Recombinant Mycobacterium tuberculosis Nicotinate-nucleotide pyrophosphorylase [carboxylating] (nadC)

What is the role of nicotinate-nucleotide pyrophosphorylase (nadC) in Mycobacterium tuberculosis metabolism?

Nicotinate-nucleotide pyrophosphorylase (nadC) is a crucial enzyme in the NAD(P) biosynthetic pathway of Mycobacterium tuberculosis. It catalyzes the conversion of quinolinic acid (pyridine-2,3-dicarboxylic acid) to nicotinate mononucleotide (NMn), which serves as a precursor molecule in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+). This reaction is a key step in the pathway that provides essential cofactors required for redox balance, energy metabolism, and the activity of the NAD-dependent DNA ligase in M. tuberculosis . The nadC gene works in concert with other genes (nadA, nadB, nadD, nadE, and ppnK) to complete the de novo NAD(P) biosynthetic pathway, which is critical for mycobacterial survival and virulence.

What are the basic methodological approaches for studying nadC function?

To study nadC function, researchers commonly employ several approaches:

• Genetic manipulation: Constructing knockout strains through allelic exchange, as demonstrated in studies where a nadA-C deletion strain (nad::hyg) was created by replacing segments of the genes with a hygromycin resistance cassette .

• Expression analysis: Using quantitative real-time PCR (qRT-PCR) to monitor gene expression under various conditions, including in vitro hypoxic environments and in vivo infection models.

• Protein purification: Employing recombinant protein expression systems with N-terminal His-tags, followed by purification using affinity chromatography, ion exchange chromatography, and size exclusion chromatography .

• Enzymatic assays: Developing in vitro assays to measure the conversion of quinolinic acid to nicotinate mononucleotide, typically using spectrophotometric or HPLC-based methods.

• Complementation studies: Reintroducing functional nadC genes into knockout strains to confirm phenotypes are due to specific gene loss rather than secondary effects.

What are the optimal conditions for expression and purification of recombinant M. tuberculosis nadC?

For optimal expression and purification of recombinant M. tuberculosis nadC, researchers should consider the following methodology based on successful approaches with similar enzymes:

• Expression system: Use E. coli C43(DE3) strain, which is particularly suitable for potentially toxic or membrane proteins. Transform cells with a pET-based vector containing the nadC gene fused to an N-terminal TEV protease-cleavable 6xHis-tag .

• Induction conditions: Cultivate transformed cells at 32°C and induce protein expression with 0.4 mM IPTG for approximately 8 hours to achieve optimal yield while maintaining protein solubility .

• Cell lysis: Resuspend harvested cells in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM β-mercaptoethanol, 1 mg/ml lysozyme, and a mixture of protease inhibitors (aprotinin, leupeptin, pepstatin, and PMSF), followed by gentle disruption through sonication .

• Purification workflow:

  • Initial purification via Ni-NTA affinity chromatography

  • Cleavage of the N-terminal His-tag with TEV protease

  • Further purification using MonoQ ion exchange chromatography

  • Final polishing via size exclusion chromatography using a HiLoad Superdex 200 column

• Quality assessment: Verify the purity of the final protein preparation using Coomassie-stained SDS-PAGE, with purified nadC typically appearing as a monomeric protein of approximately 21 kDa .

This optimized protocol typically yields approximately 15 mg of highly purified protein per liter of cultured cells, sufficient for subsequent structural and functional studies.

What crystallization approaches are most effective for structural studies of M. tuberculosis nadC?

Based on successful crystallization of related nicotinate-nucleotide pyrophosphorylases, the following approaches are recommended for M. tuberculosis nadC:

• Initial screening: Employ sitting-drop vapor diffusion method at 20°C using commercially available sparse matrix screens to identify preliminary crystallization conditions.

• Optimization conditions: For apo-enzyme crystals, optimal conditions typically include 0.1 M Hepes-KOH (pH 7.3), 25% (w/v) PEG 3350, and 0.2 M Mg(OAc)₂. For ligand-bound crystals (such as with NMn), conditions containing 0.1 M Bis-tris propane (pH 6.5), 15% (v/v) MPD, and 50 mM Na-Cacodylate have proven successful .

• Crystal handling: After obtaining diffraction-quality crystals, transfer them to a cryo-protectant solution containing 25% (v/v) glycerol in mother liquor before flash-cooling in liquid nitrogen for X-ray diffraction studies.

• Data collection parameters: Crystals of related pyrophosphorylases typically belong to the trigonal symmetry space group R32 with four monomers per asymmetric unit. Data collection to resolutions of 1.7-2.0 Å is generally achievable with synchrotron radiation .

• Structure determination: Employ molecular replacement using existing structures as search models, followed by iterative refinement using programs such as PHENIX and manual model building in COOT .

The concentration of precipitants (particularly MPD for ligand-bound complexes) appears critical for obtaining well-ordered crystals suitable for high-resolution structure determination.

How can researchers effectively study the kinetics of nadC catalytic activity?

For detailed kinetic characterization of nadC catalytic activity, researchers should implement the following methodological approach:

• Substrate preparation: Purify or synthesize quinolinic acid (pyridine-2,3-dicarboxylic acid) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to high purity (>98%) for reliable kinetic measurements.

• Assay development: Establish a continuous spectrophotometric assay monitoring either the consumption of substrates or formation of products (NMn). Alternative methods include HPLC-based assays for direct quantification of reaction components.

• Determination of optimal reaction conditions:

  • Buffer composition (typically Tris or HEPES-based buffers)

  • pH range (usually 7.0-8.0)

  • Temperature (physiologically relevant at 37°C)

  • Divalent metal ion requirements (typically Mg²⁺)

  • Ionic strength

• Kinetic parameter measurements:

  • Determine KM and Vmax for both substrates using initial velocity measurements

  • Analyze product inhibition patterns

  • Examine potential allosteric effects from metabolites in the NAD biosynthetic pathway

• Data analysis: Apply appropriate enzyme kinetic models (Michaelis-Menten, Lineweaver-Burk, or more complex models if allosteric behavior is observed) to determine kinetic constants and reaction mechanisms.

• Inhibitor screening: Develop a medium-throughput assay format to evaluate potential inhibitors, determining IC₅₀ values and inhibition mechanisms (competitive, non-competitive, uncompetitive, or mixed).

• pH and temperature dependence studies: Characterize the enzyme's activity profile across different pH values and temperatures to understand its physiological operating range and stability.

This comprehensive kinetic characterization provides essential information about nadC function and can inform structure-based drug design efforts targeting this enzyme.

What approaches can be used to assess the essentiality of nadC in different growth phases of M. tuberculosis?

To rigorously assess the essentiality of nadC across different growth phases of M. tuberculosis, researchers should implement a multi-faceted experimental design:

• Conditional expression systems: Develop strains with nadC under the control of inducible promoters (such as tetracycline-responsive systems) to regulate gene expression at specific growth phases.

• Genetic knockout with complementation: As demonstrated with related NAD pathway genes, construct a nadC knockout strain maintained with exogenous nicotinamide supplementation (10 μg/ml), then assess viability upon removal of the supplement during different growth phases .

• Transcriptional profiling: Perform RNA-seq or qRT-PCR to quantify nadC expression levels during:

  • Logarithmic growth phase

  • Stationary phase

  • Hypoxic conditions (NRP-1 and NRP-2 states)

  • In vivo infection (from macrophages and infected animal tissues)

• Metabolic labeling: Utilize ¹⁴C-labeled nicotinamide incorporation assays to assess the relative contribution of de novo synthesis versus salvage pathways during different growth phases .

• Chemical inhibition: Apply specific inhibitors targeting nadC at various growth phases and measure effects on bacterial viability and NAD/NADH ratios.

• In vivo assessment: Evaluate the survival of nadC-deficient strains in mouse models of acute and chronic tuberculosis infection to determine phase-specific requirements.

This systematic approach reveals that while nadC may be essential during active replication, M. tuberculosis demonstrates considerable flexibility in NAD synthesis pathways, potentially switching between de novo synthesis and recycling pathways depending on growth conditions .

How can researchers effectively study the interplay between nadC and other NAD biosynthetic enzymes?

To study the interplay between nadC and other NAD biosynthetic enzymes in M. tuberculosis, implement the following experimental approaches:

• Co-expression and protein-protein interaction studies:

  • Bacterial two-hybrid system to assess direct protein interactions

  • Co-immunoprecipitation with tagged versions of nadC and other pathway enzymes

  • Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) to detect interactions in living cells

• Metabolic flux analysis:

  • Use isotope-labeled precursors (¹³C or ¹⁵N) to trace the flow of metabolites through the pathway

  • Quantify intermediate metabolites using mass spectrometry to identify potential rate-limiting steps or metabolic bottlenecks

  • Compare flux patterns in wild-type vs. genetic mutants of different pathway components

• Genetic interaction mapping:

  • Construct double knockouts or knockdowns of nadC with other NAD pathway genes

  • Perform synthetic lethality screening to identify genes with redundant or complementary functions

  • Utilize CRISPR interference to simultaneously modulate multiple genes in the pathway

• Transcriptional co-regulation analysis:

  • Compare expression profiles of all NAD biosynthetic genes under various conditions

  • Identify shared transcription factor binding sites and regulatory elements

  • Perform ChIP-seq to map transcription factor binding across the pathway genes

• Enzyme kinetic coupling:

  • Develop multi-enzyme reaction systems to study substrate channeling

  • Measure kinetic parameters in combined enzyme assays versus isolated enzymes

  • Assess the impact of metabolite concentrations on the coordinated activity of pathway enzymes

This integrated approach reveals that while the de novo pathway (including nadC) is essential during active replication, there is significant plasticity in NAD metabolism, with complementary roles between de novo synthesis and recycling pathways .

What are the recommended methods for analyzing the impact of nadC mutations on M. tuberculosis viability and virulence?

For comprehensive analysis of nadC mutations on M. tuberculosis viability and virulence, researchers should implement the following methodological framework:

• Systematic mutation strategy:

  • Create point mutations at catalytic residues identified through structural analysis

  • Generate truncation variants to assess domain functionality

  • Develop a library of random mutations using error-prone PCR for unbiased screening

• Phenotypic characterization:

  • Growth curve analysis under standard conditions and stress conditions (hypoxia, nutrient limitation, oxidative stress)

  • Determination of minimum inhibitory concentrations (MICs) for various antibiotics to assess potential sensitization

  • Measurement of intracellular NAD/NADH ratios using enzymatic cycling assays or fluorescent biosensors

• In vitro macrophage infection models:

  • Assess bacterial survival in resting versus activated macrophages

  • Quantify cytokine responses to identify immunomodulatory effects

  • Perform time-course imaging to track intracellular bacterial replication and persistence

• Animal infection studies:

  • Low-dose aerosol infection of mice to evaluate bacterial burden in lungs and other organs

  • Histopathological analysis of infected tissues to assess granuloma formation and tissue damage

  • Survival analysis to determine virulence attenuation

• Complementation analysis:

  • Restore wild-type nadC expression in mutant strains to confirm phenotype specificity

  • Express mutant variants in wild-type background to identify potential dominant-negative effects

• NAD metabolite profiling:

  • Targeted metabolomics to quantify NAD, NADH, NADP, NADPH, and pathway intermediates

  • Comparative analysis between wild-type and mutant strains under various growth conditions

This approach has revealed that disruption of the de novo NAD biosynthetic pathway (which includes nadC) leads to cell death upon removal of exogenous nicotinamide during active replication in vitro. This lethality results from both cofactor starvation and disruption of cellular redox homeostasis as electron transport becomes impaired by limiting NAD .

How can structural information about nadC be leveraged for inhibitor design?

To effectively leverage structural information about M. tuberculosis nadC for rational inhibitor design, researchers should implement the following comprehensive approach:

• Structural analysis of substrate binding sites:

  • Obtain high-resolution crystal structures of nadC in complex with substrates (quinolinic acid and PRPP) and products (NMn)

  • Identify key catalytic residues and molecular interactions through structural analysis

  • Map the electrostatic and hydrophobic properties of the active site to guide inhibitor design

• Structure-based virtual screening:

  • Develop a pharmacophore model based on substrate binding mode

  • Perform molecular docking of compound libraries against the active site

  • Utilize molecular dynamics simulations to account for protein flexibility and identify transient binding pockets

• Fragment-based drug discovery:

  • Screen fragment libraries using techniques such as NMR, X-ray crystallography, or surface plasmon resonance

  • Identify weak-binding fragments that can be elaborated or linked to create potent inhibitors

  • Progress through iterative cycles of structure determination with bound fragments to guide optimization

• Structure-activity relationship (SAR) studies:

  • Synthesize analogues of identified hits with systematic modifications

  • Correlate structural changes with changes in inhibitory potency

  • Use crystallography to confirm binding modes of optimized compounds

• Analysis of species selectivity:

  • Compare structures of M. tuberculosis nadC with human homologues

  • Identify structural differences that can be exploited for selective targeting

  • Design inhibitors that preferentially bind to bacterial versus human enzyme forms

• Consideration of protein dynamics:

  • Perform hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • Use NMR to characterize protein dynamics in solution

  • Incorporate dynamic information into inhibitor design strategy

This structure-guided approach can lead to effective inhibitors of nadC, potentially contributing to the development of new antitubercular agents that could be effective against both actively growing and nonreplicating bacilli .

What are the advantages and limitations of using nadC as a potential drug target compared to other enzymes in the NAD biosynthetic pathway?

A comprehensive analysis of nadC as a potential drug target compared to other enzymes in the NAD biosynthetic pathway reveals several important considerations:

Advantages of targeting nadC:

• Pathway position: nadC catalyzes a key step in the de novo NAD biosynthetic pathway, which is essential for M. tuberculosis during active replication in vitro .

• Structural uniqueness: The enzyme structure offers potential for selective targeting compared to human homologues, reducing off-target effects.

• Metabolic importance: Inhibition disrupts NAD homeostasis, affecting multiple essential cellular processes including redox balance and energy metabolism.

• Biochemical tractability: The enzyme catalyzes a well-defined reaction amenable to high-throughput screening and rational inhibitor design.

Limitations of targeting nadC:

• Pathway redundancy: M. tuberculosis shows considerable flexibility in switching between de novo synthesis and recycling pathways to maintain NAD levels, suggesting that nadC inhibition alone might be insufficient for complete growth inhibition .

• Adaptation mechanisms: Transcriptional analysis reveals that M. tuberculosis can upregulate alternative NAD production pathways (particularly recycling pathways) under stress conditions and during in vivo infection .

• Variable essentiality: While critical during active replication, nadC may be less essential during latent infection when recycling pathways are upregulated.

Comparison with other pathway targets:

• Universal pathway enzymes (NadD and NadE): These represent more attractive targets as they are common to both de novo and recycling pathways. Inhibitors of NAD synthetase (NadE) display bactericidal effects against both actively growing and nonreplicating M. tuberculosis .

• Recycling pathway enzymes: Given the upregulation of recycling pathway genes (particularly pncB2) under hypoxic conditions and during chronic infection, these enzymes might be more relevant targets for latent tuberculosis .

• Combined targeting: A dual-inhibition strategy targeting both de novo synthesis (nadC) and recycling pathways might be more effective in preventing adaptation.

The research suggests that while nadC is a viable target, the two common enzymes shared by both pathways (NadD and NadE) may be more viable drug targets for comprehensive inhibition of NAD metabolism in M. tuberculosis .

How can researchers design experiments to understand the role of nadC in M. tuberculosis persistence during host infection?

To elucidate the role of nadC in M. tuberculosis persistence during host infection, researchers should design experiments that address the dynamic nature of NAD metabolism across infection stages:

• Temporal gene expression analysis:

  • Implement time-course RNA-seq or qRT-PCR of nadC and other NAD pathway genes during:

    • Early infection (1-2 weeks post-infection)

    • Established infection (4-8 weeks)

    • Chronic/latent phase (>12 weeks)

  • Compare expression in different infection microenvironments (granulomas vs. non-granulomatous tissue)

  • Correlate nadC expression with bacterial metabolic state markers

• Conditional gene silencing in vivo:

  • Develop tetracycline-inducible knockdown strains of nadC

  • Administer doxycycline at different time points during infection to silence nadC

  • Assess impact on bacterial persistence, tissue burden, and reactivation potential

• Metabolic labeling studies:

  • Perform ¹⁴C-nicotinamide incorporation assays in bacteria isolated directly from infected hosts

  • Compare incorporation efficiency between bacteria from different infection phases

  • Assess the relative contribution of de novo synthesis versus salvage pathways at each stage

• Infection models optimized for persistence:

  • Cornell model of latent tuberculosis with antibiotic-induced latency

  • Hypoxic granuloma models using C3HeB/FeJ mice or rabbits

  • Non-human primate models with natural latency development

• Cell-specific effects:

  • Isolate M. tuberculosis from different host cell types (alveolar macrophages, interstitial macrophages, dendritic cells)

  • Assess nadC expression and NAD metabolism in bacteria from each cellular niche

  • Correlate with host cell activation status and metabolic profile

• Integration with systems biology:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Develop predictive models of NAD metabolism during different infection phases

  • Identify potential compensatory mechanisms during nadC inhibition

These experiments should take into account the finding that genes involved in the early steps of the de novo biosynthesis pathway (including nadC) show strong repression in M. tuberculosis isolated from chronically infected mice, while recycling pathway genes (particularly pncB2) show strong upregulation under these conditions . This suggests a metabolic shift away from de novo NAD synthesis during persistent infection.

What are common challenges in expressing soluble and active recombinant M. tuberculosis nadC?

Researchers working with recombinant M. tuberculosis nadC frequently encounter several challenges that can be addressed through specific methodological adjustments:

• Expression system selection challenges:

  • Problem: Low expression levels or inclusion body formation in standard E. coli strains

  • Solution: Use specialized expression strains such as E. coli C43(DE3) that are designed for membrane proteins and toxic proteins, which has proven successful with similar pyrophosphorylases

• Protein solubility issues:

  • Problem: Formation of insoluble aggregates despite optimized expression conditions

  • Solution:

    • Reduce induction temperature to 32°C rather than standard 37°C

    • Lower IPTG concentration to 0.4 mM

    • Extend induction time to 8 hours instead of standard 3-4 hours

    • Add solubility-enhancing fusion tags (SUMO, MBP) if His-tag constructs remain problematic

• Protein stability challenges:

  • Problem: Purified protein shows rapid activity loss or precipitation

  • Solution:

    • Include reducing agents (5 mM β-mercaptoethanol) in all purification buffers

    • Add glycerol (10-15%) to final storage buffer

    • Identify optimal buffer conditions through thermal shift assays (Thermofluor)

    • Store protein at high concentration (>5 mg/ml) to prevent surface denaturation

• Enzymatic activity inconsistencies:

  • Problem: Variable or low enzymatic activity between preparations

  • Solution:

    • Include a complete protease inhibitor mixture during cell lysis

    • Verify the presence of essential divalent metals (typically Mg²⁺) in activity assays

    • Ensure substrate quality, particularly for labile PRPP

    • Consider the need for potential cofactors or activators

• Post-translational modifications:

  • Problem: Bacterial expression system lacks necessary modifications

  • Solution:

    • Test expression in mycobacterial hosts if E. coli-expressed protein lacks activity

    • Consider eukaryotic expression systems if mycobacterial modifications are suspected

These optimizations generally yield approximately 15 mg of highly purified, active protein per liter of cultured cells, enabling subsequent structural and functional studies .

How can researchers address contradictory results regarding nadC essentiality in different experimental models?

When confronting contradictory results regarding nadC essentiality across different experimental models, researchers should implement a systematic troubleshooting approach:

• Standardize genetic manipulation methods:

  • Problem: Different knockout strategies may leave varying genomic scars affecting neighboring genes

  • Solution:

    • Use precise in-frame deletion methods without antibiotic markers when possible

    • Confirm deletion boundaries with sequencing

    • Complement mutants with wild-type gene at different expression levels

    • Verify transcription of flanking genes is unaffected by the mutation

• Control for strain background effects:

  • Problem: Laboratory strain adaptation versus clinical isolate behavior

  • Solution:

    • Perform parallel studies in multiple strain backgrounds (H37Rv, CDC1551, clinical isolates)

    • Account for potential compensatory mutations in laboratory-adapted strains

    • Sequence verify strains to identify potential secondary mutations

• Normalize growth conditions:

  • Problem: Media composition affects the relative importance of de novo synthesis versus salvage pathways

  • Solution:

    • Standardize media formulations, particularly regarding NAD precursor content

    • Test multiple carbon sources that may affect redox balance and NAD requirements

    • Systematically vary oxygen tension to model different in vivo environments

• Reconcile in vitro versus in vivo discrepancies:

  • Problem: nadC appears more essential in vitro than in mouse models

  • Solution:

    • Analyze host-derived metabolites that may complement pathway deficiencies

    • Compare nadC mutant growth in defined media versus tissue homogenates

    • Trace metabolic flux using isotope-labeled precursors in different models

• Consider temporal aspects of essentiality:

  • Problem: Differential requirements during infection stages

  • Solution:

    • Implement time-course studies with inducible gene silencing

    • Distinguish between requirements for establishment versus maintenance of infection

    • Correlate gene expression levels with apparent essentiality

This comprehensive approach acknowledges that while the de novo NAD biosynthetic pathway (including nadC) appears essential during active replication in vitro, M. tuberculosis demonstrates considerable flexibility in NAD metabolism. The organism can switch between de novo synthesis and recycling of exogenously acquired nicotinamide depending on environmental conditions , explaining apparent contradictions in essentiality across different experimental models.

What methodological considerations are important when designing inhibitor screening assays for nadC?

When designing robust inhibitor screening assays for M. tuberculosis nadC, researchers should address these critical methodological considerations:

• Assay format optimization:

  • Primary considerations:

    • Select between continuous spectrophotometric assays (higher throughput) versus endpoint assays (more sensitive)

    • Determine optimal substrate concentrations relative to KM values (typically at or below KM for inhibitor identification)

    • Establish optimal enzyme concentration that provides linear reaction rates for the assay duration

  • Practical implementation:

    • Develop a 384-well format for higher throughput

    • Include positive controls (known inhibitors if available) and negative controls (vehicle only)

    • Standardize reaction times to capture initial velocity conditions

• Signal detection strategies:

  • Assay options:

    • Direct detection of pyrophosphate release using coupled enzyme systems

    • Monitoring quinolinic acid consumption via absorbance

    • Measuring NMn formation using HPLC or fluorescence-based methods

  • Optimization parameters:

    • Signal-to-background ratio (aim for >3:1)

    • Z' factor (aim for >0.5 for robust screening)

    • Limit of detection for product formation or substrate consumption

• Interference mitigation:

  • Common interferences:

    • Compound autofluorescence or absorbance at detection wavelengths

    • Direct enzyme inactivation through aggregation or non-specific effects

    • Interference with coupled enzyme systems if used

  • Countermeasures:

    • Include detergent (0.01% Triton X-100) to prevent aggregate-based inhibition

    • Perform parallel screens against coupling enzymes alone

    • Implement counterscreens with orthogonal detection methods

• Physiological relevance:

  • Buffer considerations:

    • Maintain physiologically relevant pH (typically 7.4)

    • Include appropriate ionic strength to mimic cellular conditions

    • Ensure proper divalent metal concentrations (typically 1-5 mM Mg²⁺)

  • Substrate presentation:

    • Ensure both substrates (quinolinic acid and PRPP) are present at appropriate ratios

    • Consider substrate order of addition for bisubstrate reaction mechanism

• Follow-up validation workflow:

  • Primary hit confirmation:

    • Dose-response curves with fresh compound samples

    • Determination of inhibition mechanism (competitive, non-competitive, uncompetitive)

    • Evaluation of reversibility through enzyme dilution or rapid dilution experiments

  • Secondary assays:

    • Thermal shift assays to confirm direct binding

    • Surface plasmon resonance or isothermal titration calorimetry for binding kinetics

    • Crystallography of enzyme-inhibitor complexes

This methodologically rigorous approach ensures identification of specific, mechanism-based inhibitors rather than promiscuous compounds with non-specific effects, establishing a foundation for structure-based optimization of nadC inhibitors as potential tuberculosis therapeutics.

What emerging technologies could advance our understanding of nadC function in M. tuberculosis?

Several cutting-edge technologies hold promise for deepening our understanding of nadC function in M. tuberculosis:

• CRISPR interference (CRISPRi) for temporal gene regulation:

  • Implement inducible dCas9-based systems for precise transcriptional repression of nadC

  • Create multiplexed CRISPRi to simultaneously modulate multiple NAD pathway genes

  • Apply during different growth phases to assess stage-specific requirements for nadC

• Single-cell analysis technologies:

  • Develop fluorescent biosensors for real-time NAD/NADH ratio monitoring in single bacterial cells

  • Apply microfluidic platforms to track individual bacterial responses to nadC inhibition

  • Perform single-cell RNA-seq on bacteria isolated from different microenvironments in infected tissues

• Advanced structural biology approaches:

  • Implement time-resolved crystallography to capture intermediate states during catalysis

  • Apply cryo-electron microscopy for structure determination without crystallization

  • Utilize neutron diffraction to precisely locate hydrogen atoms in the active site

• Metabolic flux analysis with stable isotopes:

  • Trace carbon flow through NAD metabolic pathways using ¹³C-labeled precursors

  • Quantify flux distributions using mass spectrometry-based metabolomics

  • Develop computational models integrating metabolic flux data with gene expression

• In situ visualization techniques:

  • Apply correlative light and electron microscopy to localize nadC within the bacterial cell

  • Utilize proximity labeling methods to identify protein-protein interactions in the native cellular context

  • Implement MALDI-imaging mass spectrometry to map NAD metabolites in infected tissues

• Host-pathogen interaction technologies:

  • Apply dual RNA-seq to simultaneously monitor host and bacterial transcriptional responses

  • Develop organoid infection models to better recapitulate human granuloma environments

  • Utilize humanized mouse models for more physiologically relevant in vivo studies

These technologies will enable researchers to move beyond the current understanding that M. tuberculosis displays considerable flexibility in NAD metabolism , helping to elucidate the precise contexts in which nadC function is critical for bacterial survival and pathogenesis.

How might research on nadC contribute to developing novel therapeutic strategies for drug-resistant tuberculosis?

Research on nadC holds significant potential for developing novel therapeutic strategies against drug-resistant tuberculosis through several mechanistic approaches:

• Exploitation of metabolic vulnerabilities:

  • Target NAD metabolism as an orthogonal pathway to current drug targets

  • Develop dual-targeting compounds affecting both nadC and other enzymes in the pathway

  • Create drug combinations specifically designed to prevent resistance development

• Rationally designed inhibitor scaffolds:

  • Utilize high-resolution structural data of nadC for structure-based drug design

  • Develop transition state analogs that mimic the reaction coordinate

  • Create bisubstrate inhibitors linking structural features of both quinolinic acid and PRPP

• Activity against non-replicating populations:

  • Exploit the finding that NAD metabolism remains essential even in nonreplicating bacilli

  • Target the universal pathway enzymes (NadD and NadE) that are required in both active and latent tuberculosis

  • Develop compounds specifically active under the hypoxic conditions characteristic of latent infection

• Novel drug delivery strategies:

  • Design prodrugs activated by mycobacterial enzymes for specificity

  • Develop nanoparticle formulations targeting host cells harboring persistent bacteria

  • Create host-directed therapies that modify the tissue microenvironment to enhance nadC inhibitor efficacy

• Combination therapy approaches:

  • Identify synergistic interactions between nadC inhibitors and existing antibiotics

  • Develop rational combinations targeting both de novo synthesis and recycling pathways

  • Create therapeutic regimens specifically designed for different disease stages

• Precision medicine applications:

  • Analyze clinical isolate genomic data to identify NAD pathway variations

  • Develop inhibitor panels effective against diverse strain backgrounds

  • Create diagnostic tools to guide selection of optimal NAD metabolism-targeting compounds

Research has already demonstrated that inhibitors of NAD synthetase (NadE), an essential enzyme common to both recycling and de novo synthesis pathways, display bactericidal effects against both actively growing and nonreplicating M. tuberculosis . This finding suggests that targeting the universal pathway of NAD metabolism represents a particularly promising approach for developing therapeutics effective against both active and latent tuberculosis, including drug-resistant strains.

What interdisciplinary approaches might yield new insights into nadC regulation and function?

Innovative interdisciplinary approaches can provide transformative insights into nadC regulation and function in M. tuberculosis:

• Systems biology integration:

  • Combine transcriptomics, proteomics, and metabolomics data to create comprehensive models of NAD metabolism

  • Implement flux balance analysis to predict metabolic adaptations to nadC perturbation

  • Develop machine learning algorithms to identify non-obvious regulatory relationships affecting nadC expression

• Synthetic biology approaches:

  • Engineer synthetic NAD biosynthetic pathways with tunable expression of individual components

  • Create reporter strains with nadC promoter fusions to fluorescent proteins for real-time monitoring

  • Develop minimal synthetic cells to identify the core requirements for NAD metabolism

• Evolutionary biology perspectives:

  • Perform comparative genomics across mycobacterial species to trace the evolution of NAD biosynthetic pathways

  • Implement experimental evolution under NAD-limiting conditions to identify adaptive mechanisms

  • Analyze clinical isolates for natural variations in nadC sequence and expression patterns

• Chemical biology tools:

  • Develop activity-based probes that covalently label active nadC

  • Create photo-switchable inhibitors for temporal control of enzyme inactivation

  • Implement chemoproteomics approaches to identify off-target interactions of nadC inhibitors

• Computational chemistry and biology:

  • Apply quantum mechanics/molecular mechanics (QM/MM) simulations to elucidate the catalytic mechanism

  • Implement enhanced sampling techniques to identify cryptic binding sites

  • Develop deep learning approaches for predicting inhibitor binding modes and efficacy

• Host immunology integration:

  • Investigate connections between NAD metabolism and mycobacterial immunomodulation

  • Explore the impact of host NAD-consuming enzymes (PARPs, sirtuins) on bacterial metabolism

  • Develop immunometabolic interventions that synergize with nadC inhibition

These interdisciplinary approaches acknowledge the remarkable plasticity of M. tuberculosis in maintaining NAD levels through different pathways and seek to develop a more holistic understanding of how nadC functions within the broader context of bacterial physiology, host-pathogen interactions, and disease progression.