Recombinant Francisella tularensis subsp. novicida Nucleoside diphosphate kinase (ndk)

What is Francisella tularensis subsp. novicida and how does it relate to other Francisella species?

Francisella tularensis subsp. novicida (F. novicida) is a close relative of F. tularensis, sharing approximately 97% nucleotide identity. Unlike F. tularensis subspecies tularensis (type A) and holarctica (type B) which cause tularemia in humans, F. novicida very rarely causes human illness, with cases primarily limited to immunocompromised individuals or those with underlying health conditions . Comparative genomics and molecular typing evidence suggest that F. novicida could be the common ancestor of F. tularensis subspecies .

Important taxonomic distinctions include:

F. novicida U112 strain is exempt from U.S. select agent regulations, making it a valuable laboratory surrogate for studying Francisella biology without requiring specialized containment facilities .

What is nucleoside diphosphate kinase (ndk) and what is its function?

Nucleoside-diphosphate kinase (ndk) is an enzyme that catalyzes the exchange of terminal phosphate between different nucleoside diphosphates (NDP) and triphosphates (NTP) in a reversible manner . The general reaction follows a ping-pong mechanism:

XDP + YTP ↔ XTP + YDP

Where X and Y represent different nitrogenous bases . This enzyme plays a critical role in:

• Maintaining equilibrium between concentrations of different nucleoside triphosphates

• Converting GTP produced in the citric acid cycle to ATP

• Supporting cell proliferation, differentiation, and development

• Facilitating signal transduction and gene expression

• Providing RNA and DNA precursors except ATP

What are the structural characteristics of bacterial ndk proteins?

Nucleoside-diphosphate kinases are typically homohexameric proteins composed of monomers approximately 152 amino acids in length with a theoretical molecular weight of 17.17 kDa . These enzymes are found in both mitochondria and the soluble cytoplasm of cells . The reaction mechanism involves:

• Phosphorylation of a conserved histidine residue in the active site

• Transfer of the terminal phosphate group (γ-phosphate) from ATP to NDP β-phosphate

• Production of a new NTP through a reversible reaction

The enzyme shows limited specificity toward nucleoside bases and can accept both nucleotides and deoxyribonucleotides as substrates or donors .

What expression systems are most effective for producing recombinant F. novicida ndk?

Based on research methodologies for related bacterial proteins, optimal expression systems for F. novicida ndk would include:

For RNA-based analysis of ndk expression, researchers commonly employ:

• T7 polymerase-based RNA amplification protocols

• RT-PCR and RT-qPCR for gene expression analysis

What purification strategies yield active recombinant F. novicida ndk?

A multi-step purification approach is recommended to obtain highly pure and active enzyme:

• Initial capture using affinity chromatography (typically His-tag based IMAC)

• Intermediate purification via ion-exchange chromatography to remove nucleic acid contaminants

• Polishing step using size-exclusion chromatography to ensure homogeneous hexameric assembly

• Buffer optimization: 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0), 100-150 mM NaCl, 5-10% glycerol

When assessing purity and activity, researchers should implement:

• SDS-PAGE analysis with Coomassie or silver staining

• Western blotting using anti-His or anti-ndk antibodies

• Enzymatic activity assays measuring phosphate transfer between nucleotides

How does F. novicida ndk activity compare with ndks from other bacterial species?

While specific data for F. novicida ndk activity is not directly available in the literature, comparative analysis of bacterial ndks suggests:

The enzymatic activity would typically be measured using coupled enzyme assays where ATP production is linked to NADH oxidation through pyruvate kinase and lactate dehydrogenase, allowing spectrophotometric monitoring at 340 nm.

How do environmental conditions affect F. novicida ndk stability and activity?

Based on general properties of bacterial ndks and the growth conditions of Francisella species, the enzyme would likely exhibit the following characteristics:

These parameters are particularly relevant when designing experiments to assess ndk function under conditions mimicking the host environment during infection.

What evidence suggests ndk involvement in Francisella virulence mechanisms?

While direct evidence for ndk's role in F. novicida virulence is limited in the search results, several factors suggest potential involvement:

• Nucleotide metabolism is essential for intracellular pathogen survival

• Related bacterial ndks contribute to stress responses during infection

• Comparative proteomics studies between Francisella subspecies have identified proteins differentially expressed in virulent strains

• The ndk enzyme may support bacterial adaptation to the nutrient-limited intracellular environment

Research approaches to investigate this question include:

• Generating ndk deletion mutants and assessing virulence in cellular and animal models

• Complementation studies to confirm phenotypes are specifically due to ndk

• Transcriptomics to identify conditions that regulate ndk expression

• Protein interaction studies to identify virulence-associated binding partners

How does ndk expression differ between F. tularensis subspecies with varying virulence?

Comparative proteomics studies have identified proteins that are uniquely expressed or up-regulated in virulent F. tularensis subspecies compared to less virulent strains like F. novicida . While ndk is not specifically mentioned among these differentially expressed proteins in the search results, research has shown:

• Virulent F. tularensis strains express unique proteins or isoforms not found in F. novicida

• Several proteins unique to subspecies tularensis, such as FTT_0607, FTT_0435, and FTT_1157, are implicated in virulence

• Expression patterns of metabolic enzymes differ between subspecies, which may include nucleotide metabolism enzymes like ndk

Methodologies to investigate differential ndk expression include:

• Two-dimensional electrophoresis (2DE) comparative proteomics

• RNA-seq transcriptomic analysis across subspecies

• Quantitative RT-PCR targeting ndk expression

• Western blot analysis with anti-ndk antibodies

How can recombinant F. novicida ndk be used to identify potential therapeutic targets?

Recombinant F. novicida ndk offers several advantages for therapeutic target identification:

• Structural analysis: Crystallographic studies can reveal unique structural features not present in human ndks

• Inhibitor screening: The purified enzyme can be used in high-throughput screens to identify selective inhibitors

• Host-pathogen interactions: Studies can identify host proteins that interact with ndk during infection

• Functional conservation: Findings may apply to virulent F. tularensis strains without requiring BSL-3 facilities

Research design considerations include:

• Ensuring that the recombinant protein retains native conformational epitopes

• Including appropriate controls like heat-inactivated enzyme and catalytically inactive mutants

• Validating findings in more virulent strains when possible

• Considering species-specific variations that might affect drug targeting

What technical challenges exist in studying F. novicida ndk function in relevant host-pathogen models?

Research with F. novicida ndk presents several methodological challenges:

Researchers should consider implementing the T7 polymerase-based RNA amplification protocol that has been optimized for samples with low bacterial RNA content .

How can crystallographic studies of F. novicida ndk inform structure-based drug design?

Structural studies of F. novicida ndk could provide valuable insights for developing inhibitors:

• Active site architecture: Detailed mapping of substrate binding pockets could reveal differences from human ndks

• Allosteric sites: Identification of regulatory sites unique to bacterial ndks

• Hexamer assembly: Understanding interfaces that could be disrupted by small molecules

• Conformational changes: Characterizing protein dynamics during catalysis

Critical methodological considerations include:

• Ensuring homogeneity of the protein preparation

• Testing multiple crystallization conditions with various substrates/analogs

• Obtaining sufficient resolution to identify water molecules and metal binding sites

• Validating structural insights with mutagenesis and activity assays

What controls are essential when measuring F. novicida ndk enzymatic activity?

Robust experimental design for ndk activity measurement requires several controls:

Additionally, researchers should consider time-course measurements to ensure linearity of the reaction and appropriate enzyme concentration to avoid substrate depletion.

What methodologies are recommended for studying ndk expression during F. novicida infection?

For analyzing ndk expression during infection, researchers should implement:

• RNA isolation and amplification:

  • Extract RNA using optimized protocols for infected tissues

  • Implement T7 polymerase-based RNA amplification for low-abundance samples

  • Validate RNA quality using bioanalyzer or formaldehyde gel electrophoresis

• Expression analysis:

  • Perform RT-qPCR with validated reference genes for normalization

  • Design primers with specificity for F. novicida ndk

  • Include no-RT controls to detect genomic DNA contamination

• Protein detection:

  • Develop specific antibodies against F. novicida ndk

  • Implement Western blotting with appropriate loading controls

  • Consider targeted mass spectrometry for absolute quantification

• Data analysis:

  • Calculate relative transcript abundance (RTA) with appropriate statistical tests

  • Perform time-course analysis to capture expression dynamics

  • Compare results across multiple infection models

How should researchers address potential contradictions in ndk functional data between in vitro and in vivo studies?

When confronting discrepancies between in vitro enzyme studies and in vivo observations:

• Systematic comparison of conditions:

  • Test whether buffer components, pH, or ionic strength affect enzyme behavior

  • Consider whether post-translational modifications present in vivo are absent in recombinant protein

  • Evaluate the impact of macromolecular crowding using crowding agents

• Methodological validation:

  • Confirm antibody specificity using knockout controls

  • Validate activity assays with multiple methodologies

  • Ensure recombinant protein maintains native oligomeric state

• Biological context:

  • Consider compartmentalization effects in vivo

  • Evaluate regulatory mechanisms that may not be recapitulated in vitro

  • Assess interactions with other proteins in the cellular environment

• Reporting and resolution:

  • Document all experimental conditions meticulously

  • Present both datasets with appropriate caveats

  • Design experiments specifically to address the mechanistic basis of discrepancies