.

What are the potential therapeutic applications of targeting RimM in oral pathogens?

Targeting RimM in oral pathogens presents several therapeutic opportunities with unique advantages over conventional approaches:

• Novel Antimicrobial Development:

  • RimM inhibitors would target a pathway distinct from existing antibiotics

  • Small molecule inhibitors could be designed to interfere with:

    • RimM-RNA binding interfaces

    • RimM-protein interactions essential for function

    • Allosteric sites affecting RimM conformation

  • Advantages include potential activity against antibiotic-resistant strains due to the novel target

• Anti-virulence Strategy:

  • Partial inhibition of RimM could attenuate virulence without strong selection for resistance

  • This approach may:

    • Reduce virulence factor production

    • Impair biofilm formation

    • Limit interspecies interactions within the Red Complex

  • Benefits include reduced disruption of commensal microbiota compared to broad-spectrum antibiotics

• Combination Therapies:

  • RimM inhibitors could sensitize T. denticola to conventional antibiotics

  • Potential synergistic effects with:

    • Ribosome-targeting antibiotics

    • Cell wall-active compounds

    • Host-defense peptides

  • This approach might allow lower doses of conventional agents, reducing side effects

• Delivery Considerations for Periodontal Application:

  • Local delivery systems could include:

    • Controlled-release polymer films

    • Hydrogels for subgingival application

    • Nanoparticle formulations for penetration into biofilms

  • Target product profile would emphasize sustained release and retention in the periodontal pocket

This research direction would benefit from screening approaches to identify lead compounds, followed by medicinal chemistry optimization and testing in relevant biofilm and animal models of periodontitis.

How might comparative genomics of RimM across oral spirochetes inform evolutionary adaptations in ribosome assembly?

Comparative genomics of RimM across oral spirochetes can provide valuable insights into evolutionary adaptations in ribosome assembly mechanisms:

• Sequence Conservation Analysis:

  • Multiple sequence alignment of RimM proteins from diverse spirochetes (Treponema denticola, T. socranskii, T. vincentii, etc.)

  • Identification of:

    • Core conserved domains indicative of essential functions

    • Variable regions suggesting species-specific adaptations

    • Signature motifs unique to oral spirochetes

• Structural Bioinformatics:

  • Homology modeling of spirochete RimM proteins

  • Comparison with solved structures from model organisms

  • Analysis of:

    • RNA-binding surfaces

    • Protein-protein interaction interfaces

    • Spirochete-specific structural elements

• Genomic Context Analysis:

  • Examination of gene neighborhoods around rimM

  • Identification of conserved or variable operonic structures

  • Detection of potential co-evolution patterns with other ribosome assembly factors

• Evolutionary Rate Analysis:

  • Calculation of dN/dS ratios to identify regions under selective pressure

  • Identification of positively selected sites that may indicate adaptation to specific niches

  • Correlation of evolutionary rates with pathogenicity potential

• Host-Adaptation Signatures:

  • Comparison of RimM between host-associated and free-living spirochetes

  • Identification of features potentially related to:

    • Adaptation to inflammatory environments

    • Interaction with host translation machinery

    • Resistance to host defense mechanisms targeting translation

This research would not only illuminate the evolution of ribosome assembly in this important bacterial phylum but could also identify spirochete-specific features of RimM that might be exploited for targeted therapeutic development.

What roles might RimM play in coordinating ribosome assembly with other cellular processes in T. denticola?

RimM likely serves as a regulatory node coordinating ribosome assembly with other cellular processes in T. denticola through several interconnected mechanisms:

• Stress Response Integration:

  • RimM may function as a conditional assembly factor whose activity is modulated during stress

  • Research approach: Compare RimM expression and localization under various stress conditions (oxidative, nutritional, pH) using fluorescence microscopy with tagged constructs

  • Expected findings: Potential redistribution of RimM during stress, suggesting altered ribosome assembly priorities

• Growth Phase-Dependent Regulation:

  • RimM function may vary with growth phase to adjust ribosome production to metabolic demands

  • Methodology: Time-course analysis of RimM-dependent ribosome assembly during different growth phases using quantitative mass spectrometry of ribosomal proteins

  • Hypothesis: RimM activity may be highest during exponential growth and modulated during stationary phase

• Nutrient Sensing Pathways:

  • RimM activity could be linked to nutrient availability sensors like (p)ppGpp-mediated stringent response

  • Experimental approach: Examine RimM function in stringent response mutants using ribosome profiling

  • Potential mechanism: Post-translational modifications of RimM in response to nutritional status

• Cell Division Coordination:

  • RimM-mediated ribosome assembly may be synchronized with cell division

  • Research strategy: Single-cell analysis of RimM localization throughout the cell cycle using super-resolution microscopy

  • Expected pattern: Potential co-localization with division machinery or asymmetric distribution during cell division

• Virulence Regulation Networks:

  • RimM may interface with virulence regulatory networks to coordinate translation capacity with virulence factor production

  • Approach: ChIP-seq analysis to identify potential interactions between RimM and virulence regulators, coupled with transcriptome analysis of virulence genes in RimM-depleted strains

  • Relevance: Could explain how T. denticola coordinates resource allocation between growth and virulence

These interconnections would position RimM as more than just a ribosome assembly factor, but as a potential integrator of cellular states that helps T. denticola adapt to the dynamic environment of the periodontal pocket.