Recombinant Treponema denticola Phosphonates import ATP-binding protein PhnC (phnC)

What is PhnC and what is its role in Treponema denticola?

PhnC (UniProt Q73P71) is a phosphonates import ATP-binding protein in Treponema denticola with an EC classification of 3.6.3.28. As an ATP-binding cassette (ABC) transporter component, it likely participates in the import of phosphonate compounds into T. denticola cells. PhnC functions within the context of T. denticola's role as an oral pathogen implicated in periodontal disease . The protein consists of 256 amino acids and contains characteristic nucleotide-binding domains typical of ABC transporter proteins .

How does PhnC relate to T. denticola's pathogenicity?

While direct evidence connecting PhnC to T. denticola's virulence mechanisms is limited in the current literature, T. denticola is recognized as a keystone pathogen in periodontitis and serves as a model organism for studying Treponema physiology and host-microbe interactions . As a nutrient acquisition protein, PhnC likely contributes to bacterial survival in the competitive oral microenvironment. T. denticola expresses various virulence factors that interact with host proteins and mediate tissue destruction, including surface-expressed and secreted proteins that interact with extracellular matrix components and innate immune system components .

What are phosphonates and why is their transport significant in bacterial systems?

Phosphonates are organophosphorus compounds characterized by direct carbon-phosphorus (C-P) bonds that resist hydrolysis. Their transport is particularly significant in bacterial systems because they serve as alternative phosphorus sources when inorganic phosphate is limited. In T. denticola, the phosphonate import system (of which PhnC is a component) may provide a competitive advantage in the phosphate-limited environment of dental plaque. The ATP-binding capability of PhnC (EC 3.6.3.28) suggests it provides energy for active transport of these compounds across the bacterial membrane .

How is recombinant PhnC typically produced for research purposes?

Recombinant PhnC can be produced in expression systems such as yeast, as indicated in the product information . The production typically involves:

• Cloning the full-length phnC gene (encoding all 256 amino acids) into an appropriate expression vector

• Transforming the construct into a compatible expression host

• Inducing protein expression under optimized conditions

• Purifying the protein using affinity chromatography (facilitated by an appropriate tag)

• Confirming purity (>85%) via SDS-PAGE analysis

• Proper storage at -20°C/-80°C, with glycerol addition recommended for long-term stability

How does the structure of T. denticola PhnC compare to other bacterial phosphonate transporters?

T. denticola PhnC shares structural characteristics with other bacterial ABC-type phosphonate transporters, containing the conserved sequence motifs typical of the ATP-binding cassette superfamily. The protein sequence (MILELKNISK TYPSGRRALQ SISFKIEEGE ILAIIGLSGA GKSTMLRCIN RLVEPDEGEV IFLGEKINKL KGKKLRQYRS KIGMIFQNYN LVERLNAVEN VLHGCLGSIP SYRGALGLYT EEEKEKAFAL LQTVGMEEFA FQRCSELSGG QKQRIGIARA LMQSPKLLLC DEPIASLDPQ SSETVLNYIK EFAVNKNIAC LISLHQMEAA KKYADRIIAL NNGKIVFDGI PDSLNDEVLH KEIFTNVSID SGEKSL) includes the characteristic Walker A motif (G-K-S/T) at positions 48-50 and likely contains the Walker B motif and signature ABC transporter sequence . Unlike surface proteins such as Msp in T. denticola, which forms oligomeric complexes and interacts directly with host molecules like fibronectin , PhnC likely functions primarily in nutrient acquisition rather than direct host interaction.

What experimental approaches can distinguish between different conformational states of PhnC during its catalytic cycle?

To distinguish between different conformational states of PhnC during its ATP binding and hydrolysis cycle, researchers could employ:

• Nucleotide-state trapping experiments using:

  • Non-hydrolyzable ATP analogs (AMP-PNP, ATP-γ-S)

  • Vanadate to trap the transition state

  • Beryllium fluoride (BeF₃⁻) to mimic the ATP-bound state

  • Aluminum fluoride (AlF₄⁻) to mimic the transition state

• Biophysical techniques:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions with altered solvent accessibility

  • Differential scanning fluorimetry (DSF) to assess thermal stability changes

  • Förster resonance energy transfer (FRET) with strategically placed fluorophores to monitor conformational changes

• Structural biology approaches:

  • X-ray crystallography with different nucleotide-bound states

  • Cryo-electron microscopy (cryo-EM) to capture multiple conformational states

  • Nuclear magnetic resonance (NMR) spectroscopy for solution-state dynamics

How might the presence of dentilisin protease activity affect PhnC function in T. denticola?

Dentilisin, a major protease produced by T. denticola, could potentially influence PhnC function through several mechanisms:

• Regulatory interactions: Dentilisin-mediated proteolysis might participate in post-translational regulation of membrane transport systems including the phosphonate import system.

• Modification of extracellular environment: Dentilisin's degradation of host proteins, particularly extracellular matrix components like fibronectin , could release phosphorus-containing compounds that require PhnC-mediated transport for utilization.

• Stress response coordination: T. denticola dentilisin activates TLR2/MyD88 signaling pathways , potentially triggering host cell responses that alter the nutrient environment, necessitating adaptive responses in bacterial transport systems.

• Compartmental protection: While dentilisin is known to degrade extracellular proteins, membrane-associated transport proteins like PhnC may be protected from its activity through spatial separation or structural features, allowing simultaneous function of both virulence and transport systems .

What is the potential evolutionary relationship between T. denticola PhnC and similar transporters in other oral pathogens?

The evolutionary relationship between T. denticola PhnC and homologous transporters in other oral pathogens likely reflects both vertical inheritance and horizontal gene transfer events. As T. denticola is considered a model organism for studying Treponema physiology , comparative analysis of PhnC might reveal:

• Conservation patterns among spirochetes: High sequence similarity would be expected with other Treponema species like T. pallidum, which shares many homologous proteins with T. denticola .

• Functional adaptation signatures: Unique sequence features compared to phosphonate transporters in non-spirochete oral pathogens might indicate adaptation to specific niches within the oral microbiome.

• Horizontal gene transfer evidence: Unusually high similarity to distantly related species might suggest horizontal acquisition, particularly relevant in the diverse oral microbiome where genetic exchange is common.

• Selective pressure indicators: Ratio analysis of synonymous to non-synonymous mutations can reveal whether PhnC has undergone positive selection, potentially indicating adaptation to specific phosphonate sources in the oral environment .

What are the critical factors for maintaining PhnC stability during purification and storage?

Based on recommended handling practices for recombinant PhnC, critical stability factors include:

• Temperature management:

  • Store lyophilized protein at -20°C/-80°C (12-month shelf life)

  • Store reconstituted protein at -20°C/-80°C (6-month shelf life)

  • Working aliquots can be maintained at 4°C for up to one week

• Buffer composition:

  • Addition of glycerol (recommended 5-50%, with 50% as default) for cryoprotection

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

• Physical handling:

  • Brief centrifugation prior to opening vials

  • Avoidance of repeated freeze-thaw cycles

  • Proper aliquoting for single-use applications

• Potential stabilizing additives:

  • Reducing agents to prevent disulfide formation

  • Protease inhibitors to prevent degradation

  • Appropriate salt concentration to maintain protein solubility

What expression systems are most effective for producing functional recombinant T. denticola PhnC?

While the product information indicates yeast as a source for recombinant PhnC production , several expression systems might be considered:

• Yeast systems (e.g., Pichia pastoris, Saccharomyces cerevisiae):

  • Advantages: Post-translational modifications, proper protein folding, high yield

  • Considerations: Codon optimization, signal sequence selection, purification strategy

• Bacterial systems (e.g., Escherichia coli):

  • Advantages: Rapid growth, high yield, simple genetics

  • Considerations: Potential toxicity (as observed with other T. denticola proteins) , inclusion body formation, lack of post-translational modifications

• Insect cell systems:

  • Advantages: Post-translational modifications, complex protein folding

  • Considerations: Longer production time, more complex methodology

• Cell-free systems:

  • Advantages: Avoids toxicity issues, rapid production

  • Considerations: Lower yield, higher cost

Selection factors include protein yield requirements, downstream applications, budget constraints, and available laboratory infrastructure .

How can researchers overcome the challenges of T. denticola genetic manipulation to study PhnC in its native context?

Genetic manipulation of T. denticola presents several challenges, but researchers can employ these strategies:

• Addressing restriction barriers:

  • Unlike other T. denticola genes that contain TdeIII restriction sites (such as msp with its 467GGGCCC473 sequence) , researchers should analyze the phnC sequence for such sites and design mutation strategies accordingly

  • Use of DNA methylation to protect constructs from restriction systems

• Transformation optimization:

  • Electroporation parameter optimization specific for T. denticola

  • Treatment of cells to increase competence

  • Use of shuttle vectors with appropriate origin of replication and selection markers

• Gene replacement strategies:

  • Homologous recombination with adequate flanking sequences

  • CRISPR-Cas9 system adapted for T. denticola

  • Site-directed mutagenesis approaches

• Expression verification:

  • Development of specific antibodies against PhnC

  • Addition of epitope tags that don't interfere with function

  • qRT-PCR to verify transcription levels

What assays can accurately measure PhnC ATPase activity and phosphonate transport?

To effectively measure PhnC functionality, researchers can employ:

• ATPase activity assays:

  • Colorimetric phosphate release assays (malachite green, molybdate)

  • Coupled enzyme assays (pyruvate kinase/lactate dehydrogenase system)

  • Radiometric assays using [γ-32P]ATP

  • Bioluminescence assays measuring ATP consumption

• Transport assays:

  • Radiolabeled phosphonate uptake measurements

  • Fluorescently labeled phosphonate analogs

  • Phosphonate depletion from medium (measured by appropriate analytical methods)

  • Liposome reconstitution systems with purified components

• Binding assays:

  • Isothermal titration calorimetry (ITC) for nucleotide and substrate binding

  • Surface plasmon resonance (SPR) for interaction kinetics

  • Microscale thermophoresis for binding affinity measurements

  • Fluorescence anisotropy with labeled ligands

• Whole-cell approaches:

  • Growth assays with phosphonates as sole phosphorus source

  • Competition assays with phosphonate analogs

  • Transport inhibition studies

How does the amino acid sequence of PhnC inform predictions about its functional domains?

Analysis of the PhnC amino acid sequence (MILELKNISK TYPSGRRALQ SISFKIEEGE ILAIIGLSGA GKSTMLRCIN RLVEPDEGEV IFLGEKINKL KGKKLRQYRS KIGMIFQNYN LVERLNAVEN VLHGCLGSIP SYRGALGLYT EEEKEKAFAL LQTVGMEEFA FQRCSELSGG QKQRIGIARA LMQSPKLLLC DEPIASLDPQ SSETVLNYIK EFAVNKNIAC LISLHQMEAA KKYADRIIAL NNGKIVFDGI PDSLNDEVLH KEIFTNVSID SGEKSL) reveals key functional domains characteristic of ABC transporter nucleotide-binding domains:

• Walker A motif: The sequence GSGAGKS (amino acids 45-51) matches the canonical GxxGxGKS/T pattern essential for ATP binding and phosphate coordination.

• Walker B motif: A hydrophobic sequence followed by aspartate residues (likely in the region around positions 170-180) is responsible for Mg²⁺ coordination and catalysis.

• ABC signature motif: The sequence LSGG (positions 189-192) is part of the LSGGQ motif characteristic of ABC transporters, which interacts with the γ-phosphate of ATP.

• Q-loop and H-loop: Conserved glutamine and histidine residues (positions not explicitly identified in the sequence) that participate in water activation for ATP hydrolysis.

• N-terminal and C-terminal regions: These regions likely mediate interactions with the transmembrane domains of the phosphonate transport system .

What insights can be gained from comparing PhnC to the Major Surface Protein (Msp) in terms of membrane association and exposure?

Comparing PhnC to T. denticola's Major Surface Protein (Msp) reveals important differences in membrane association and exposure:

• Structural organization:

  • Msp: Forms oligomeric outer membrane complexes with porin-like β-barrel structure and has surface-exposed epitopes accessible to proteinase K treatment

  • PhnC: Likely associates with the inner membrane as part of an ABC transporter complex, with minimal surface exposure

• Host interactions:

  • Msp: Directly interacts with host proteins like fibronectin and has cytotoxic pore-forming activity

  • PhnC: Primarily functions in nutrient acquisition rather than direct host interaction

• Protein modifications:

  • Msp: Contains N-glycosylation and surface-exposed epitopes

  • PhnC: May undergo less extensive post-translational modification due to its cytoplasmic/inner membrane localization

• Mutagenesis considerations:

  • Msp: Terminal regions and surface epitopes are critical for oligomer formation, with deletion of as few as three C-terminal amino acids disrupting surface expression

  • PhnC: Mutagenesis likely affects ATP binding and catalysis rather than oligomerization and surface exposure

What role might PhnC play in T. denticola's adaptation to the periodontal environment?

In the context of periodontal disease, PhnC likely contributes to T. denticola's adaptation through:

• Nutrient acquisition: The periodontal pocket is a competitive, nutrient-limited environment where alternative phosphorus sources become important for bacterial survival. PhnC may enable T. denticola to utilize phosphonates when inorganic phosphate is limited .

• Environmental sensing: ABC transporters often function in environmental sensing, potentially allowing T. denticola to detect and respond to changes in the periodontal microenvironment during disease progression.

• Metabolic flexibility: The ability to import and metabolize phosphonates provides metabolic flexibility that contributes to T. denticola's role as a keystone pathogen in periodontitis .

• Indirect virulence contribution: While not a direct virulence factor like dentilisin (which triggers TLR2/MyD88 activation and tissue destruction) , PhnC supports bacterial growth and persistence, indirectly contributing to the long-term tissue damage characteristic of periodontal disease .

What are the most promising future research directions for understanding PhnC's role in T. denticola biology?

The most promising future research directions include:

• Structural biology: Determining the three-dimensional structure of PhnC alone and in complex with its transport partners would provide insights into its mechanism and potential for targeted inhibition.

• Host-pathogen interface: Investigating how phosphonate metabolism relates to T. denticola's interactions with other oral microbiome members and host cells during periodontal disease progression.

• Systems biology: Integration of PhnC function within the broader metabolic network of T. denticola to understand its contribution to survival and virulence in changing environmental conditions.

• Therapeutic targeting: Exploration of PhnC as a potential target for novel anti-spirochete therapeutics, potentially addressing both T. denticola in periodontal disease and related pathogens like T. pallidum .

• Comparative analysis: Further characterization of PhnC relative to its homologs in other oral and non-oral pathogens to understand specialized adaptations in phosphonate utilization strategies .