Recombinant Yersinia pestis bv. Antiqua Nucleoside diphosphate kinase (ndk)

What is Nucleoside diphosphate kinase (ndk) in Yersinia pestis bv. Antiqua?

Nucleoside diphosphate kinase (ndk) in Yersinia pestis bv. Antiqua is a 142-amino acid enzyme (UniProt No. Q1C5I4) that catalyzes the transfer of terminal phosphate groups from nucleoside triphosphates to nucleoside diphosphates (EC 2.7.4.6). The enzyme has alternative names including "Nucleoside-2-P kinase" and "NDP kinase" . The recombinant protein is typically expressed in E. coli expression systems and can be purified to >85% purity as determined by SDS-PAGE analysis . The protein is part of the essential nucleotide metabolism pathway in Y. pestis, which remains a significant pathogen of interest due to its historical impact as the causative agent of plague and its potential use in bioterrorism .

How does ndk function in bacterial metabolism and what is its enzymatic mechanism?

Nucleoside diphosphate kinase catalyzes the reversible phosphoryl transfer reaction: N₁TP + N₂DP ↔ N₁DP + N₂TP, where N₁ and N₂ represent different nucleosides. This reaction is critical for maintaining balanced nucleotide pools in bacterial cells. The enzyme transfers the γ-phosphate from nucleoside triphosphates (typically ATP) to nucleoside diphosphates through a ping-pong mechanism involving a phospho-enzyme intermediate.

In Y. pestis specifically, ndk likely plays crucial roles in:

• Supporting nucleotide metabolism during rapid bacterial replication

• Maintaining balanced NTP pools for DNA and RNA synthesis

• Contributing to metabolic adaptations during the transition between flea vector and mammalian host environments

Research into Y. pestis metabolism has shown that the bacterium relies heavily on carbohydrate metabolism during host colonization . While ndk is not directly mentioned in the carbohydrate utilization pathways described in the search results, its role in energy metabolism through nucleotide interconversion would support these essential metabolic processes.

How does ndk expression vary during different stages of Y. pestis infection?

While the search results don't specifically address ndk expression patterns, research on Y. pestis gene expression during infection provides context for understanding potential regulation. Y. pestis undergoes significant metabolic adaptation when transitioning between its flea vector and mammalian hosts. Studies have identified numerous genes that are upregulated in vivo during mammalian infection .

Y. pestis requires carbohydrate metabolism when colonizing mammalian hosts, and several metabolic genes show altered expression patterns during infection . Given ndk's role in nucleotide metabolism and energy transfer, its expression may be coordinated with other metabolic genes to support bacterial replication during infection. The bacterium has been shown to rely on anaerobic respiration to cause plague, suggesting that ndk might function under various oxygen tension conditions throughout the infection cycle .

What are the optimal storage conditions for recombinant Yersinia pestis ndk?

For optimal stability of recombinant Yersinia pestis ndk, researchers should follow these evidence-based storage guidelines:

The stability is influenced by multiple factors including "storage state, buffer ingredients, storage temperature and the stability of the protein itself" . For working with the protein, it's recommended to briefly centrifuge the vial prior to opening to bring contents to the bottom . Repeated freezing and thawing should be strictly avoided to maintain protein integrity and enzymatic activity .

How should recombinant Yersinia pestis ndk be reconstituted for experimental use?

For optimal reconstitution of recombinant Yersinia pestis ndk, follow this methodological approach:

• Briefly centrifuge the vial containing lyophilized protein to collect all material at the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For long-term storage, add glycerol to a final concentration of 5-50% (the manufacturer's default is 50%)

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store reconstituted protein according to the temperature guidelines in section 2.1

This protocol ensures maximum retention of enzymatic activity and structural integrity. For applications requiring specific buffer conditions, further buffer exchange may be performed after initial reconstitution using dialysis or desalting columns to maintain protein stability.

What analytical methods are most effective for characterizing recombinant Yersinia pestis ndk?

Multiple complementary analytical techniques should be employed for comprehensive characterization of recombinant Y. pestis ndk:

For structural studies specifically, researchers have successfully used capillary electrophoresis to characterize recombinant Y. pestis proteins . Additionally, experimental approaches that examine ndk under conditions mimicking the in vivo environment during infection may provide insights into its functional adaptations during host colonization.

What are the key considerations when designing experiments to study ndk's role in Y. pestis virulence?

When investigating ndk's potential role in Y. pestis virulence, researchers should consider these methodological approaches:

• Genetic manipulation strategies:

  • Generate precise ndk deletion mutants using techniques similar to those used for other Y. pestis genes

  • Create complemented strains to verify phenotypes are specifically due to ndk disruption

  • Consider conditional expression systems to study essential gene functions

• Virulence assessment models:

  • Use established rodent models of bubonic plague as employed in previous Y. pestis virulence studies

  • Implement competitive index assays comparing wild-type and ndk mutants

  • Apply per-pool screening methods that have successfully identified other virulence factors

• Host-pathogen interaction analyses:

  • Examine bacterial survival in macrophages, as intracellular survival is important for plague pathogenesis

  • Assess lymph node colonization efficiency, a critical step in plague development

  • Monitor ndk expression patterns during different infection stages

• Controls and validation:

  • Include well-characterized virulence mutants as comparative controls

  • Verify that phenotypes are not due to general growth defects by comparing in vitro and in vivo growth

  • Use multiple independent mutants to confirm results

Research has shown that intracellular survival during the early stage of infection is important for plague pathogenesis , suggesting that studying ndk's potential role in this process would be particularly valuable.

How does ndk contribute to Y. pestis adaptation to different host environments?

Nucleoside diphosphate kinase likely plays important roles in Y. pestis adaptation to diverse host environments through several mechanisms:

• Metabolic flexibility support:

  • Facilitates nucleotide interconversion to support metabolic adaptations when transitioning between flea vector and mammalian host

  • Contributes to the carbohydrate metabolism pathways known to be essential during mammalian host colonization

  • May support anaerobic respiration processes that Y. pestis relies upon during infection

• Stress response mechanisms:

  • Helps maintain nucleotide homeostasis during exposure to host defense mechanisms

  • May support bacterial responses to oxidative stress encountered within macrophages

  • Contributes to metabolic adaptations during nutrient limitation in host tissues

• Replication support:

  • Ensures adequate nucleotide supplies for rapid bacterial division during infection

  • Maintains energy transfer processes critical for bacterial growth

  • Supports nucleic acid synthesis necessary for virulence factor production

Research into Y. pestis metabolism has revealed that the bacterium relies on specific carbohydrate uptake systems and metabolic pathways during infection . Understanding ndk's integration with these essential metabolic networks could provide insights into bacterial adaptation mechanisms during plague pathogenesis.

What is the significance of studying ndk in relation to Y. pestis evolution and virulence?

Studying ndk in Y. pestis offers significant insights into plague pathogen evolution and virulence for several reasons:

• Evolutionary context:

  • Y. pestis evolved from Yersinia pseudotuberculosis, with key evolutionary steps occurring during the Late Neolithic/Early Bronze Age period

  • Comparing ndk sequence and function across Yersinia species could reveal adaptive changes

  • Analysis could identify whether ndk belongs to the core genome maintained during Y. pestis evolution or shows lineage-specific adaptations

• Virulence factor research:

  • Y. pestis produces several major virulence factors, including the capsular F1 antigen

  • Understanding how ndk supports the expression and function of established virulence factors could reveal metabolic dependencies

  • Research could determine if ndk has acquired additional functions in Y. pestis compared to non-pathogenic relatives

• Plague biology insights:

  • Y. pestis causes different forms of plague (bubonic, pneumonic) with varying transmission routes

  • Studies of ndk could reveal metabolic adaptations specific to different infection scenarios

  • May contribute to understanding how Y. pestis rapidly dispersed across continents historically

The comprehensive study of Y. pestis genes has already identified previously uncharacterized genes required for virulence, including some acquired by horizontal gene transfer . Similar approaches could determine if ndk has unique properties in Y. pestis that contribute to its remarkable virulence and host adaptation.

How can recombinant ndk be utilized in the development of plague countermeasures?

Recombinant Y. pestis ndk has several potential applications in plague countermeasure development:

• Vaccine research:

  • While F1 and V antigens are the primary focus of current plague vaccine development , metabolic proteins like ndk could serve as additional targets

  • Recombinant ndk could be evaluated as a component in multi-antigen vaccine formulations

  • Plant-based expression systems that have successfully produced other Y. pestis antigens could be employed for ndk production

• Therapeutic target identification:

  • Structure-function studies of ndk could reveal unique features amenable to selective inhibition

  • High-throughput screening against purified recombinant ndk could identify novel inhibitors

  • Combination approaches targeting multiple metabolic enzymes including ndk might overcome bacterial resistance

• Diagnostic development:

  • Recombinant ndk could serve as a positive control in molecular diagnostic assays

  • Anti-ndk antibodies could be incorporated into immunoassays for Y. pestis detection

  • Structural studies might reveal Y. pestis-specific epitopes for selective detection

• Research tool applications:

  • Purified ndk provides a valuable reagent for studying Y. pestis metabolism

  • Fluorescently tagged ndk could monitor protein localization during infection

  • Immobilized ndk could identify interaction partners in pull-down assays

Modern approaches to plague countermeasures have focused on recombinant subunit vaccines and plant biotechnology for antigen production . Similar technologies could be applied to metabolic proteins like ndk if research establishes their value as therapeutic or diagnostic targets.

What metabolic pathways interact with ndk function in Y. pestis during infection?

Nucleoside diphosphate kinase in Y. pestis integrates with multiple metabolic pathways that are critical during infection:

• Carbohydrate metabolism:

  • Y. pestis relies on glucose, gluconate, and maltose uptake during mammalian host colonization

  • ndk likely supports energy production from these carbohydrates by maintaining ATP pools

  • The terminal part of the glycolysis pathway is essential for Y. pestis virulence , suggesting ndk may interact with these processes

• Anaerobic respiration:

  • Y. pestis appears to rely on anaerobic respiration during infection rather than the TCA cycle

  • DMSO reductase (DmsABC) and glycerol-3P dehydrogenase (GlpABC) are important for virulence

  • ndk may provide nucleotides needed for expression of these anaerobic respiration components

• Nucleotide salvage pathways:

  • During infection, Y. pestis may encounter nucleotide limitation in certain host environments

  • ndk would play a crucial role in recycling available nucleotides for essential functions

  • This recycling minimizes energy expenditure for de novo nucleotide synthesis

• Stress response mechanisms:

  • Host-imposed stresses trigger specific metabolic adaptations in Y. pestis

  • ndk's role in maintaining nucleotide homeostasis supports these adaptive responses

  • Integration with stress response pathways would enhance bacterial survival during infection

The complex metabolic adaptations of Y. pestis during mammalian infection highlight the importance of understanding how ndk functions within these integrated metabolic networks to support plague pathogenesis .

What are the key challenges in determining ndk's specific contribution to Y. pestis virulence?

Investigating ndk's role in Y. pestis virulence presents several significant challenges:

• Genetic manipulation constraints:

  • If ndk is essential for bacterial viability, traditional knockout approaches would be ineffective

  • Conditional expression systems would be required to study function in vivo

  • Separation of catalytic and potential moonlighting functions requires precise mutation strategies

• Functional redundancy:

  • Other enzymes may partially compensate for ndk activity

  • Multiple pathways for nucleotide metabolism could mask phenotypes

  • Different host environments may reveal distinct requirements for ndk

• Technical limitations:

  • Detecting subtle virulence defects requires sensitive in vivo models

  • Metabolic flux analysis in infection settings presents technical difficulties

  • Temporal regulation of ndk during infection is challenging to monitor

• Translational relevance assessment:

  • Connecting biochemical function to in vivo phenotypes requires multiple approaches

  • Determining if ndk is a viable therapeutic target needs comprehensive validation

  • Strain-specific variations may affect generalizability of findings

Researchers have successfully addressed similar challenges in studying other Y. pestis genes by employing per-pool virulence screening methods and targeted functional studies . Similar multidisciplinary approaches combining genetics, biochemistry, and in vivo models would be necessary to elucidate ndk's specific contributions to plague pathogenesis.

How might recombinant ndk be used to study potential interactions with host cellular components?

Recombinant Y. pestis ndk can be utilized in several sophisticated approaches to investigate potential host interactions:

• Protein-protein interaction studies:

  • Affinity purification mass spectrometry using immobilized ndk to capture host binding partners

  • Yeast two-hybrid screening against human protein libraries

  • Surface plasmon resonance to characterize binding kinetics with candidate host targets

  • Biolayer interferometry to measure real-time interactions

• Host cell response analysis:

  • Exogenous application of purified ndk to host cells followed by transcriptomic analysis

  • Phosphoproteomics to identify potential host substrates of ndk phosphotransferase activity

  • Immunofluorescence microscopy to track ndk localization in infected cells

  • Live-cell imaging using fluorescently labeled ndk to monitor cellular entry and trafficking

• Structural biology approaches:

  • Co-crystallization of ndk with host protein partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Molecular dynamics simulations to predict host-pathogen protein interactions

  • NMR spectroscopy to characterize dynamic interactions

• Functional validation methods:

  • siRNA knockdown of identified host targets to confirm functional significance

  • Competitive inhibition assays using ndk-derived peptides

  • Mutational analysis of interaction interfaces identified through structural studies

Understanding potential moonlighting functions of bacterial metabolic enzymes has revealed important virulence mechanisms in other pathogens, suggesting this approach could provide valuable insights into Y. pestis pathogenesis as well.

What cutting-edge technologies could advance our understanding of ndk's role in Y. pestis metabolism?

Several emerging technologies could significantly enhance our understanding of ndk's metabolic functions in Y. pestis:

• Advanced metabolomics approaches:

  • Stable isotope labeling to track nucleotide flux through ndk-catalyzed reactions

  • Single-cell metabolomics to capture heterogeneity in bacterial populations

  • Spatial metabolomics to map metabolite distributions during infection

  • Real-time metabolic monitoring during host-pathogen interactions

• Systems biology integration:

  • Multi-omics data integration combining transcriptomics, proteomics, and metabolomics

  • Genome-scale metabolic modeling incorporating ndk-specific parameters

  • Network analysis to position ndk within Y. pestis' metabolic architecture

  • Machine learning approaches to predict metabolic adaptations during infection

• High-resolution imaging techniques:

  • Cryo-electron microscopy of ndk complexes with metabolic partners

  • Super-resolution microscopy to visualize ndk localization during infection

  • Correlative light and electron microscopy to connect function with ultrastructure

  • Mass spectrometry imaging to map metabolite distributions related to ndk activity

• Genetic engineering innovations:

  • CRISPR interference for tunable ndk expression

  • Optogenetic control of ndk activity during specific infection phases

  • Biosensor development to monitor real-time ndk activity in live bacteria

  • Directed evolution approaches to probe ndk function through enhanced variants

These technologies could reveal how ndk integrates with the broader metabolic networks that Y. pestis requires for mammalian host colonization, particularly the carbohydrate utilization and anaerobic respiration pathways identified as essential for virulence .

What structural and functional studies would advance our understanding of Y. pestis ndk as a potential therapeutic target?

To evaluate Y. pestis ndk as a potential therapeutic target, the following structural and functional studies would be valuable:

• Comprehensive structural characterization:

  • High-resolution X-ray crystallography of Y. pestis ndk in multiple conformational states

  • Mapping of the active site architecture and catalytic mechanism

  • Identification of allosteric sites that could be targeted by small molecules

  • Comparative structural analysis with human NDK to identify pathogen-specific features

• Structure-based drug design approaches:

  • Virtual screening against identified binding pockets

  • Fragment-based drug discovery to identify chemical starting points

  • Structure-activity relationship studies of identified inhibitors

  • Crystallization of ndk-inhibitor complexes to guide optimization

• Functional validation in disease-relevant contexts:

  • Testing candidate inhibitors against Y. pestis in macrophage infection models

  • Evaluation of inhibitor efficacy in rodent plague models

  • Assessment of resistance development potential

  • Combination studies with established antibiotics

• Target validation studies:

  • Generating point mutations that confer inhibitor resistance

  • Creating dominant-negative ndk variants to validate phenotypic effects

  • Chemical genetic approaches to establish on-target activity

  • Testing inhibitors against closely related pathogens to assess specificity

The development of novel plague countermeasures remains important given concerns about antibiotic resistance and biological warfare potential . Metabolic enzymes like ndk could represent a new class of targets for anti-virulence therapeutics if research establishes their essential role in Y. pestis pathogenesis.