Recombinant Treponema pallidum subsp. pallidum Nucleoside diphosphate kinase (ndk)

What is the function of Nucleoside Diphosphate Kinase (NDK) in Treponema pallidum?

Nucleoside diphosphate kinases represent an important class of enzymes responsible for maintaining cellular metabolism by providing pools of nucleotide triphosphates (NTPs) and deoxynucleotide triphosphates (dNTPs) for various energetic processes . In spirochetes like T. pallidum, NDK produces NTPs and dNTPs from NDPs and dNDPs via a catalytic reversible phosphate-transfer mechanism, similar to the process observed in Borrelia burgdorferi . This reaction proceeds through a ping-pong mechanism using a phosphorylated histidine intermediate . For organisms with restricted metabolic capabilities like spirochetes, NDK activity is particularly crucial because these bacteria cannot support the Krebs cycle, oxidative phosphorylation, or de novo biosynthesis pathways for nutrients such as amino acids, carbohydrates, or lipids .

How does NDK catalytic activity contribute to T. pallidum pathogenesis?

Based on studies with related spirochetes, NDK function appears critical for establishing robust infections. Infectivity studies with ndk transposon mutants in B. burgdorferi demonstrated that NDK function was important for establishing robust infection in mice, providing a rationale for therapeutic targeting of this enzyme . For T. pallidum, which has extremely limited metabolic capabilities and relies heavily on host-derived nutrients, NDK likely plays an essential role in maintaining nucleotide balance during infection . T. pallidum undergoes intense antigenic responses during all stages of infection, suggesting the importance of proteins involved in basic cellular functions like NDK .

What expression systems are most effective for producing recombinant T. pallidum NDK?

For recombinant expression of T. pallidum proteins, E. coli-based systems with hexahistidine tags have proven successful for related proteins. The insertion of PCR-amplified DNA into E. coli expression vectors results in high-level expression of treponemal antigens . N-terminal hexahistidine sequences allow efficient purification of fusion proteins by metal chelate affinity chromatography . This approach has been successfully applied to several T. pallidum proteins and could be adapted for NDK expression. For structural studies, the Seattle Structural Genomics Center for Infectious Diseases (SSGCID) protocols for recombinant protein expression could be employed, as used successfully with B. burgdorferi NDK .

What are the key structural features of T. pallidum NDK?

While specific structural data for T. pallidum NDK is not extensively documented, comparisons with other bacterial NDKs, particularly from B. burgdorferi, suggest it likely shares similar primary, secondary, tertiary, and quaternary structures . Based on structural genomics studies of B. burgdorferi NDK, T. pallidum NDK would be expected to:

• Form a hexameric quaternary structure

• Contain conserved residues forming the catalytic pocket

• Use a histidine residue (equivalent to His132 in B. burgdorferi) as the phosphohistidine-transfer residue

• Demonstrate conformational changes between apo and ligand-bound states

These structural features would support the phosphoryl-transfer function that characterizes NDK enzymes across species.

What crystallization approaches are most successful for obtaining T. pallidum NDK structures?

Based on successful crystallization of B. burgdorferi NDK, the following protocol would likely be effective for T. pallidum NDK:

This approach has yielded high-resolution (2.2 Å) structures of B. burgdorferi NDK in both apoenzyme and transition-state complex forms , and would likely be transferable to T. pallidum NDK.

What methods are most reliable for assessing T. pallidum NDK enzymatic activity?

For reliable assessment of T. pallidum NDK enzymatic activity, the following methods are recommended:

• Spectrophotometric coupled-enzyme assay: This approach couples NDK activity to pyruvate kinase and lactate dehydrogenase reactions, monitoring NADH oxidation at 340 nm as NDP is converted to NTP.

• Radioactive phosphoryl transfer assay: Using [γ-32P]ATP as donor and measuring transfer to various NDP acceptors.

• Bioluminescence ATP detection: For measuring ATP production when GDP is used as phosphate acceptor.

The spectrophotometric method offers advantages in real-time monitoring while the radioactive method provides higher sensitivity. Additionally, the rate of phosphoryl transfer can be measured in the presence and absence of Mg2+, as this cofactor significantly increases the reaction rate but is not absolutely required .

How do mutations in the active site affect T. pallidum NDK catalytic function?

Based on structure-function relationships in bacterial NDKs, mutations in key residues would affect T. pallidum NDK function in the following ways:

Site-directed mutagenesis targeting the catalytic histidine residue (equivalent to His132 in B. burgdorferi NDK) would be expected to abolish enzymatic activity completely, while mutations affecting the binding pocket architecture would likely alter substrate specificity .

How can structural insights into T. pallidum NDK inform drug development against syphilis?

Structural insights into T. pallidum NDK provide compelling opportunities for therapeutic development through several approaches:

• Transition state analog design: The solved structures of NDK bound to ADP and vanadate from related spirochetes offer templates for designing transition state analogs that could selectively inhibit the enzyme .

• Active site targeting: The catalytic pocket featuring the conserved histidine residue presents a druggable target for small molecule inhibitors .

• Allosteric regulation exploitation: Conformational changes observed between apo and ligand-bound states suggest potential for allosteric inhibition strategies .

• Structure-guided fragment screening: Using the solved structures to conduct in silico or experimental fragment-based screening to identify starting points for inhibitor development.

Given that NDK function appears important for establishing robust infection in mouse models of related spirochetal diseases, inhibiting this enzyme represents a promising therapeutic strategy for treating syphilis .

What is the role of T. pallidum NDK in the host-pathogen interface?

The role of T. pallidum NDK at the host-pathogen interface likely extends beyond its intracellular catalytic function, though direct evidence specific to T. pallidum is still emerging. Based on studies of related bacterial NDKs, potential roles include:

• Immune modulation: Bacterial NDKs can sometimes be secreted or surface-exposed, potentially interacting with host immune components.

• Adaptation to host environment: NDK ensures adequate nucleotide pools under the resource-limited conditions T. pallidum faces during infection.

• Persistence mechanisms: The enzyme may contribute to bacterial survival during long-term infection by maintaining essential metabolic functions.

This is particularly relevant given the restricted metabolic capabilities of T. pallidum, which cannot support the Krebs cycle, oxidative phosphorylation, or de novo biosynthesis pathways . Unlike many bacterial pathogens, T. pallidum has few surface-exposed outer membrane proteins , making internal metabolic enzymes like NDK potentially important therapeutic targets.

How does T. pallidum NDK compare between different strain variants?

Comparative analysis between T. pallidum strains reveals important insights about protein conservation and variation. While specific NDK comparison data is not extensively documented, proteomic analyses of T. pallidum SS14 and Nichols strains show:

• Approximately two-thirds of all proteins are detectable and quantifiable across strains

• 95% of high-abundance proteins are conserved between strains

• Most strain-to-strain amino acid sequence differences are located in surface-exposed regions

For metabolic enzymes like NDK, conservation is typically high between strains, with any variations more likely to occur in surface-exposed regions rather than in the catalytic core . This conservation pattern suggests that therapeutic approaches targeting NDK would potentially be effective across different T. pallidum strains.

How can recombinant T. pallidum NDK be utilized in syphilis diagnostics?

While NDK itself has not been the primary focus of syphilis diagnostics to date, the methodologies established for other T. pallidum recombinant proteins could be applied to NDK. Based on successful approaches with other T. pallidum antigens:

• Enzyme-linked immunosorbent assays (ELISAs): Recombinant NDK could be used to coat plates for detecting anti-NDK antibodies in patient sera, following protocols established for other T. pallidum antigens .

• Multiplex protein arrays: NDK could be incorporated into panels alongside other T. pallidum antigens to improve diagnostic sensitivity and specificity.

• Point-of-care tests: If NDK proves immunogenic enough, it could potentially contribute to rapid diagnostic test development.

The successful application of recombinant T. pallidum lipoproteins TpN17, TpN47, and TpN44.5 in ELISAs with high antibody titers suggests a methodological framework that could be adapted for NDK if it proves sufficiently immunogenic .

What optimizations are needed for high-yield production of recombinant T. pallidum NDK?

For high-yield production of recombinant T. pallidum NDK, the following optimizations would be recommended:

These optimizations would follow the successful approach used for other T. pallidum antigens, where PCR-amplified DNA was inserted into E. coli expression vectors resulting in high-level expression , and N-terminal hexahistidine sequences allowed efficient purification by metal chelate affinity chromatography .

What novel approaches might enhance inhibitor development against T. pallidum NDK?

Several innovative approaches could enhance inhibitor development targeting T. pallidum NDK:

• Covalent inhibitor design: Developing compounds that form covalent bonds with the catalytic histidine residue of NDK.

• Allosteric inhibitor screening: Identifying compounds that bind outside the active site but prevent the conformational changes necessary for catalysis.

• Computational discovery pipelines: Using virtual screening, molecular dynamics simulations, and quantum mechanics calculations to identify and optimize novel inhibitors.

• Fragment-based drug design: Building inhibitors from small molecular fragments that bind to different regions of the enzyme.

• Selective targeting strategies: Exploiting structural differences between bacterial and human NDKs to achieve selectivity.

These approaches build upon structural insights from related bacterial NDKs, where ADP and vanadate complexes have been used to model the transition state of phosphoryl-transfer reactions, demonstrating pocket closure upon ADP binding .

How might genomic and proteomic insights enhance our understanding of T. pallidum NDK function?

Integrating genomic and proteomic approaches could significantly advance our understanding of T. pallidum NDK:

• Comparative genomics: Analysis across T. pallidum strains and other spirochetes to identify conserved and variable regions of NDK.

• Transcriptomics: RNA-seq studies to determine expression patterns of NDK under different conditions.

• Protein-protein interaction networks: Identifying NDK binding partners to better understand its cellular role beyond catalytic function.

• Post-translational modification analysis: Mass spectrometry to identify modifications that might regulate NDK activity.

• In vivo expression studies: Confirming NDK expression levels during different stages of infection.

Recent proteomic analysis of T. pallidum SS14 and Nichols strains provides a methodological template for such work, having characterized approximately two-thirds of all proteins and confirmed the expression of 259 proteins for the first time .