Recombinant Listeria monocytogenes serotype 4b L-lactate dehydrogenase 2 (ldh2)

What is Listeria monocytogenes serotype 4b ldh2 and what distinguishes it from other lactate dehydrogenases?

L-lactate dehydrogenase 2 (ldh2) from Listeria monocytogenes serotype 4b (strain F2365) is a 311-amino acid protein that catalyzes the interconversion of pyruvate to lactate using NADH/NAD+ as cofactors. This enzyme is critical for redox homeostasis in L. monocytogenes, particularly under anaerobic conditions.

Unlike ldh1, which is 313 amino acids long and has been more extensively characterized, ldh2 exhibits distinct expression patterns and potentially different kinetic properties. Both enzymes are part of L. monocytogenes' respiro-fermentative metabolism that enables adaptation to diverse environmental conditions, including different oxygen levels .

Distinguishing characteristics of serotype 4b ldh2:

• Belongs specifically to lineage I of L. monocytogenes

• Contributes to strain-specific metabolic adaptations

• Shows sequence variations that may affect catalytic efficiency

• May play differential roles in virulence compared to ldh1

What is the genetic organization and sequence conservation of the ldh2 gene in L. monocytogenes serotype 4b?

The ldh2 gene in L. monocytogenes serotype 4b shows high conservation within lineage I strains. Based on multilocus sequence typing (MLST) studies, ldh is one of several housekeeping genes that exhibit limited allelic variation within serotypes but significant variation between serotypes.

When analyzing the sequence diversity:

• The ldh gene has been used successfully in MLST schemes, indicating sufficient but not excessive polymorphism

• The dN/dS ratio for ldh is significantly lower than 1, demonstrating it is under purifying selection rather than diversifying selection

• The chromosomal location of ldh is unlinked to other housekeeping genes used in MLST, with sufficient distance to make joint horizontal transfer unlikely

Sequence analysis shows that ldh is one of the genes that helps distinguish the three major evolutionary lineages of L. monocytogenes, with serotype 4b strains (including those expressing ldh2) belonging predominantly to lineage I .

What are the optimal expression systems and purification protocols for recombinant L. monocytogenes serotype 4b ldh2?

Expression Systems:
The optimal expression system for producing recombinant L. monocytogenes serotype 4b ldh2 is Escherichia coli, though alternative systems include yeast, baculovirus, or mammalian cells depending on specific experimental requirements .

Recommended Expression Protocol:

• Clone the ldh2 gene (aa 1-311) into an expression vector with an appropriate tag

• Transform into an E. coli expression strain (BL21(DE3) or similar)

• Culture at 37°C until reaching OD600 of 0.6-0.8

• Induce with IPTG (0.1-1.0 mM) at reduced temperature (16-25°C)

• Harvest cells after 4-16 hours

Purification Protocol:

• Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors

• Clarify lysate by centrifugation at 15,000 × g for 30 minutes

• Apply to appropriate affinity column based on tag

• Elute using tag-specific methods

• Perform size exclusion chromatography for higher purity

• Verify purity by SDS-PAGE (>85% purity is standard)

Storage Recommendations:

• Short-term: 4°C for up to one week

• Long-term: -20°C/-80°C in buffer containing 50% glycerol

• Avoid repeated freeze-thaw cycles

How can researchers verify the enzymatic activity and structural integrity of purified recombinant ldh2?

Enzymatic Activity Assays:

• Spectrophotometric NADH Oxidation Assay:

  • Measure decrease in absorbance at 340 nm as NADH is oxidized to NAD+

  • Reaction buffer: 50 mM Tris-HCl (pH 7.5), 0.2 mM NADH, varying concentrations of pyruvate

  • Calculate specific activity and determine kinetic parameters (Km, Vmax)

• Coupled Enzymatic Assay:

  • Couple LDH reaction with another enzymatic reaction for increased sensitivity

  • Useful for low concentrations of enzyme or inhibitor studies

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-260 nm): Secondary structure content

  • Near-UV CD (250-350 nm): Tertiary structure fingerprint

• Thermal Shift Assay:

  • Determine protein stability and melting temperature

  • Use fluorescent dye (SYPRO Orange) to monitor protein unfolding

• Size Exclusion Chromatography:

  • Confirm monomeric state or appropriate oligomeric assembly

  • Detect potential aggregation

Activity Verification Controls:

• Include commercial LDH as positive control

• Compare with published kinetic parameters

• Use specific LDH inhibitors to confirm specificity

How does recombinant L. monocytogenes serotype 4b ldh2 contribute to bacterial redox homeostasis and metabolic adaptation?

L. monocytogenes ldh2 plays a crucial role in maintaining redox balance under various environmental conditions. Recent research has revealed that:

• NAD+/NADH Homeostasis:

  • ldh2 regenerates NAD+ during fermentative metabolism, essential when oxygen is limited

  • This process is critical for continued glycolytic activity and ATP production

• Metabolic Adaptation:

  • L. monocytogenes employs a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions

  • ldh2 contributes to this metabolic flexibility, allowing growth across diverse ecological niches

• Interaction with Electron Transport Components:

  • Research suggests that ldh2 may interact with components like NADH dehydrogenase Ndh2

  • This interaction represents an additional layer of metabolic adaptability in L. monocytogenes

• Relationship with Menaquinone Pathway:

  • Studies of 1,4-dihydroxy-2-naphthoate (DHNA) demonstrate that L. monocytogenes can maintain redox homeostasis through alternative mechanisms

  • Specifically, the NADH dehydrogenase Ndh2 can utilize DHNA independent of its role in extracellular electron transport

This metabolic flexibility contributes to L. monocytogenes' ability to survive in diverse environments, potentially including low oxygen conditions encountered during host infection.

What roles does ldh2 play in L. monocytogenes stress responses, particularly to osmotic and cold stress?

Research has demonstrated that lactate dehydrogenases contribute significantly to L. monocytogenes' ability to withstand environmental stresses:

• Cold Stress Response:

  • L. monocytogenes is remarkably capable of growth at refrigeration temperatures

  • Lactate dehydrogenase activity is upregulated during cold stress

  • The enzyme contributes to maintaining energy metabolism at low temperatures

  • This adaptation is particularly important given that L. monocytogenes accumulates glycine betaine under chill stress

• Osmotic Stress Adaptation:

  • L. monocytogenes is notably resistant to osmotic stress

  • LDH enzymes help maintain redox balance during osmotic challenge

  • This function complements the accumulation of osmolytes like glycine betaine

• Integration with Other Stress Responses:

  • LDH activity appears to be coordinated with other stress response mechanisms

  • This includes potential interaction with systems involved in:

    • pH homeostasis

    • Oxidative stress defense

    • Cell wall stress responses

• Impact on Survival Under Extreme Conditions:

  • LDH activity contributes to the remarkable thermal tolerance of L. monocytogenes

  • The organism shows exceptional survival even at temperatures above 100°C when in low water activity environments

  • Specific D-values (decimal reduction times) range from minutes to hours depending on temperature and substrate

These stress response roles may explain why ldh is classified among the 394 open reading frames required for growth under standard laboratory conditions, highlighting its essential nature for L. monocytogenes survival .

How does serotype 4b ldh2 compare with ldh enzymes from other L. monocytogenes serotypes and related bacterial species?

Comparative analysis of ldh2 from L. monocytogenes serotype 4b with other lactate dehydrogenases reveals important evolutionary and functional relationships:

Sequence Comparison with Other L. monocytogenes Serotypes:

Functional Comparisons:

• The ldh genes have been useful in multilocus sequence typing (MLST) due to their balance between conservation and variability

• Sequence analysis shows dN/dS ratios significantly lower than 1, indicating purifying selection across all serotypes

• Despite sequence differences, the core catalytic function appears conserved across variants

Evolutionary Context:

This comparison highlights that while ldh2 maintains its essential enzymatic function across serotypes, the specific sequence variations may contribute to the distinctive metabolic characteristics of serotype 4b strains.

What are the optimal approaches for creating ldh2 knockouts or mutations to study its function in L. monocytogenes serotype 4b?

Creating effective ldh2 knockouts or mutations requires careful consideration of methodological approaches:

Genetic Modification Strategies:

• Homologous Recombination:

  • Design targeting constructs with ~1 kb homology arms flanking ldh2

  • Include selectable markers (antibiotic resistance) between homology arms

  • Transform using penicillin G transformation protocols established for L. monocytogenes

  • Screen transformants by PCR and confirm by Southern blot analysis

• CRISPR-Cas9 System:

  • Design sgRNAs targeting ldh2-specific sequences

  • Use temperature-sensitive plasmids for transient expression

  • Include homology-directed repair templates for precise modifications

  • Verify edits by sequencing

Verification Protocols:

• Genotypic Confirmation:

  • PCR screening with primers specific to the 5' and 3' ends of the target region

  • Southern blot analysis using probes against ldh2 and the inserted marker

  • Whole genome sequencing to confirm single integration and absence of off-target effects

• Phenotypic Validation:

  • Enzymatic assays to confirm absence of LDH2 activity

  • Growth curve analysis under standard and stress conditions

  • Complementation studies to restore wildtype phenotype

Potential Challenges and Solutions:

Controls to Include:

• Wild-type strain

• Single gene complementation

• Chromosomal versus episomal complementation (which can produce different results)

• Related gene knockouts (e.g., ndh1, ndh2) for comparative analysis

How can researchers develop accurate kinetic models of ldh2 activity under different environmental conditions relevant to L. monocytogenes pathogenesis?

Developing accurate kinetic models for ldh2 requires systematic characterization across multiple environmental parameters:

Experimental Design Framework:

• Enzyme Purification and Basic Kinetics:

  • Purify recombinant ldh2 to >95% homogeneity

  • Determine baseline kinetic parameters (Km, kcat, kcat/Km) for forward and reverse reactions

  • Study cofactor preferences (NADH vs. NADPH)

  • Establish substrate specificity profiles

• Environmental Parameter Testing:

  • Temperature Range: 4°C to 45°C (covering refrigeration to host temperature)

  • pH Range: 4.5 to 8.0 (covering intraphagosomal to cytosolic environments)

  • Salt Concentration: 0-10% NaCl (reflecting osmotic stress conditions)

  • Oxygen Levels: Aerobic, microaerobic, and anaerobic conditions

• Kinetic Model Development:

  • Use appropriate software (e.g., DynaFit, KinTek Explorer) for model fitting

  • Apply square-root models for temperature dependence:
    √μmax = b(T - Tmin), where μmax is maximum reaction rate, b is slope, T is temperature, and Tmin is theoretical minimum temperature for activity

  • For pH modeling, use multiplicative models that incorporate terms for each ionizable group

  • For combined effects, apply response surface methodology or non-linear logistic regression

Validation Methods:

• In Vitro Cellular Assays:

  • Compare model predictions with enzyme activity in cellular extracts

  • Validate under different growth conditions (temperature, pH, osmolarity)

• Intracellular Activity Measurement:

  • Use FRET-based NAD+/NADH sensors to monitor redox changes in living cells

  • Correlate with ldh2 activity during infection models

• Model Refinement:

  • Perform sensitivity analysis to identify critical parameters

  • Iterate model with experimental data under combined stress conditions

  • Validate with independent datasets

Data Presentation Format:
Present kinetic parameters in comprehensive tables, for example:

This approach enables a comprehensive understanding of ldh2 activity across the diverse environmental conditions encountered by L. monocytogenes during pathogenesis.

How might recombinant ldh2 be utilized in the development of novel detection methods or control strategies for L. monocytogenes?

Recombinant ldh2 offers several promising avenues for developing improved detection and control methods:

Novel Detection Approaches:

• Enzyme-Based Biosensors:

  • Immobilize recombinant ldh2 on electrodes to detect NADH/NAD+ conversion

  • Couple with electrochemical detection systems for rapid, sensitive detection

  • Design for field-portable applications in food safety monitoring

• Antibody Development:

  • Use recombinant ldh2 as an immunogen to develop serotype-specific antibodies

  • Implement in lateral flow immunoassays or ELISA formats

  • Target detection of serotype 4b strains with higher clinical relevance

• Aptamer-Based Detection:

  • Select DNA/RNA aptamers with high specificity for ldh2

  • Develop label-free detection systems based on conformational changes

  • Integrate with nanomaterial-based amplification for improved sensitivity

Control Strategy Development:

• Inhibitor Discovery:

  • Screen compound libraries for selective ldh2 inhibitors

  • Focus on molecules that exploit structural differences between bacterial and human LDH

  • Evaluate inhibitors against multiple serotypes to determine specificity

• Anti-Virulence Approaches:

  • Develop strategies that don't kill bacteria but reduce pathogenicity

  • Target metabolic bottlenecks that specifically affect intracellular survival

  • Combine with conventional antibiotics for synergistic effects

• Vaccine Development:

  • Evaluate recombinant ldh2 as a component of subunit vaccines

  • Consider fusion with adjuvant molecules for enhanced immunogenicity

  • Test protective efficacy in relevant animal models

Research Priorities and Challenges:

Ultimately, the unique properties of recombinant ldh2 make it a promising candidate for improving both detection sensitivity and control strategy specificity, particularly for clinically relevant serotype 4b strains.

What are the current gaps in understanding the structure-function relationships of L. monocytogenes ldh2 and how might these be addressed?

Several significant knowledge gaps exist in our understanding of L. monocytogenes ldh2 structure-function relationships:

Current Knowledge Gaps:

• Structural Determinants of Catalysis:

  • Limited structural data specific to L. monocytogenes ldh2

  • Unclear substrate-binding pocket differences between ldh1 and ldh2

  • Insufficient information on quaternary structure and oligomerization

• Regulatory Mechanisms:

  • Unknown allosteric regulators specific to ldh2

  • Limited understanding of post-translational modifications

  • Incomplete knowledge of transcriptional control under various conditions

• Protein-Protein Interactions:

  • Uncharacterized potential interactions with other metabolic enzymes

  • Limited data on potential moonlighting functions

  • Unknown interactions with host cellular components during infection

Methodological Approaches to Address These Gaps:

Research Hypotheses to Test:

• Different substrate specificities between ldh1 and ldh2 contribute to metabolic adaptation under varied environmental conditions

• Ldh2 may interact with specific cellular components during intracellular infection

• The structural stability of ldh2 contributes to L. monocytogenes' adaptation to temperature stress

• Serotype-specific variations in ldh2 structure may correlate with virulence differences

Potential Experimental Design:

Begin with comprehensive structural determination using X-ray crystallography or cryo-EM, followed by systematic mutagenesis of key residues identified in the structure. Complement with functional assays under various environmental conditions, and validate findings in cellular models using gene replacement strategies with mutant variants.

These approaches would significantly advance our understanding of how ldh2's structure relates to its function in L. monocytogenes metabolism and virulence.