Recombinant Treponema denticola Probable inorganic polyphosphate/ATP-NAD kinase (ppnK)

What is the function of ppnK in Treponema denticola?

The ppnK gene (TDE_0514) encodes a probable inorganic polyphosphate/ATP-NAD kinase that catalyzes the phosphorylation of NAD to NADP+, using either ATP or inorganic polyphosphate [poly(P)] as phosphoryl donors. This enzyme is crucial for NADP+ production, which is essential for various anabolic reactions, particularly biosynthetic pathways and oxidative stress response mechanisms in T. denticola . The ability to utilize both ATP and poly(P) distinguishes poly(P)/ATP-NADKs from ATP-specific NADKs, potentially giving T. denticola metabolic flexibility in the nutrient-limited and fluctuating environment of the periodontal pocket.

How is ppnK genetically conserved across T. denticola strains?

Multilocus sequence analysis of T. denticola strains has included ppnK as one of seven conserved protein-encoding genes used for phylogenetic analysis. Studies have shown that ppnK demonstrates approximately 8-19% sequence polymorphism across different T. denticola strains . This level of conservation suggests that ppnK plays an important functional role in T. denticola metabolism. Researchers have identified 6 distinct clonal lineages of T. denticola present in strains isolated from subjects in Asia, Europe, and North America, with several lineages demonstrating global distribution .

What methodology is typically used to clone and express recombinant T. denticola ppnK?

Recombinant T. denticola ppnK is typically produced using the following protocol:

• PCR amplification of the ppnK gene from T. denticola genomic DNA using gene-specific primers containing appropriate restriction sites

• Restriction digestion and ligation into an expression vector (commonly pET-based vectors for E. coli expression)

• Transformation into a competent E. coli expression strain (typically BL21(DE3) or derivatives)

• Induction of protein expression using IPTG (commonly 0.1-1 mM)

• Cell lysis by sonication or mechanical disruption

• Purification using affinity chromatography (typically His-tag purification)

• Further purification using size exclusion and/or ion exchange chromatography

• Confirmation of purity by SDS-PAGE and activity by enzymatic assays

When working with T. denticola proteins, researchers should consider the differences in codon usage between T. denticola and E. coli, which may necessitate codon optimization or use of E. coli strains supplemented with rare tRNAs.

How can the enzymatic activity of recombinant T. denticola ppnK be measured?

The enzymatic activity of recombinant ppnK can be assessed using several methods:

• Spectrophotometric coupled assay: This approach measures NADP+ production through a coupled reaction with an NADP+-dependent enzyme such as glucose-6-phosphate dehydrogenase (G6PDH). The reaction mixture typically contains:

  • Purified recombinant ppnK (1-10 μg)

  • NAD+ (0.5-2 mM)

  • ATP or poly(P) (1-5 mM)

  • MgCl₂ (5-10 mM)

  • G6PDH (1-2 U)

  • Glucose-6-phosphate (2-5 mM)

  • Buffer (typically Tris-HCl or HEPES, pH 7.5-8.0)

  The rate of NADPH formation is measured by monitoring absorbance at 340 nm.

• HPLC analysis: This method directly quantifies NAD+ and NADP+ concentrations. The reaction is stopped at defined time points by heat inactivation or acid precipitation, and the nucleotides are separated by reverse-phase or ion exchange HPLC.

• Luciferase-based ATP consumption assay: For measuring ATP-dependent activity, researchers can monitor ATP consumption using commercial luciferase-based ATP detection kits.

When comparing ATP versus poly(P) as phosphoryl donors, researchers should use equivalent phosphoryl donor concentrations and standardize reaction conditions to directly compare the kinetic parameters.

What are the optimal conditions for studying recombinant T. denticola ppnK activity?

Optimal reaction conditions for T. denticola ppnK typically include:

Researchers should conduct preliminary optimization experiments to determine the exact optimal conditions for their specific recombinant protein preparation.

How does the structure of T. denticola ppnK compare to other bacterial NAD kinases?

While the specific structure of T. denticola ppnK has not been fully characterized, comparative analysis with other bacterial NADKs provides insights:

The ability to utilize both ATP and poly(P) as phosphoryl donors suggests that T. denticola ppnK belongs to the poly(P)/ATP-NADK family, which is typically found in Gram-positive bacteria and Archaea, rather than the ATP-specific NADKs common in Gram-negative bacteria . This is notable because T. denticola is a Gram-negative spirochete, suggesting potential horizontal gene transfer or unique evolutionary adaptation.

Studies of other bacterial NADKs indicate that the phosphoryl donor specificity is often determined by a single amino acid residue. In γ-proteobacterial NADKs, a specific amino acid substitution can confer the ability to utilize poly(P) . Structural analysis would likely reveal:

• A Rossmann-fold NAD-binding domain typical of NADKs

• A distinct ATP-binding pocket

• A specific region or residue(s) that accommodates poly(P) binding

• Potential oligomerization interfaces (many NADKs function as dimers or tetramers)

Researchers investigating T. denticola ppnK structure should consider using X-ray crystallography, cryo-electron microscopy, or homology modeling based on other bacterial NADKs with resolved structures.

What is the role of ppnK in T. denticola stress response and virulence?

NADP+ production via ppnK is critical for:

• Oxidative stress response: NADPH (produced from NADP+ by various dehydrogenases) is essential for maintaining redox homeostasis and detoxifying reactive oxygen species (ROS). T. denticola encounters oxidative stress in the periodontal pocket due to host immune responses.

• Biosynthetic pathways: NADPH is required for anabolic processes, including fatty acid and nucleotide biosynthesis, which are crucial for bacterial growth and adaptation.

• Energy metabolism: The ability to use poly(P) as a phosphoryl donor may provide metabolic flexibility, especially under ATP-limited conditions that might occur in periodontal pockets.

Research has shown that during T. denticola infection, stressors can trigger changes in expression of various metabolic genes. A study investigating differential gene expression in T. denticola mutants found that certain enzymes involved in stress response were upregulated, including desulfoferrodoxin/neelaredoxin (TDE1754), RecA (TDE0872), DNA topoisomerase I (TopA; TDE1208), and transcription termination factor Rho (TDE1503) . While ppnK was not specifically mentioned, these findings highlight the importance of stress response mechanisms in T. denticola.

To explore ppnK's role in virulence, researchers should consider:

• Creating ppnK knockout or knockdown T. denticola strains and assessing changes in virulence in cell culture and animal models

• Examining ppnK expression under various stress conditions (oxidative stress, nutrient limitation, pH shifts)

• Investigating potential interactions between ppnK and known virulence factors like Msp (major surface protein) and dentilisin

How is ppnK expression regulated in T. denticola under different environmental conditions?

Regulation of ppnK expression in T. denticola is not well characterized, but several regulatory mechanisms likely influence its expression:

• Two-component systems (TCS): T. denticola possesses several TCS that sense environmental changes and regulate gene expression accordingly. The AtcSR TCS has been implicated in regulating T. denticola virulence factors and motility . Research shows that AtcS (the histidine kinase component) deletion results in significant changes in the T. denticola transcriptome, particularly affecting genes involved in motility and the dentilisin protease complex .

• Stringent response: T. denticola encodes small alarmone synthetase (Tde-SAS) and hydrolase (Tde-SAH) proteins responsible for synthesizing and hydrolyzing alarmones such as ppGpp, which regulate bacterial adaptation to nutrient limitation . These alarmones may influence ppnK expression under stress conditions.

• Transcriptional regulators: Several transcriptional regulators have been identified in T. denticola, including TDE_0127 (DNA-binding protein), TDE_0814 (transcriptional regulator), and TDE_0344 (AbrB family transcriptional regulator), which show altered expression in dentilisin and Msp-deficient mutants . These regulators may directly or indirectly influence ppnK expression.

Researchers studying ppnK regulation should consider:

• qRT-PCR analysis of ppnK expression under various environmental conditions (pH changes, oxygen tension, nutrient availability)

• Promoter analysis to identify potential regulatory elements

• Chromatin immunoprecipitation (ChIP) to identify transcription factors that bind to the ppnK promoter region

• Analysis of ppnK expression in various regulatory mutants (e.g., TCS deletion strains)

How does phosphoryl donor preference of T. denticola ppnK compare with enzymes from other oral pathogens?

Comparative analysis of phosphoryl donor preference is crucial for understanding metabolic adaptations among oral pathogens:

The ability of T. denticola ppnK to utilize poly(P) is particularly interesting since poly(P) serves as an energy storage molecule in many bacteria. This capability may reflect adaptation to the periodontal pocket environment, where nutrient availability fluctuates.

To study phosphoryl donor preference experimentally, researchers should:

• Express recombinant ppnK from multiple oral pathogens using identical expression systems

• Conduct parallel enzyme assays under standardized conditions with varying concentrations of ATP versus poly(P)

• Determine kinetic parameters (Km, Vmax, kcat) for each phosphoryl donor

• Correlate these biochemical properties with ecological niches and metabolic strategies

What insights can be gained from the kinetic parameters of T. denticola ppnK?

Kinetic parameter analysis provides critical insights into enzyme function and adaptation:

When analyzing kinetic data for T. denticola ppnK, researchers should consider:

• Km values for NAD⁺: Typically in the range of 0.1-1 mM for bacterial NADKs. Lower Km values suggest higher affinity for NAD⁺ and potentially more efficient NADP⁺ production under NAD⁺-limited conditions.

• Km values for phosphoryl donors: Comparative analysis of Km values for ATP versus poly(P) reveals preference. Similar Km values would suggest true dual-specificity, while significantly different values would indicate a preference for one donor.

• Catalytic efficiency (kcat/Km): This parameter allows direct comparison of efficiency with different substrates. Higher catalytic efficiency with poly(P) might indicate evolutionary adaptation to utilize this phosphoryl donor.

• pH and temperature optima: These parameters can provide insights into adaptation to the periodontal pocket environment, which exhibits pH fluctuations and a temperature around 37°C.

• Inhibitor sensitivity: Sensitivity to product inhibition (by NADP⁺) or feedback regulation can reveal regulatory mechanisms affecting enzyme activity in vivo.

Hypothetical kinetic data for T. denticola ppnK might look like:

How can discrepancies in ppnK activity data between different studies be reconciled?

When encountering discrepancies in published ppnK activity data, researchers should systematically analyze potential sources of variation:

• Protein preparation differences:

  • Expression systems (E. coli strains, vector types)

  • Purification methods (affinity tags, chromatography steps)

  • Protein storage conditions (buffer composition, glycerol percentage)

  • Presence/absence of tag cleavage

• Assay condition variations:

  • Buffer composition and pH

  • Temperature

  • Divalent cation type and concentration

  • NAD⁺ concentration ranges

  • Phosphoryl donor type (ATP vs. poly(P)) and concentration

  • Poly(P) chain length (which can significantly affect activity)

• Detection method differences:

  • Direct vs. coupled assays

  • Spectrophotometric vs. HPLC-based detection

  • Endpoint vs. continuous monitoring

• Data analysis approaches:

  • Linear vs. non-linear regression for kinetic parameter determination

  • Inclusion/exclusion of initial lag phases

  • Methods for handling substrate inhibition

To reconcile discrepancies, researchers should:

• Directly compare experimental methods and conditions

• If possible, obtain and test the same protein preparation using different assay methods

• Consider enzyme stability and the potential for different oligomeric states

• Evaluate whether differences reflect true biological variation (different strains) or methodological variation

What is the potential contribution of ppnK to T. denticola's role in periodontal disease progression?

The contribution of ppnK to periodontal disease likely involves several mechanisms:

• Support for survival in the hostile periodontal environment: The periodontal pocket is characterized by fluctuating oxygen levels, pH changes, and nutrient limitations. ppnK-generated NADP⁺ (converted to NADPH) supports detoxification systems that protect against host-derived reactive oxygen species.

• Metabolic adaptation during infection: T. denticola must adapt its metabolism during different stages of periodontal disease progression. The ability to utilize different phosphoryl donors (ATP vs. poly(P)) may provide metabolic flexibility as the microenvironment changes.

• Support for virulence factor production: Major virulence factors of T. denticola include the major surface protein (Msp) and the chymotrypsin-like protease dentilisin . NADPH-dependent biosynthetic pathways may be required for the production and processing of these proteins.

• Contribution to community dynamics: T. denticola exists in polymicrobial biofilms with other periodontal pathogens, notably Porphyromonas gingivalis and Tannerella forsythia (collectively known as the "red complex") . Metabolic capabilities conferred by ppnK may influence T. denticola's interactions with these species.

Studies have shown that T. denticola suppresses host immune responses, including the expression of human β-defensins (HBDs) in gingival epithelial cells and activates mitogen-activated protein kinase signaling via Toll-like receptor 2 (TLR2) . While a direct connection between these processes and ppnK has not been established, the metabolic support provided by ppnK may be essential for these immune modulatory activities.

To study ppnK's role in periodontal disease, researchers should consider:

• Creating ppnK knockdown strains and evaluating their virulence in cell culture and animal models

• Examining ppnK expression during different stages of biofilm formation and in different periodontal microenvironments

• Investigating correlations between ppnK sequence variants and disease severity across clinical isolates

What are the key challenges in producing active recombinant T. denticola ppnK, and how can they be overcome?

Researchers face several challenges when producing recombinant T. denticola ppnK:

• Protein solubility issues:

  • Challenge: Recombinant expression often leads to inclusion body formation

  • Solutions:

    • Use lower induction temperatures (16-20°C)

    • Employ solubility-enhancing fusion tags (MBP, SUMO, TrxA)

    • Optimize expression conditions (IPTG concentration, induction time)

    • Screen multiple E. coli expression strains (BL21(DE3), Rosetta, ArcticExpress)

• Codon usage bias:

  • Challenge: T. denticola has different codon preferences than E. coli

  • Solutions:

    • Use codon-optimized synthetic genes

    • Express in E. coli strains supplying rare tRNAs (Rosetta, CodonPlus)

    • Identify and optimize rare codon clusters in the ppnK sequence

• Protein stability concerns:

  • Challenge: Recombinant ppnK may have limited stability in solution

  • Solutions:

    • Screen stabilizing buffer conditions (pH, salt, additives)

    • Add glycerol (10-20%) to storage buffer

    • Include reducing agents (DTT, β-mercaptoethanol) to prevent oxidation

    • Store at -80°C in small aliquots to avoid freeze-thaw cycles

• Activity verification:

  • Challenge: Ensuring the recombinant enzyme has native-like activity

  • Solutions:

    • Compare kinetic parameters with those of other bacterial NADKs

    • Verify both ATP and poly(P) utilization capabilities

    • Confirm oligomeric state by size exclusion chromatography

    • Use multiple activity assay methods for validation

• Post-translational modifications:

  • Challenge: E. coli may not reproduce native post-translational modifications

  • Solutions:

    • Investigate whether activity requires specific modifications

    • Consider alternative expression systems if necessary

    • Use mass spectrometry to identify any modifications in native enzyme

What approaches can be used to study ppnK function in T. denticola when genetic manipulation is challenging?

Studying ppnK function in T. denticola presents unique challenges due to the difficulty of genetic manipulation in this organism. Alternative approaches include:

• Antisense RNA/CRISPR interference:

  • When gene deletion is not feasible, antisense RNA or CRISPR interference can reduce gene expression

  • Design antisense oligonucleotides or guide RNAs targeting ppnK

  • Introduce via electroporation or conjugation

  • Verify knockdown by qRT-PCR and enzymatic assays

• Chemical inhibition:

  • Identify specific inhibitors of ppnK through in vitro screening

  • Test inhibitor specificity against recombinant enzyme

  • Apply to growing cultures to assess phenotypic effects

  • Confirm target engagement through metabolomic analysis

• Heterologous expression systems:

  • Express T. denticola ppnK in more genetically tractable bacteria

  • Assess impact on host metabolism and stress response

  • Compare with effects of expressing mutated versions

  • Use complementation of NADK-deficient strains to confirm function

• Cell-free systems:

  • Develop T. denticola cell extracts that retain ppnK activity

  • Manipulate extract components to study pathway interactions

  • Add recombinant proteins or inhibitors to modulate activity

  • Monitor metabolite changes using mass spectrometry

• Comparative genomics and transcriptomics:

  • Compare ppnK sequences across clinical isolates with different virulence

  • Correlate expression levels with environmental conditions

  • Identify genes co-regulated with ppnK under various stresses

  • Infer function from integration with metabolic network models