Recombinant Treponema denticola SsrA-binding protein (smpB)

What is SsrA-binding protein (SmpB) and what is its fundamental role in bacterial systems?

SmpB is a unique RNA-binding protein that serves as an essential component of the bacterial quality control system. It works in conjunction with SsrA RNA (also known as tmRNA or 10Sa RNA) to form a ribonucleoprotein complex that recognizes ribosomes stalled on defective mRNAs . This system plays a critical role in:

• Rescuing stalled ribosomes

• Tagging incomplete proteins for degradation

• Preventing accumulation of potentially toxic protein fragments

Structurally, SmpB contains approximately 160 amino acids and is reasonably basic with 36 lysine, histidine, and arginine residues balanced against 18 aspartic acid and glutamic acid residues. It is predominantly a β-sheet protein, as indicated by its circular dichroism spectrum .

In experimental settings, SmpB has been demonstrated to bind specifically and with high affinity to SsrA RNA with a Kd of approximately 20 nM under physiological conditions, showing approximately 400-fold higher specificity for SsrA RNA compared to bulk tRNA .

How is the SsrA-SmpB system experimentally studied in periodontal pathogens?

When studying the SsrA-SmpB system in periodontal pathogens like Treponema denticola, researchers typically employ several methodological approaches:

Genetic approaches:

• Gene deletion studies: Constructing SmpB-defective strains by deleting >80% of the smpB gene, similar to approaches used in E. coli studies. These deletion mutants can be examined for phenotypic changes compared to wild-type strains .

• Complementation assays: Testing whether SmpB from T. denticola can functionally complement SmpB-deficient E. coli strains to determine functional conservation.

Biochemical approaches:

• Protein purification: Recombinant SmpB can be overexpressed, purified, and characterized through sequential Edman degradation to confirm the N-terminal sequence .

• RNA-binding assays: Gel-mobility shift assays can be used to investigate the interaction between recombinant SmpB and SsrA RNA, measuring binding affinity and specificity .

• Ribosome association analysis: Fractionation of cell lysates followed by Northern blot hybridization to monitor the localization of SsrA RNA in wild-type versus SmpB mutant strains .

How does the structure of recombinant T. denticola SmpB compare to SmpB proteins from other bacteria?

Comparative structural features:

SmpB is considered to be a unique protein family present universally in bacteria, with no significant homologs in eukaryotic or archaeal organisms . This makes it an interesting target for studying bacterial-specific processes.

What methods are used to express and purify recombinant T. denticola SmpB?

The expression and purification of recombinant T. denticola SmpB presents several technical challenges that researchers should address through specific methodological approaches:

Expression strategies:

• Vector selection: Tightly regulated T7 RNA polymerase expression systems are recommended as SmpB expression can be toxic to E. coli when the entire gene is present .

• Co-expression: Co-expressing SmpB with SsrA RNA can result in production of a soluble complex, addressing the issue of inclusion body formation when SmpB is expressed alone .

• Expression constructs: Using a pET28b expression vector to add a His6-tag to the N-terminus of SmpB facilitates purification and detection .

Purification protocol:

• Metal affinity chromatography: Purify His6-tagged SmpB using Ni²⁺-NTA chromatography.

• Size-exclusion chromatography: Further purify using gel filtration (e.g., Sephacryl S300) in buffer containing 200 mM KCl .

• Protein folding verification: Assess proper folding using circular dichroism spectroscopy, which typically shows a predominantly β-sheet structure for properly folded SmpB .

Purity assessment:

• SDS-PAGE and silver staining should show a band corresponding to the expected molecular weight (~21 kDa for His6-SmpB) .

• Recombinant SmpB purity should exceed 90% for functional studies .

How can researchers experimentally determine if T. denticola SmpB forms a larger ribonucleoprotein complex similar to that observed in other bacteria?

To investigate whether T. denticola SmpB forms a larger ribonucleoprotein complex with additional protein partners, researchers can employ a systematic multi-technique approach:

Co-immunoprecipitation studies:

• Express His6-tagged SmpB in T. denticola or E. coli.

• Use the tagged SmpB as "bait" to capture associated proteins.

• Analyze co-precipitated proteins by SDS-PAGE, silver staining, and mass spectrometry.

This approach has previously identified a larger SsrA-SmpB ribonucleoprotein complex in E. coli that contains ribosomal protein S1, phosphoribosyl pyrophosphate synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB . A comparable analysis in T. denticola might reveal spirochete-specific complex components.

RNA-dependent associations:
Researchers should test whether observed protein associations are direct or mediated by RNA:

• Treatment of purified complexes with RNase or hydroxide to disrupt RNA-dependent interactions

• Northern blot analysis using probes specific for SsrA RNA and ribosomal RNAs to identify associated RNA species

Size estimation of native complexes:

• Gel filtration chromatography to determine the approximate molecular weight of the native complex

• Blue native PAGE to analyze intact membrane-protein complexes

• Gradient ultracentrifugation to separate complexes based on size and density

What are the functional differences between SmpB in T. denticola and other periodontal pathogens, and how can these be experimentally determined?

To investigate functional differences between SmpB in T. denticola and other bacteria, researchers can employ several comparative experimental approaches:

Cross-species complementation assays:

• Generate SmpB deletion mutants in E. coli and T. denticola.

• Introduce plasmids expressing SmpB from different species.

• Measure the ability to restore phenotypes such as:

  • Phage development (λ immP22 hybrid phage plating)

  • SsrA-mediated tagging of model substrates

  • Ribosome association of SsrA RNA

Substrate specificity analysis:
The substrate-binding domains of Lon proteases in different bacteria show considerable variation, suggesting they may recognize divergent substrates . Similarly, SmpB proteins might show species-specific substrate preferences:

• Construct hybrid SmpB proteins with domains from different species.

• Test binding to various model substrates and SsrA RNAs from different species.

• Analyze binding kinetics using surface plasmon resonance or microscale thermophoresis.

Structural biology approaches:

• X-ray crystallography or cryo-EM to determine structures of SmpB-SsrA complexes from different species

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• NMR spectroscopy to analyze domain dynamics and interactions

What experimental strategies can be employed to examine the role of T. denticola SmpB in virulence and host-pathogen interactions?

To investigate the potential role of T. denticola SmpB in virulence and host-pathogen interactions, researchers can implement several experimental strategies:

Genetic manipulation approaches:

• Gene deletion: Generate SmpB-deficient T. denticola mutants using recombination cassettes constructed via PCR-based splicing by overlap-extension (SOE) methods .

• Complementation studies: Re-introduce wild-type or mutant SmpB to determine which domains are essential for virulence phenotypes.

• Domain mapping: Create targeted mutations in functional domains to determine their specific roles in virulence.

Host interaction models:

• Epithelial cell co-culture: Assess cytotoxicity, adhesion, and invasion capabilities of wild-type versus SmpB mutant T. denticola.

• Immune cell response: Measure neutrophil chemotaxis inhibition, similar to studies with Msp protein .

• Multispecies biofilm models: Evaluate the role of SmpB in synergistic biofilm formation with other oral pathogens .

In vivo models:
Previous studies have shown that disruption of the smpB gene decreased the virulence of Salmonella typhimurium in mice and reduced bacterial survival within macrophages . Similar approaches could be applied to T. denticola:

• Animal periodontitis models to compare wild-type and SmpB-deficient strains

• Competitive index assays to measure relative fitness in vivo

• Histopathological analysis to assess tissue damage and immune cell recruitment

How can researchers design experiments to investigate the potential involvement of T. denticola SmpB in stress response and antibiotic resistance?

The SsrA-SmpB system plays a critical role in bacterial quality control and stress response. To investigate its potential involvement in T. denticola stress response and antibiotic resistance, researchers can implement these experimental designs:

Stress response analysis:

• Growth curve analysis: Compare growth of wild-type and SmpB-deficient T. denticola under various stress conditions:

  • Temperature shifts (heat shock)

  • Oxidative stress (H₂O₂ exposure)

  • Nutrient limitation

  • pH fluctuations (acid stress)

• Transcriptome analysis (RNA-Seq):

  • Examine global gene expression changes in wild-type vs. SmpB-deficient strains

  • Identify stress-response pathways affected by SmpB deficiency

  • Compare transcriptional profiles under normal and stress conditions

Antibiotic susceptibility testing:

• Minimum inhibitory concentration (MIC) determination for various antibiotics

• Time-kill kinetics to assess the rate of bacterial death

• Persister cell formation assays following antibiotic exposure

Proteome quality control assessment:
Since the SsrA-SmpB system targets incomplete proteins for degradation, researchers can examine protein aggregation and turnover:

• Monitor accumulation of protein aggregates using fluorescent reporters

• Measure degradation rates of model substrates with or without SsrA tags

• Examine production of specific stress proteins under antibiotic exposure

What are the current methodological challenges in studying T. denticola SmpB and potential solutions?

Researchers face several significant challenges when studying T. denticola SmpB:

Challenge 1: Genetic manipulation difficulties
T. denticola genetic manipulation is complicated by:

• The presence of restriction-modification systems (like TdeIII) that recognize specific DNA sequences and prevent transformation

• Toxic effects when expressing full-length proteins (such as Msp) in E. coli

Solutions:

• Use host strains lacking restriction systems (e.g., T. denticola 35405/ΔTDE0911)

• Employ tightly regulated expression systems with inducible promoters

• Construct complete deletion mutants first, then introduce modified genes

• Use PCR-based methods like splicing by overlap-extension (SOE) and FastCloning for mutagenesis

Challenge 2: Protein expression and folding issues

• Formation of inclusion bodies when overexpressing SmpB alone

• Obtaining soluble, properly folded protein for functional studies

Solutions:

• Co-express SmpB with SsrA RNA to improve solubility

• Add solubility tags or fusion partners

• Use mild detergents or optimize buffer conditions

• Express protein in spirochete-specific cell-free systems

Challenge 3: Functional assay limitations

• Difficulty in developing assays specific to T. denticola SmpB function

• Limited availability of T. denticola-specific tools and reagents

Solutions:

• Develop reporter systems using SsrA-tagged fluorescent proteins

• Create in vitro translation systems derived from T. denticola

• Establish dual-species assays to detect cross-talk with other oral bacteria

How can researchers develop novel therapeutic strategies targeting the T. denticola SmpB-SsrA system for periodontal disease treatment?

The SmpB-SsrA system represents a potential therapeutic target for periodontal disease treatment due to its essential role in bacterial quality control. Researchers can explore several strategies to develop novel therapeutics:

Target validation approaches:

• Demonstrate essentiality: Determine whether SmpB is essential for T. denticola survival or virulence through conditional knockdown systems

• Phenotypic characterization: Compare biofilm formation, host cell interactions, and virulence between wild-type and SmpB-deficient strains

• In vivo significance: Validate the importance of SmpB in animal models of periodontitis

Drug discovery strategies:

• High-throughput screening:

  • Develop fluorescence-based assays to monitor SmpB-SsrA binding

  • Screen compound libraries for inhibitors of this interaction

  • Validate hits using secondary biochemical and cellular assays

• Structure-based drug design:

  • Determine crystal structures of T. denticola SmpB alone and in complex with SsrA

  • Identify binding pockets suitable for small molecule inhibition

  • Use virtual screening and molecular docking to identify lead compounds

Therapeutic development approaches:

• Peptide inhibitors: Design peptides that mimic SsrA RNA binding sites to competitively inhibit SmpB function

• RNA aptamers: Develop aptamers that specifically bind to SmpB and prevent SsrA interaction

• CRISPR-Cas targeting: Design CRISPR-Cas systems to specifically target the smpB gene in T. denticola

Delivery considerations:

• Oral biofilm penetration: Formulate therapeutics to penetrate dental biofilms

• Selectivity: Design molecules that specifically target T. denticola without disrupting beneficial oral microbiota

• Combination approaches: Develop multi-target strategies combining SmpB inhibitors with conventional antimicrobials