Recombinant Listeria monocytogenes serotype 4b Probable nicotinate-nucleotide adenylyltransferase (nadD)

What is the biochemical function of nicotinate-nucleotide adenylyltransferase (nadD) in Listeria monocytogenes?

Nicotinate-nucleotide adenylyltransferase (nadD) catalyzes the adenylation of nicotinate mononucleotide (NaMN) to form nicotinate adenine dinucleotide (NaAD), representing a critical step in NAD biosynthesis. This reaction requires ATP and magnesium as cofactors. In L. monocytogenes, this enzyme is essential for maintaining NAD+ levels, which serve as critical cofactors for numerous metabolic processes including redox reactions, energy production, and potentially cell wall biosynthesis.

For experimental validation of nadD function, researchers should:

• Generate conditional knockdown strains rather than direct knockouts (as nadD is likely essential)

• Perform enzyme activity assays measuring the conversion of NaMN to NaAD

• Quantify NAD+ levels in wild-type versus nadD-depleted conditions

• Assess growth phenotypes under various metabolic stress conditions

How is L. monocytogenes serotype 4b classified within the broader taxonomy of Listeria?

L. monocytogenes serotype 4b strains predominantly belong to lineage I, although some serotype 4b strains have been identified in lineage III as well . Lineage I typically includes serotypes associated with epidemic human listeriosis, making serotype 4b particularly important for clinical research.

Molecular typing methods reveal that serotype 4b strains from different lineages possess distinct genetic features:

• Lineage I serotype 4b strains react positively with serotype-specific ORF2110 PCR primers

• Lineage III serotype 4b strains are negative for ORF2110 and lmo1134 primers

• Lineage III strains form two separate groups based on their reactions with virulence-specific lmo2821 primers

These molecular distinctions highlight the genetic diversity within serotype 4b strains, which may influence metabolic enzyme function, including nadD.

What expression systems are optimal for producing recombinant L. monocytogenes serotype 4b nadD protein?

For optimal expression of recombinant L. monocytogenes serotype 4b nadD, researchers should consider the following methodological approach:

• Vector design considerations:

  • Include an N-terminal His6 or MBP tag for purification

  • Consider codon optimization for the expression host

  • Include a precision protease cleavage site (TEV or 3C) between tag and target protein

  • Select a vector with tunable expression control (T7 or similar inducible system)

• Expression host selection:

  • E. coli BL21(DE3) is suitable for initial trials

  • E. coli Rosetta strain for addressing potential rare codon issues

  • Bacillus subtilis systems for challenging cases (being a Gram-positive host like Listeria)

  • Avoid expression systems requiring secretion as this may affect enzyme folding

• Optimization parameters (suggested experimental design):

• Purification strategy:

  • IMAC (Ni-NTA) for initial capture of His-tagged protein

  • Ion exchange chromatography for intermediate purification

  • Size exclusion chromatography for final polishing

  • Include 5-10% glycerol and 1-5 mM DTT in all buffers to maintain stability

What methods can accurately measure the enzymatic activity of purified nadD?

The activity of purified nadD can be assessed through several complementary approaches:

• Direct spectrophotometric assay:

  • Reaction components: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM ATP, 0.5 mM NaMN, purified nadD

  • Monitor decrease in ATP concentration by coupling to ATP-consuming enzymes

  • Calculate initial reaction rates at varying substrate concentrations

  • Determine kinetic parameters (Km, Vmax, kcat) for both NaMN and ATP

• HPLC-based product quantification:

  • Reaction carried out as above, but stopped at defined timepoints with EDTA

  • Separate NaMN, NaAD, ATP, and AMP by reverse-phase HPLC

  • Quantify product formation against standard curves

  • Advantage: direct measurement without coupling enzymes

• Continuous coupled assay system:

  • Link NaAD formation to a detectable signal through coupled enzymes

  • For example, couple to NAD synthetase and NADH-producing dehydrogenase

  • Monitor NADH formation at 340 nm continuously

  • Provides real-time measurement capability

• Isothermal titration calorimetry:

  • Measure heat released during substrate binding and catalysis

  • Determine thermodynamic parameters of the reaction

  • Assess substrate binding order and potential cooperativity

  • Advantage: provides both binding and catalytic information

When comparing nadD from different bacterial sources or assessing mutants, standardize conditions and include appropriate controls for accurate comparisons.

How does nadD function integrate with cell wall biosynthesis in L. monocytogenes?

While direct evidence linking nadD to cell wall biosynthesis in L. monocytogenes is limited, several potential connections can be experimentally investigated:

• Metabolic intersections:

  • NAD+ serves as a cofactor for several enzymes involved in peptidoglycan precursor synthesis

  • NAD+/NADH ratio affects redox state, potentially influencing cell wall structure

  • ATP generated through NAD+-dependent pathways provides energy for cell wall biosynthesis

• Experimental approach to investigate connections:

  • Compare cell wall integrity in nadD-depleted vs. wild-type conditions using osmotic shock sensitivity

  • Analyze peptidoglycan composition quantitatively using HPLC

  • Measure expression correlation between nadD and cell wall synthesis genes under various stresses

  • Test sensitivity to cell wall-targeting antibiotics when nadD expression is modulated

• Findings from related studies:

  • L. monocytogenes GlmR functions as an accessory uridyltransferase involved in UDP-GlcNAc synthesis

  • UDP-GlcNAc is an essential precursor for both peptidoglycan and wall teichoic acid synthesis

  • Disruption of cell wall precursor synthesis pathways leads to increased sensitivity to lysozyme

  • Metabolic pathways supporting cell wall synthesis are critical for virulence

These connections suggest that proper NAD metabolism, facilitated by nadD, may be crucial for maintaining cell wall integrity, particularly during stress conditions or intracellular growth.

What is known about nadD's role in L. monocytogenes virulence and pathogenesis?

The contribution of nadD to L. monocytogenes virulence can be examined through several experimental approaches:

• Conditional expression studies:

  • Generate strains with inducible or repressible nadD expression

  • Assess virulence phenotypes in cell culture and animal models when nadD is depleted

  • Compare transcriptomes of wild-type and nadD-depleted bacteria during infection

  • Measure metabolic profiles to identify downstream effects of nadD deficiency

• In vitro infection models:

  • Intracellular growth assays in macrophages and epithelial cells

  • Measurement of phagosomal escape efficiency

  • Cell-to-cell spread quantification

  • Survival under conditions mimicking host environments (ROS, RNS, acidic pH)

• In vivo infection studies:

  • Utilize established murine listeriosis models with defined endpoints

  • Compare bacterial burdens in liver and spleen between wild-type and nadD-attenuated strains

  • Assess immune responses, particularly IFN-γ production against the different strains

  • Implement competitive index assays to directly compare fitness in vivo

• Mechanisms potentially connecting nadD to virulence:

  • Maintenance of redox balance during oxidative stress encountered in phagosomes

  • Energy production required for virulence factor synthesis and secretion

  • Potential metabolic adaptations needed for cytosolic replication

  • Integration with stress response pathways activated during infection

While nadD itself may not be a virulence factor per se, its essential metabolic function likely indirectly contributes to pathogenesis by supporting bacterial survival and replication in hostile host environments.

How can structural biology approaches be used to investigate nadD from L. monocytogenes serotype 4b?

Structural characterization of L. monocytogenes serotype 4b nadD provides critical insights into enzyme function and potential inhibitor design:

• X-ray crystallography workflow:

  • High-purity protein preparation (>95% by SDS-PAGE)

  • Crystal screening using commercial sparse matrix screens

  • Optimization of crystallization conditions (pH, temperature, additives)

  • Data collection at synchrotron radiation sources

  • Structure determination by molecular replacement using homologous structures

• Protein NMR approaches:

  • Expression of 15N/13C-labeled protein in minimal media

  • Sequential backbone assignment using triple-resonance experiments

  • Ligand binding studies to identify substrate interaction sites

  • Dynamics measurements to identify flexible regions important for catalysis

  • Chemical shift perturbation experiments with substrate analogues

• Computational structural analysis:

  • Homology modeling if experimental structures prove challenging

  • Molecular dynamics simulations to explore conformational flexibility

  • Docking studies with substrates and potential inhibitors

  • Conservation analysis mapped onto structural models

  • Prediction of functionally important residues for mutagenesis

• Complementary biophysical techniques:

  • Circular dichroism to assess secondary structure content

  • Thermal shift assays to identify stabilizing conditions

  • Small-angle X-ray scattering to determine solution structure

  • Analytical ultracentrifugation to characterize oligomeric state

The structural information obtained can guide rational mutagenesis studies to probe catalytic mechanisms and inform structure-based inhibitor design targeting specific features of L. monocytogenes nadD.

What site-directed mutagenesis strategies can elucidate critical functional residues in nadD?

A systematic mutagenesis approach can identify key functional residues in L. monocytogenes nadD:

• Target residue selection based on:

  • Sequence conservation across bacterial nadD enzymes

  • Structural predictions of catalytic and substrate-binding sites

  • Homology to characterized residues in nadD from other organisms

  • Predicted metal coordination sites essential for activity

• Comprehensive mutagenesis strategy:

• Functional characterization of mutants:

  • Expression and purification under identical conditions

  • Enzyme activity measurements (initial velocity, Km, kcat)

  • Substrate binding analysis using isothermal titration calorimetry

  • Thermal stability assessment using differential scanning fluorimetry

  • Structural integrity verification by circular dichroism

• Structure-function correlation:

  • Map activity changes to structural models

  • Identify residues essential for catalysis versus substrate binding

  • Determine the role of specific residues in metal coordination

  • Classify residues involved in maintaining structural integrity

• In vivo relevance testing:

  • Complementation studies using mutant alleles in nadD-depleted strains

  • Assessment of growth phenotypes under various conditions

  • Measurement of intracellular NAD+ levels with mutant nadD variants

  • Evaluation of virulence properties when expressing critical mutants

This systematic approach provides a comprehensive understanding of residues critical for nadD function and identifies potential sites for targeted inhibitor development.

How does nadD from L. monocytogenes serotype 4b compare with homologous enzymes from other bacterial pathogens?

Comparative analysis of nadD across bacterial species provides evolutionary insights and identifies unique features of the L. monocytogenes enzyme:

• Sequence and structural comparison:

  • Multiple sequence alignment across diverse bacterial pathogens

  • Phylogenetic analysis to determine evolutionary relationships

  • Conservation mapping of catalytic residues and substrate binding sites

  • Identification of lineage-specific insertions or deletions

• Functional comparative analysis:

  • Expression and purification of nadD from multiple bacterial species

  • Standardized kinetic parameter determination (Km, kcat, inhibitor profiles)

  • Cross-complementation experiments to test functional equivalence

  • Biochemical characterization under varying conditions (pH, temperature, salt)

• Comparative data for selected bacterial pathogens:

• Structural basis for functional differences:

  • Species-specific active site geometry affecting substrate binding

  • Differences in oligomerization states influencing catalytic efficiency

  • Surface properties affecting protein-protein interactions

  • Allosteric regulation sites present in specific bacterial lineages

• Evolutionary implications:

  • Evidence for horizontal gene transfer versus vertical inheritance

  • Selection pressures on nadD in pathogenic versus environmental bacteria

  • Co-evolution with other NAD biosynthesis pathway components

  • Adaptation to host environments in pathogenic species

This comparative approach identifies conserved features critical for enzyme function as well as species-specific adaptations that might be exploited for selective inhibitor development.

How do serotype-specific variations in L. monocytogenes influence nadD function?

Analyzing nadD variation across L. monocytogenes serotypes provides insights into potential functional adaptations:

• Genetic variation analysis:

  • Sequence comparison of nadD across all 13 L. monocytogenes serotypes

  • Special focus on differences between serotype 4b lineage I versus lineage III strains

  • Analysis of promoter regions to identify regulatory differences

  • Examination of genetic linkage with virulence-associated genes

• Expression pattern differences:

  • RT-qPCR quantification of nadD expression across serotypes

  • Western blot analysis to compare protein levels

  • Transcriptomic analysis under various stress conditions

  • Correlation with serotype-specific virulence traits

• Functional implications of variation:

  • Recombinant expression of nadD variants from different serotypes

  • Comparative enzyme kinetics under standardized conditions

  • Protein stability and activity under stress conditions

  • Cross-complementation studies in different serotype backgrounds

• Structure-function relationships:

  • Homology modeling of nadD variants to predict functional consequences

  • Identification of surface variations that might affect protein interactions

  • Analysis of potential serotype-specific post-translational modifications

  • Substrate specificity differences that might reflect metabolic adaptations

• Correlation with virulence:

  • Comparison between epidemic-associated serotypes (primarily 4b) and others

  • Infection studies using isogenic strains with nadD variants

  • Assessment of NAD+ levels during infection with different serotypes

  • Integration with known serotype-specific virulence mechanisms

Understanding serotype-specific variations in nadD may explain differences in metabolic adaptation and potentially contribute to the enhanced virulence observed in certain L. monocytogenes serotypes, particularly serotype 4b strains associated with listeriosis outbreaks.

How can L. monocytogenes infection models be adapted to study the role of nadD in pathogenesis?

Established L. monocytogenes infection models can be modified to investigate nadD's contribution to pathogenesis:

• Cell culture infection model adaptations:

  • Generate L. monocytogenes strains with inducible/repressible nadD expression

  • Measure intracellular bacterial growth curves in macrophages and epithelial cells

  • Assess phagosomal escape efficiency using fluorescence microscopy

  • Quantify cell-to-cell spread through plaque formation assays

  • Monitor NAD+ levels in bacteria during different infection stages

• Mouse infection model considerations:

  • Use established protocols for intraperitoneal infection as described in literature

  • Calculate appropriate bacterial inoculum (10^5 CFU for studying innate responses)

  • Select appropriate mouse strain (C57BL/6J recommended for immune studies)

  • Time measurements appropriately for different response phases

  • Include proper controls (wild-type, complemented strains)

• Specific experimental approaches:

  • Competitive index assays comparing wild-type and nadD-conditional strains

  • Histopathological examination of infected tissues

  • Flow cytometry analysis of immune cell responses to infection

  • Biochemical measurement of NAD+ levels in bacteria recovered from tissues

  • Transcriptomic analysis of bacteria isolated from host tissues

• Integration with metabolomic approaches:

  • Measure NAD+/NADH ratios in bacteria during infection

  • Track isotope-labeled NAD+ precursors in infected models

  • Assess metabolic adaptations when nadD expression is modulated

  • Correlate metabolic changes with virulence phenotypes

• Examining host responses:

  • Measure IFN-γ production by innate and adaptive immune cells

  • Assess inflammatory cytokine profiles in response to infection

  • Compare host cell metabolic reprogramming during infection

  • Evaluate bacterial clearance rates with wild-type versus nadD-modified strains

These adapted models provide comprehensive insights into how nadD contributes to L. monocytogenes pathogenesis through its role in bacterial metabolism and survival.

What techniques can measure the impact of nadD inhibition on L. monocytogenes survival in experimental infection models?

Assessing the effects of nadD inhibition requires specialized techniques spanning from in vitro to in vivo approaches:

• Chemical biology approaches:

  • Design substrate-competitive or mechanism-based inhibitors targeting nadD

  • Validate target engagement using thermal shift assays or activity measurements

  • Determine inhibitor specificity profiles across multiple enzymes

  • Optimize inhibitor properties for cellular penetration in L. monocytogenes

• In vitro bacterial culture measurements:

  • Growth inhibition assays in rich and minimal media

  • Time-kill kinetics under various inhibitor concentrations

  • NAD+ level quantification using enzymatic cycling assays

  • Resistance development monitoring through serial passage

• Cell culture infection model measurements:

  • Bacterial intracellular replication quantification (CFU counting, GFP reporters)

  • Live cell imaging to track bacterial growth and spread

  • Host cell viability assessment to monitor cytotoxicity

  • Dose-response relationships at different infection stages

• In vivo efficacy measurements:

  • Bacterial burden quantification in liver and spleen

  • Survival to endpoints assessment using established protocols

  • Histopathological evaluation of infected tissues

  • Pharmacokinetic and pharmacodynamic parameter determination

• Data analysis and interpretation:

  • Correlation between nadD inhibition and antibacterial effects

  • Comparison with established antibiotics (benchmark controls)

  • Evaluation of resistance mechanisms and frequency

  • Integration of in vitro potency with in vivo efficacy data

These techniques provide a comprehensive assessment of whether nadD represents a viable therapeutic target and how its inhibition affects L. monocytogenes survival and virulence in relevant model systems.

How might CRISPR-Cas9 technologies be applied to study nadD function in L. monocytogenes?

CRISPR-Cas9 technologies offer powerful approaches for investigating nadD's role in L. monocytogenes:

• Genome editing applications:

  • Creation of conditional expression systems for essential genes like nadD

  • Introduction of point mutations to study structure-function relationships

  • Generation of reporter fusions to monitor expression patterns

  • Domain swapping between L. monocytogenes nadD and homologues from other species

• Technical methodology:

  • Design of sgRNAs targeting specific regions of nadD with minimal off-target effects

  • Optimization of transformation protocols for L. monocytogenes

  • Selection of appropriate Cas9 variants (SpCas9, SaCas9) for efficient editing

  • Development of scarless editing techniques to avoid polar effects

• CRISPRi application for nadD functional studies:

  • Targeted repression of nadD using catalytically inactive dCas9

  • Creation of inducible knockdown systems to titrate nadD expression

  • Temporal control of repression to study nadD requirement at different infection stages

  • Multiplexed targeting of nadD along with interacting partners

• Genome-wide interaction studies:

  • CRISPR screens to identify synthetic lethal interactions with nadD

  • Identification of compensatory pathways activated when nadD is depleted

  • Screening for mutations that alter sensitivity to nadD inhibition

  • Discovery of novel functional connections through suppressor screens

• In vivo applications:

  • Generation of strains with tunable nadD expression for animal studies

  • Creation of fluorescent reporter strains to track nadD expression during infection

  • Development of CRISPR-based antimicrobials targeting nadD gene sequences

  • High-throughput screening platforms to identify nadD inhibitors

These CRISPR-based approaches provide unprecedented precision in manipulating nadD expression and function, enabling detailed mechanistic studies even for an essential enzyme.

What emerging technologies can advance understanding of nadD's role in L. monocytogenes metabolism and pathogenesis?

Cutting-edge technologies offer new opportunities to elucidate nadD's functions:

• Single-cell technologies:

  • Single-cell RNA-seq to capture heterogeneity in nadD expression during infection

  • Single-cell metabolomics to measure NAD+ levels in individual bacteria

  • Microfluidic systems to track nadD activity in real-time at the single-cell level

  • Correlative light and electron microscopy to localize nadD within bacterial cells

• Advanced structural biology approaches:

  • Cryo-electron microscopy for high-resolution structural determination

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Integrative structural biology combining multiple data sources

  • Time-resolved structural techniques to capture catalytic intermediates

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Flux balance analysis to quantify metabolic changes when nadD is perturbed

  • Network modeling to predict cellular responses to nadD inhibition

  • Machine learning applications to identify patterns in large-scale datasets

• Advanced in vivo imaging:

  • Intravital microscopy to track L. monocytogenes in real-time during infection

  • PET imaging with radiolabeled NAD+ precursors to track metabolism in vivo

  • MALDI-imaging mass spectrometry for spatial metabolomics in infected tissues

  • Bioluminescence resonance energy transfer (BRET) sensors for NAD+ detection

• High-throughput functional genomics:

  • Transposon sequencing (Tn-seq) to identify genes synthetically lethal with nadD

  • Ribosome profiling to assess translational regulation of nadD

  • CRISPR interference screens to map genetic interactions

  • Paired genome-wide association studies with clinical isolates to correlate nadD variants with virulence

These emerging technologies will provide unprecedented insights into nadD function and potentially identify novel approaches for therapeutic intervention targeting L. monocytogenes metabolism.