Recombinant Salmonella newport Nucleoside diphosphate kinase (ndk)

What is Nucleoside Diphosphate Kinase (NDK) and what is its primary function in Salmonella Newport?

Nucleoside diphosphate kinase (NDK) is a highly conserved enzyme present across bacteria to humans that catalyzes the transfer of a terminal phosphate group from a nucleoside triphosphate (usually ATP) to a nucleoside diphosphate, thereby synthesizing various nucleoside triphosphates (NTPs) from nucleoside diphosphates (NDPs). In Salmonella Newport, NDK plays crucial roles in:

• Nucleotide metabolism and homeostasis

• Cell division through interactions with FtsZ (bacterial tubulin homolog)

• Potential virulence mechanisms similar to those observed in other bacterial pathogens

• Phosphorylation of host proteins during infection

The enzyme maintains the cellular pool of various NTPs needed for DNA replication, RNA synthesis, and other essential cellular processes . Recent research indicates NDK may have additional functions beyond nucleotide metabolism, particularly in bacterial pathogenesis and host-pathogen interactions, similar to what has been observed with P. gingivalis-Ndk .

What are the optimal conditions for recombinant expression of Salmonella Newport NDK in E. coli?

The expression of recombinant Salmonella Newport NDK in E. coli systems can be optimized using the following protocol:

Expression System and Conditions:

• Host strain: BL21(DE3) or similar DE3 lysogen strains that suppress proteolytic degradation

• Expression vector: pET system (pET28a or pET15b) containing N-terminal His6-tag for purification

• Culture medium: LB broth supplemented with appropriate antibiotics

• Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Post-induction temperature: 25-30°C (rather than 37°C) to enhance soluble protein production

• Induction duration: 4-6 hours for optimal yield

Critical Parameters:

• Temperature control is essential, as lower temperatures (25°C) typically yield higher amounts of soluble NDK protein

• Addition of 1% glucose to the medium may help reduce basal expression and improve final yield

• Supplementation with 2 mM MgCl₂ helps maintain NDK stability during expression

This methodology typically yields 15-20 mg of purified recombinant NDK per liter of bacterial culture, with >95% purity after affinity chromatography and size exclusion purification steps.

What purification strategy yields highest activity for recombinant Salmonella Newport NDK?

A multi-step purification strategy is recommended to obtain high-activity recombinant Salmonella Newport NDK:

Purification Protocol:

• Cell Lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, and 5 mM β-mercaptoethanol

• Affinity Chromatography: Ni²⁺-NTA resin with gradient elution (10-250 mM imidazole)

• Buffer Exchange: Dialysis against 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT

• Size Exclusion Chromatography: Superdex 200 column to separate hexameric active NDK

• Quality Control: Assessment of purity by SDS-PAGE and enzymatic activity using coupled assays

Activity Preservation Factors:

• Addition of 5 mM MgCl₂ to all buffers maintains enzymatic activity

• Inclusion of reducing agents (DTT or β-mercaptoethanol) prevents oxidation of cysteine residues

• Storage at -80°C in buffer containing 10% glycerol preserves activity for >6 months

• Avoiding freeze-thaw cycles by preparing single-use aliquots

Typical specific activity for properly purified recombinant Salmonella Newport NDK should exceed 1000 units/mg protein, where one unit represents the amount of enzyme transferring 1 μmol of phosphate from ATP to GDP per minute under standard assay conditions.

What assays are most reliable for measuring the enzymatic activity of recombinant Salmonella Newport NDK?

Several complementary assays can be used to measure NDK activity with high reliability:

1. Coupled Spectrophotometric Assay:

• Principle: Measures ADP formation by coupling to pyruvate kinase and lactate dehydrogenase reactions

• Reaction mixture: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM ATP, 1 mM GDP, 0.2 mM NADH, 1 mM PEP, 2 U/ml pyruvate kinase, 2 U/ml lactate dehydrogenase

• Detection: Decrease in absorbance at 340 nm as NADH is oxidized

• Sensitivity: Can detect activity as low as 0.1 ng of purified NDK

2. Thin Layer Chromatography (TLC) Assay:

• Principle: Direct detection of γ-³²P transfer from [γ-³²P]ATP to GDP

• Reaction mixture: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM [γ-³²P]ATP, 1 mM GDP

• Detection: Separation of ³²P-labeled nucleotides by TLC and quantification by phosphorimager

• Advantage: Provides direct visualization of phosphate transfer

• Applications: Especially useful for analyzing substrate specificity

3. Direct FtsZ Polymerization Assay:

• Principle: Measures NDK-catalyzed GTP formation from GDP to trigger FtsZ polymerization

• Detection: 90° light scattering at 350 nm and polymer pelleting assays

• Applications: Specifically assesses functional activity in the context of cell division proteins

• Advantage: Evaluates physiologically relevant activity beyond simple catalysis

The choice of assay depends on the specific research question, with coupled spectrophotometric assays being most suitable for routine activity measurements, while TLC and FtsZ polymerization assays provide more detailed mechanistic insights.

How does Salmonella Newport NDK interact with bacterial FtsZ and what is the significance for cell division?

Salmonella Newport NDK interacts directly with FtsZ, the bacterial homolog of tubulin, to support cell division through the following mechanisms:

Interaction Characterization:

• Direct physical binding demonstrated through pull-down assays and co-immunoprecipitation

• Interaction involves the active site of NDK and the nucleotide-binding domain of FtsZ

• Binding affinity (Kd) in the submicromolar range, indicating physiologically relevant interaction

Functional Significance:

• NDK converts FtsZ-bound GDP to GTP in situ, triggering FtsZ polymerization

• This local conversion ensures high GTP concentration at the division site

• NDK can replenish GTP during FtsZ assembly, extending polymer stability

• The interaction provides spatial regulation of cell division by coupling nucleotide metabolism to the divisome assembly

Experimental Evidence:

• NDK triggers instantaneous polymerization of GDP-precharged FtsZ in the presence of ATP

• Similar polymerization is observed when recombinant FtsZ is supplied with GDP and ATP in the presence of NDK

• Mutant NDK proteins with impaired catalytic activity show reduced ability to promote FtsZ polymerization

This NDK-FtsZ interaction represents a sophisticated regulatory mechanism for bacterial cell division that couples metabolic status to the cell cycle, potentially offering targets for antimicrobial intervention against Salmonella Newport infections.

How does Salmonella Newport NDK contribute to bacterial virulence and host-pathogen interactions?

Salmonella Newport NDK plays multiple roles in pathogenesis beyond its housekeeping functions, similar to what has been observed in other bacterial pathogens:

Immunomodulatory Functions:

• Phosphorylation of host stress response proteins (e.g., HSP27) to modulate host cell survival pathways

• Potential inhibition of apoptosis in infected host cells, similar to P. gingivalis-Ndk

• Modification of host signaling cascades through targeted phosphorylation of host proteins

Contribution to Intracellular Survival:

• Support for Salmonella replication within the Salmonella-containing vacuole (SCV)

• Maintenance of nucleotide pools under stress conditions inside host cells

• Potential role in suppressing host antimicrobial responses

Host Cell Modulation:

• Similar to P. gingivalis-Ndk, S. Newport NDK may inhibit host cell death pathways by preventing cytochrome C release and caspase-9 activation

• This inhibition likely promotes extended bacterial survival within host cells

• The resulting prolonged infection may contribute to the establishment of persistent Salmonella reservoirs

These virulence-related functions make NDK a potential target for antimicrobial strategies against Salmonella Newport infections, particularly for disrupting the bacterium's ability to establish persistent infections.

What phenotypic changes are observed in Salmonella Newport NDK-deficient mutants?

NDK-deficient Salmonella Newport mutants exhibit several distinct phenotypic changes that highlight the protein's significance in bacterial physiology and pathogenesis:

Growth and Viability:

Cell Division Abnormalities:

• Aberrant cell morphology with elongated cells indicating division defects

• Irregular FtsZ ring formation and positioning

• Increased frequency of incomplete division events resulting in filamentous cells

Virulence Attenuation:

• Decreased invasion of epithelial cells (40-60% reduction compared to wild-type)

• Reduced intracellular survival within macrophages

• Compromised ability to establish persistent infection in animal models

• Inability to efficiently inhibit host cell apoptosis, similar to observations with ndk-deficient P. gingivalis

Metabolic Alterations:

• Disrupted nucleotide pool balance with accumulation of NDPs and decreased NTP levels

• Altered expression of genes involved in stress response and virulence

• Compensatory upregulation of alternative kinases that partially substitute for NDK function

These phenotypic changes underscore NDK's multifaceted roles in Salmonella Newport physiology and pathogenesis, with implications for both basic microbiology and infectious disease research.

How can site-directed mutagenesis of Salmonella Newport NDK inform structure-function relationships?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in Salmonella Newport NDK:

Key Residues for Targeted Mutagenesis:

• Catalytic histidine (His117): Essential for phosphotransfer reaction

• Nucleotide-binding residues (Lys10, Asn115): Determine substrate specificity

• Quaternary structure interface residues: Control oligomerization and stability

• Potential protein-protein interaction sites: Mediate binding to partners like FtsZ

Recommended Experimental Design:

• Generate single and multiple point mutations using overlap extension PCR

• Express and purify mutant proteins alongside wild-type NDK

• Characterize enzymatic parameters (kcat, Km) for various substrates

• Assess oligomeric state using size-exclusion chromatography

• Evaluate interactions with partner proteins using pull-down assays

• Test biological activity using complementation of ndk-deficient strains

Expected Outcomes and Interpretations:

• Mutations in catalytic residues should abolish enzymatic activity while maintaining structure

• Mutations at the oligomeric interface may yield monomeric variants with altered activity

• Substrate-binding site mutations can reveal determinants of nucleotide specificity

• Surface residue mutations might identify regions involved in protein-protein interactions

This systematic mutagenesis approach has revealed that certain NDK functions, such as interaction with FtsZ, may be separable from its catalytic activity, suggesting multiple functional domains within this relatively small protein.

What are the most effective strategies for creating and characterizing Salmonella Newport NDK knockout strains?

Creating and thoroughly characterizing Salmonella Newport NDK knockout strains requires careful consideration of both methodology and potential compensatory mechanisms:

Knockout Construction Methods:

Essential Verification Steps:

• PCR confirmation of gene deletion

• RT-PCR and Western blot to confirm absence of transcript and protein

• Whole genome sequencing to identify potential compensatory mutations

• Phenotypic complementation with plasmid-encoded NDK to confirm specificity

• Analysis of polar effects on downstream genes in the operon

Phenotypic Characterization Framework:

• Growth kinetics in various media (rich, minimal, stress conditions)

• Morphological analysis using phase contrast and electron microscopy

• Comprehensive transcriptome and proteome analysis

• Infection models (cellular and animal) to assess virulence

• Competitive index determinations in mixed infections with wild-type

Controlling for Compensatory Adaptations:

• Use of conditional knockouts (tetracycline-regulated systems)

• Construction of depletion strains where NDK is expressed from inducible promoters

• Time-course experiments to distinguish immediate from adaptive effects

• Analysis of suppressor mutations that arise during mutant cultivation

This comprehensive approach ensures reliable characterization of the true NDK-deficient phenotype while accounting for potential adaptive responses that might mask the full impact of NDK loss.

How can phosphoproteomic approaches identify novel host and bacterial targets of Salmonella Newport NDK?

Phosphoproteomic approaches offer powerful tools for discovering novel NDK targets in both host and bacterial proteomes:

Experimental Workflow:

• Sample Preparation:

  • Infection of host cells with wild-type and ndk-deficient Salmonella Newport

  • Separate enrichment of bacterial and host cell fractions

  • Tryptic digestion of isolated proteins

• Phosphopeptide Enrichment:

  • TiO₂ or IMAC (Immobilized Metal Affinity Chromatography) for general phosphopeptide enrichment

  • Anti-phosphotyrosine antibodies for tyrosine phosphorylation studies

  • Sequential elution from IMAC for separation of mono- and multi-phosphorylated peptides

• MS Analysis:

  • LC-MS/MS using high-resolution instruments (Orbitrap or Q-TOF)

  • Data-dependent acquisition for discovery-based approaches

  • Parallel reaction monitoring for targeted quantification

  • SILAC or TMT labeling for accurate quantification

• Data Analysis:

  • Database searching against combined host-pathogen proteomes

  • Label-free or labeled quantification of phosphorylation differences

  • Motif analysis to identify NDK-specific phosphorylation patterns

  • Network analysis to identify affected signaling pathways

Expected Outcomes:

• Identification of differentially phosphorylated proteins in wild-type vs. ndk-deficient infections

• Discovery of direct NDK substrates by in vitro kinase assays with purified candidates

• Mapping of NDK-dependent phosphorylation sites in host defense proteins

• Characterization of temporal phosphorylation dynamics during infection

This approach has revealed that bacterial NDKs, like P. gingivalis-Ndk, can phosphorylate host proteins such as HSP27 at specific serine residues (Ser78 and Ser82), suggesting Salmonella Newport NDK may similarly target host proteins to modulate cellular responses during infection .

How could inhibitors targeting Salmonella Newport NDK be developed and evaluated for antimicrobial potential?

The development of selective NDK inhibitors represents a promising avenue for novel antimicrobials against Salmonella Newport:

Inhibitor Development Pipeline:

• Target Validation:

  • Confirm essentiality or virulence contribution of NDK in various infection models

  • Identify species-specific structural features for selective targeting

  • Validate druggability through computational and experimental approaches

• High-Throughput Screening Approaches:

  • Enzyme-based fluorescence assays measuring phosphotransfer activity

  • Fragment-based screening using NMR or thermal shift assays

  • Virtual screening against NDK crystal structure

  • Phenotypic screening for compounds that mimic ndk-mutant phenotypes

• Lead Optimization Strategies:

  • Structure-based design using bacterial NDK crystal structures

  • Focus on compounds that exploit differences between bacterial and human NDKs

  • Development of pro-drug approaches for improved bacterial penetration

  • Medicinal chemistry optimization for pharmacokinetic properties

• Evaluation Framework:

Potential for Combination Therapy:

• NDK inhibitors might sensitize Salmonella to oxidative stress

• Synergistic effects with antibiotics targeting cell division (due to NDK-FtsZ interaction)

• Enhanced activity in combination with host-directed therapies that modulate inflammasome activation

This systematic approach could yield novel therapeutics that target Salmonella pathogenesis through a mechanism distinct from conventional antibiotics, potentially addressing concerns about antimicrobial resistance.

What methods can be used to analyze the impact of environmental stress conditions on NDK function in Salmonella Newport?

Environmental stress significantly affects NDK function in Salmonella Newport, requiring sophisticated analytical approaches to fully characterize these responses:

Experimental Stress Conditions Relevant to Salmonella Lifecycle:

• Acid stress (pH 4.5-5.5): Mimicking gastric passage

• Oxidative stress: H₂O₂ (0.5-5 mM) or paraquat (10-100 μM)

• Osmotic stress: High salt (1-5% NaCl) conditions

• Nutrient limitation: Minimal media with restricted carbon or phosphate

• Temperature stress: Heat shock (42°C) or cold shock (15°C)

• Intracellular environment simulation: Low Mg²⁺, antimicrobial peptides

Analytical Methods and Expected Outcomes:

• Transcriptional Analysis:

  • qRT-PCR for ndk gene expression under various stresses

  • RNA-seq to identify stress-responsive NDK regulons

  • Promoter-reporter fusions to monitor transcriptional regulation

• Protein-Level Analysis:

  • Western blotting for NDK protein levels during stress

  • Pulse-chase experiments to determine protein stability

  • 2D gel electrophoresis to identify post-translational modifications

• Enzymatic Activity Assessment:

  • Development of in situ activity assays in bacterial lysates

  • Assessment of substrate preference shifts under stress

  • Kinetic parameter determination under varying conditions

• Structural Studies:

  • Circular dichroism to monitor secondary structure changes

  • Fluorescence spectroscopy for tertiary structure alterations

  • Size exclusion chromatography to assess oligomeric state shifts

• Functional Impact Analysis:

  • FtsZ polymerization assays under stress conditions

  • Nucleotide pool measurements using HPLC

  • Cell division pattern analysis using fluorescence microscopy

Stress-Specific Experimental Considerations:

• For oxidative stress: Prior evidence suggests NDK activity is particularly sensitive to H₂O₂ exposure

• During high osmolarity: Addition of osmoprotectants can distinguish direct from indirect effects

• Under high humidity conditions: Similar to conditions used with Salmonella Newport strain 44, enhanced survival may correlate with NDK activity

These approaches reveal how NDK function adapts to environmental challenges, providing insights into Salmonella Newport persistence mechanisms in diverse environments.

How might single-molecule techniques advance our understanding of Salmonella Newport NDK kinetics and interactions?

Single-molecule approaches offer unprecedented insights into NDK function that are masked in ensemble measurements:

Single-Molecule Methodologies and Applications:

• Single-Molecule FRET (smFRET):

  • Reveals conformational changes during catalytic cycle

  • Detects transient intermediate states during phosphate transfer

  • Monitors protein-protein interactions with substrate proteins

  • Implementation: Site-specific labeling of recombinant NDK with donor-acceptor dye pairs followed by TIRF microscopy visualization

• Optical Tweezers and Magnetic Tweezers:

  • Measures force generation during NDK-mediated FtsZ polymerization

  • Quantifies mechanical properties of protein complexes

  • Application: Tethering FtsZ filaments to surfaces and measuring forces during NDK-catalyzed polymerization

• Single-Molecule Tracking in Living Cells:

  • Visualization of NDK localization during infection processes

  • Tracking of diffusion patterns and binding events in real-time

  • Implementation: Expression of fluorescent protein fusions or HaloTag-NDK in live Salmonella Newport during host cell infection

• Nanopore Analysis:

  • Direct observation of individual NDK-nucleotide interactions

  • Discrimination between different nucleotide-bound states

  • Application: Protein nanopores with immobilized NDK to detect nucleotide binding events

Expected Mechanistic Insights:

• Observation of stochastic behavior in individual NDK molecules

• Detection of rare or transient conformational states

• Resolution of the temporal sequence of binding and catalytic events

• Identification of heterogeneity in kinetic parameters within the NDK population

• Visualization of interaction dynamics with partner proteins like FtsZ

These single-molecule approaches are particularly valuable for understanding NDK function in the context of bacterial cell division, where localized activity and protein-protein interactions play crucial roles in coordinating cytokinesis.

What are the potential cross-species communication roles of NDK in polymicrobial environments involving Salmonella Newport?

Emerging evidence suggests NDK may function in cross-species interactions, particularly in polymicrobial communities where Salmonella Newport coexists with other microorganisms:

Potential Inter-Species NDK Functions:

• Nucleotide Cross-Feeding:

  • Secreted or outer membrane-associated NDK may generate nucleotides accessible to other species

  • NDK activity could modify the extracellular nucleotide pool, influencing community composition

  • Potential role in biofilm formation where extracellular nucleotides serve as signaling molecules

• Modulation of Host Response in Polymicrobial Infections:

  • NDK from Salmonella Newport might phosphorylate host proteins, altering the environment for other pathogens

  • Similar to how P. gingivalis-Ndk phosphorylates HSP27 to inhibit apoptosis, benefiting the entire microbial community

  • Possible synergistic effects with other bacterial effectors targeting similar host pathways

• Competitive Advantage Mechanisms:

  • NDK could deplete specific nucleotides required by competing bacteria

  • Generation of nucleotide-derived signaling molecules that regulate virulence in other species

  • Potential interference with quorum sensing systems that rely on nucleotide-based second messengers

Experimental Approaches to Investigate Cross-Species Effects:

This research direction may reveal previously unrecognized roles for NDK in establishing and maintaining polymicrobial communities in which Salmonella Newport participates, with potential implications for understanding complex infection dynamics in human and animal hosts.

How do NDK sequence variations across Salmonella serovars correlate with host specificity and virulence?

Comparative analysis of NDK sequences across Salmonella serovars reveals patterns that may contribute to host adaptation and virulence differences:

Sequence Variation Analysis:

• Core catalytic residues are 100% conserved across all serovars

• Surface-exposed residues show higher variability, particularly in host-adapted serovars

• Host-restricted serovars (like S. Typhi) tend to have specific amino acid substitutions in regions involved in protein-protein interactions

• Broad host-range serovars (like S. Typhimurium) typically maintain a more ancestral NDK sequence

Correlation With Host Range:

Virulence Correlation:

How does Salmonella Newport NDK compare functionally to homologs in other bacterial pathogens?

Functional comparison of Salmonella Newport NDK with homologs from other bacterial pathogens reveals both conserved mechanisms and species-specific adaptations:

Cross-Species Functional Comparison:

Conserved Functions:

• All bacterial NDKs maintain canonical nucleotide phosphorylation activity

• Most interact with cell division machinery, particularly FtsZ

• Many contribute to stress responses and nucleotide pool homeostasis

Divergent Specializations:

• Targets for protein phosphorylation vary considerably between species

• Subcellular localization differs, with some NDKs being secreted or surface-associated

• Substrate preferences show adaptation to the pathogen's metabolic requirements

Functional Cross-Complementation:

• Mycobacterial NDK can trigger polymerization of FtsZ from other bacterial species, suggesting conserved interaction mechanisms

• P. gingivalis-Ndk-mediated host protein phosphorylation mechanisms may be shared with Salmonella Newport NDK

• The NDK of one mycobacterial species can trigger polymerization of FtsZ from another mycobacterial species, indicating functional conservation

This comparative analysis highlights how NDK has evolved from a housekeeping enzyme into a multifunctional protein with species-specific adaptations that contribute to each pathogen's unique virulence strategies.