Recombinant Campylobacter concisus Nucleoside diphosphate kinase (ndk)

What is Campylobacter concisus and why is it significant for NDK studies?

Campylobacter concisus is a Gram-negative, curved bacterium that requires microaerobic or anaerobic conditions enriched with H₂ for growth. Cells measure (0.5-1) × (2-6) μm and are motile via a single polarized flagellum . The organism primarily colonizes the human oral cavity, with detection rates of 97-100% in saliva samples from healthy individuals . C. concisus has gained research significance due to its association with inflammatory bowel disease (IBD), including both Crohn's disease and ulcerative colitis.

Nucleoside diphosphate kinase (ndk) from C. concisus represents an important research target because:

• As a housekeeping enzyme involved in nucleotide metabolism, NDK is essential for bacterial growth and survival

• NDK proteins in other bacterial pathogens have been implicated in virulence mechanisms

• Studying C. concisus NDK may provide insights into its metabolic adaptations and potential contribution to pathogenesis

How do the genomic characteristics of C. concisus affect NDK research?

C. concisus strains are classified into two distinct genomospecies (GS):

• GS1: Represented by the type strain ATCC 33237ᵀ

• GS2: Represented by strain CCUG 19995

These genomospecies show significant genetic differences:

• G+C content differs with medians of 37.56% for GS1 and 39.51% for GS2

• Intra-GS Average Nucleotide Identity (ANI) values range between 93% and 96%

• Inter-GS ANI values range between 88% and 91%

These genomic differences may result in variation in the NDK protein between strains from different genomospecies. Researchers should consider isolate selection carefully, as genomospecies differences may impact experimental outcomes, including recombinant protein expression, structure, and function.

What is the relationship between C. concisus detection methods and research reliability?

Detection methods significantly impact C. concisus research outcomes, as demonstrated by comparative studies:

When amplifying the NDK gene, researchers should employ multiple detection methodologies, as standard PCR may significantly underestimate prevalence. Additionally, collection of multiple biopsies greatly increases detection rates compared to single biopsies .

How should researchers approach strain selection for C. concisus NDK studies?

When selecting C. concisus strains for NDK studies, researchers should consider:

• Genomospecies classification: Clearly identify and report whether strains belong to GS1 (ATCC 33237ᵀ-like) or GS2 (CCUG 19995-like) to account for potential genetic variations in the NDK gene .

• Source of isolation: Oral isolates may differ from intestinal isolates, potentially affecting NDK characteristics . Protein profiling studies have shown that only some oral strains match intestinal strain profiles .

• Clinical context: Consider whether strains were isolated from healthy individuals or IBD patients, as this may correlate with genomospecies distribution and potentially NDK properties.

• Comparative approach: Ideally, analyze NDK from multiple strains representing both genomospecies to identify potential structural or functional differences that may correlate with different clinical manifestations.

• Validation: Sequence verification of the NDK gene is essential, especially when using clinical isolates, due to the potential presence of mixed Campylobacter species in IBD patients (20.3% in UC vs. 4.6% in controls) .

What expression systems are optimal for producing recombinant C. concisus NDK?

The selection of an appropriate expression system should consider C. concisus-specific characteristics:

• E. coli-based systems:

  • BL21(DE3) derivatives with rare codon supplementation (Rosetta, CodonPlus) are recommended due to the low G+C content of C. concisus (37.56-39.51%)

  • Consider reduced-temperature expression (16-25°C) to improve protein solubility

  • Evaluate both aerobic and microaerobic induction conditions, as C. concisus is microaerophilic

• Alternative expression hosts:

  • Campylobacter jejuni expression systems may provide more native-like conditions

  • Cell-free expression systems can be advantageous if the protein forms inclusion bodies

• Fusion tags strategy:

  • N-terminal solubility-enhancing tags (MBP, SUMO) may improve expression

  • Include protease cleavage sites for tag removal

  • Consider both N- and C-terminal tagging approaches

• Expression verification:

  • Western blotting with anti-His or anti-NDK antibodies

  • Activity assays to confirm functional expression

  • Mass spectrometry to verify protein identity

What purification challenges are specific to recombinant C. concisus NDK?

Several C. concisus-specific considerations affect purification strategies:

• Oxygen sensitivity: As C. concisus requires microaerobic or anaerobic conditions for growth , its proteins may be sensitive to oxidation. Consider:

  • Addition of reducing agents (DTT, β-mercaptoethanol) in buffers

  • Performing purification steps under nitrogen atmosphere

  • Including oxygen scavengers in final storage buffers

• Oligomeric state: Bacterial NDKs typically form hexamers, but this should be confirmed for C. concisus NDK using:

  • Size exclusion chromatography

  • Native PAGE

  • Dynamic light scattering

• Stability considerations:

  • Test pH stability across a range of conditions (pH 6.0-8.5)

  • Evaluate thermal stability using differential scanning fluorimetry

  • Determine appropriate storage conditions (buffer composition, temperature)

• Contaminant removal:

  • Include additional purification steps (ion exchange, hydrophobic interaction chromatography)

  • Test for endotoxin contamination if the protein will be used in immunological studies

  • Confirm purity by SDS-PAGE (aim for >95% purity)

What are the established methods for assessing NDK enzymatic activity?

Comprehensive characterization of C. concisus NDK activity requires multiple complementary approaches:

• Coupled spectrophotometric assay:

  • Principle: NDK transfers phosphate from NTP to NDP, with the generated NTP used by pyruvate kinase to convert phosphoenolpyruvate to pyruvate, which is then reduced by lactate dehydrogenase with concomitant oxidation of NADH

  • Detection: Decrease in NADH absorbance at 340 nm

  • Advantages: Continuous monitoring, high sensitivity

  • Considerations: Potential interference from coupling enzymes

• Direct nucleotide conversion assay:

  • Principle: Direct monitoring of NDP to NTP conversion

  • Methods: HPLC separation and quantification of nucleotides

  • Advantages: Direct measurement of substrate and product

  • Considerations: Requires specialized equipment, not continuous

• Phosphate release assay:

  • Principle: Measurement of inorganic phosphate released during reaction

  • Methods: Malachite green or other phosphate detection reagents

  • Advantages: Simple, colorimetric detection

  • Considerations: Endpoint assay, less specific

Parameters to optimize include:

• pH (typically 7.0-8.0)

• Divalent cation concentration (Mg²⁺, Mn²⁺)

• Temperature (30-37°C)

• Substrate concentrations for kinetic analysis

How can researchers investigate substrate specificity of C. concisus NDK?

A comprehensive substrate specificity profile is essential for characterizing C. concisus NDK:

• Donor nucleotide screening:

  • Test all common NTPs (ATP, GTP, CTP, UTP)

  • Determine relative efficiency as phosphate donors

  • Measure kinetic parameters (Km, kcat, kcat/Km) for each

• Acceptor nucleotide screening:

  • Test all common NDPs (ADP, GDP, CDP, UDP)

  • Determine relative efficiency as phosphate acceptors

  • Measure kinetic parameters for each

• Non-canonical substrate testing:

  • Deoxynucleotides (dADP, dGDP, dCDP, dTDP)

  • Modified nucleotides (e.g., methylated, oxidized)

  • Nucleotide analogs with potential inhibitory activity

• Data presentation format:

  Donor	Acceptor	Km (μM)	kcat (s⁻¹)	kcat/Km (M⁻¹s⁻¹)	Relative Efficiency (%)
  ATP	GDP	[value]	[value]	[value]	[value]
  ATP	CDP	[value]	[value]	[value]	[value]
  [etc]	[etc]	[value]	[value]	[value]	[value]

• Comparative analysis:

  • Compare substrate profiles between GS1 and GS2 NDK variants

  • Compare with NDK from other Campylobacter species

  • Correlate differences with structural features

What controls are essential for reliable C. concisus NDK activity assessment?

Robust experimental design requires comprehensive controls:

• Enzyme controls:

  • Active enzyme: Freshly purified recombinant C. concisus NDK

  • Negative control: Heat-inactivated (95°C, 10 min) enzyme preparation

  • Positive control: Commercial NDK (e.g., from bovine or E. coli)

  • Catalytic mutant: If available, a site-directed mutant of a key catalytic residue

• Substrate controls:

  • No-donor control: Reaction mixture lacking NTP donor

  • No-acceptor control: Reaction mixture lacking NDP acceptor

  • Background hydrolysis: Monitor NTP hydrolysis in absence of enzyme

  • Substrate purity: Verify nucleotide purity by HPLC

• Assay validation controls:

  • Linear response: Verify linearity with enzyme concentration

  • Time course: Ensure measurements in initial velocity range

  • Reproducibility: Perform at least three independent experiments

  • Buffer controls: Test effects of buffer components on activity

• Data analysis considerations:

  • Blank subtraction: Account for background signals

  • Normalization: Express activity per μg or nmol of enzyme

  • Statistical analysis: Apply appropriate statistical tests

  • Outlier identification: Use systematic approaches to identify outliers

How can structural studies enhance understanding of C. concisus NDK function?

Structural characterization provides fundamental insights into C. concisus NDK:

• Homology modeling approach:

  • Use existing bacterial NDK structures as templates

  • Generate separate models for GS1 and GS2 variants

  • Validate models using energy minimization and Ramachandran analysis

  • Compare active site architectures between variants

• X-ray crystallography:

  • Crystallization screening with purified protein (>95% purity)

  • Co-crystallization with substrate analogs or product

  • Structure determination and refinement

  • Comparative analysis with related NDK structures

• Key structural features to analyze:

  • Active site architecture and catalytic residues

  • Substrate binding pocket

  • Oligomerization interfaces

  • Surface electrostatic properties

  • Potential unique features compared to other bacterial NDKs

• Structure-function correlations:

  • Map conservation between GS1 and GS2 variants onto structure

  • Identify potential genomospecies-specific structural differences

  • Design structure-guided mutations to test functional hypotheses

  • Correlate structural features with substrate specificity profiles

How might C. concisus NDK contribute to bacterial pathogenesis?

NDK proteins in other bacterial pathogens have demonstrated roles in virulence, suggesting potential pathogenic mechanisms for C. concisus NDK:

• Potential intracellular functions:

  • Nucleotide pool maintenance during intestinal colonization

  • Adaptation to stress conditions (oxidative stress, nutrient limitation)

  • Contribution to DNA repair mechanisms

  • Metabolic adaptation between oral and intestinal environments

• Potential extracellular functions:

  • Modulation of extracellular nucleotide concentrations

  • Interference with host purinergic signaling

  • Modification of intestinal epithelial cell responses

  • Contribution to immune evasion strategies

• Host response considerations:

  • Potential immunogenicity of C. concisus NDK

  • Detection of anti-NDK antibodies in IBD patients

  • Role in triggering inflammatory responses

  • Differential responses between genomospecies variants

• Research approaches:

  • Gene knockout studies in C. concisus (challenging due to limited genetic tools)

  • Recombinant protein addition to epithelial cell models

  • Protein-protein interaction studies with host factors

  • Comparison of NDK properties between strains with different virulence

How can C. concisus NDK research contribute to diagnostic developments?

C. concisus detection shows significant association with IBD, suggesting potential diagnostic applications:

• NDK-based detection strategies:

  • Development of NDK-specific PCR primers for improved detection

  • Quantitative PCR targeting the NDK gene for bacterial load estimation

  • Design of primers distinguishing between GS1 and GS2 NDK variants

  • Multiple-biopsy sampling protocols to improve detection sensitivity

• Serological approaches:

  • Development of anti-NDK antibody detection assays

  • Correlation of antibody levels with disease activity

  • Distinction between antibody responses to GS1 versus GS2 NDK

  • Potential prognostic value of anti-NDK antibody patterns

• Activity-based detection:

  • Development of NDK activity assays in clinical samples

  • Correlation of activity levels with bacterial load

  • Inhibitor screening for specific C. concisus NDK inhibition

  • Potential for rapid colorimetric detection methods

• Clinical correlation studies:

  • Association of specific NDK variants with disease phenotypes

  • Longitudinal studies of NDK detection and disease progression

  • Relationship between NDK detection and treatment response

  • Correlation with other inflammatory or microbiome markers

What comparative genomic approaches could advance C. concisus NDK research?

Genomic analysis can provide essential context for NDK studies:

• Comprehensive NDK sequence comparison:

  • Analysis across C. concisus genomospecies (GS1 vs. GS2)

  • Comparison with NDK from other Campylobacter species

  • Identification of conserved vs. variable regions

  • Analysis of selection pressure on the NDK gene

• Genomic context analysis:

  • Examination of NDK gene neighborhood across strains

  • Identification of potential co-regulated genes

  • Analysis of promoter regions and regulatory elements

  • Investigation of horizontal gene transfer evidence

• Multi-locus approaches:

  • Include NDK in multi-locus sequence typing schemes

  • Compare phylogenetic trees based on NDK versus whole-genome data

  • Evaluate NDK as a potential marker for strain typing

  • Correlation of NDK variants with virulence-associated genes

• Transcriptomic integration:

  • Analysis of NDK expression under different conditions

  • Correlation with expression of other virulence factors

  • Differential expression between GS1 and GS2 strains

  • Response to host-derived signals or stressors

What technological advances could enhance C. concisus NDK research?

Emerging technologies offer new opportunities for NDK research:

• CRISPR-Cas9 approaches:

  • Development of genetic manipulation tools for C. concisus

  • Precise editing of the NDK gene

  • Creation of reporter strains for NDK expression studies

  • Generation of catalytic mutants for function validation

• Single-cell techniques:

  • Single-cell RNA sequencing of C. concisus from clinical samples

  • Analysis of NDK expression heterogeneity

  • Correlation with host cell interactions

  • Spatial transcriptomics in intestinal biopsies

• Advanced protein analysis:

  • Hydrogen-deuterium exchange mass spectrometry for dynamic studies

  • Native mass spectrometry for oligomeric state analysis

  • Protein-protein interaction network mapping

  • In situ structural studies using cryo-electron tomography

• High-throughput screening:

  • Development of NDK activity-based screening assays

  • Identification of specific inhibitors

  • Drug repurposing approaches targeting NDK

  • Screening for compounds that differentially affect GS1 vs. GS2 NDK

How can interdisciplinary approaches enhance C. concisus NDK research?

Integration across disciplines will advance understanding of C. concisus NDK:

• Clinical-basic science collaborations:

  • Access to well-characterized patient cohorts

  • Correlation of NDK variants with clinical phenotypes

  • Longitudinal studies linking NDK detection with disease progression

  • Evaluation of potential diagnostic applications

• Microbiome integration:

  • Analysis of C. concisus and NDK in the context of microbiome dysbiosis

  • Interactions between C. concisus and other microbiota members

  • Effect of microbial metabolites on NDK expression and activity

  • Competition for nucleotides in complex microbial communities

• Immunological perspectives:

  • Investigation of innate immune responses to C. concisus NDK

  • Analysis of adaptive immunity and antibody responses

  • Evaluation of NDK as a potential vaccine component

  • Roles in immunomodulation during chronic inflammation

• Systems biology approaches:

  • Integration of genomic, transcriptomic, and proteomic data

  • Metabolic modeling of nucleotide metabolism in C. concisus

  • Network analysis of NDK interactions in cellular processes

  • Predictive modeling of NDK contributions to pathogenesis