Recombinant Listeria monocytogenes serotype 4b 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (menD), partial

What is Listeria monocytogenes and why is it significant as a recombinant expression system?

Listeria monocytogenes (LM) is a Gram-positive bacterium with unique cellular properties that make it valuable for recombinant applications. It can enter host cells, escape from endocytic vesicles, multiply within the cytoplasm, and spread directly between cells without exposure to extracellular environments. These properties allow proteins secreted by recombinant LM to efficiently enter the major histocompatibility complex (MHC) class I antigen processing and presentation pathway . Genetic systems have been developed for stable site-specific integration of expression cassettes into the LM genome, enabling the expression and secretion of foreign antigens by recombinant strains .

What is menD enzyme and what role does it play in bacterial metabolism?

The menD enzyme (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase) catalyzes the first committed step in the classical menaquinone (MQ) biosynthesis pathway. Menaquinone functions as an essential electron carrier for membrane electron transport in many bacteria, making it critical for energy generation during both anaerobic and aerobic respiration . Given its central role in bacterial energy metabolism, menD represents a promising target for antimicrobial development, particularly against pathogens like Mycobacterium tuberculosis .

How does serotype 4b of L. monocytogenes differ from other serotypes for recombinant expression?

Serotype 4b strains are clinically significant as they are frequently associated with human listeriosis outbreaks . When using this serotype for recombinant expression, researchers must consider:

The selection of serotype 4b for menD expression may impact the immune response to recombinant proteins and potentially affect experimental outcomes in immunological studies.

How can recombinant L. monocytogenes expressing menD be utilized in vaccine development?

Recombinant L. monocytogenes has demonstrated significant potential as a live vaccine vehicle through its ability to induce strong cellular immune responses. The system allows for:

• Efficient delivery of antigens to the MHC class I pathway, generating robust CD8+ T cell responses

• Potential dual delivery of menD-derived peptides alongside other target antigens

Previous research has shown that immunization with LM vaccine strains expressing foreign antigens can confer protection against heterologous pathogens, with CD8+ T cells playing a crucial role in this protective immunity . For example, LM strains expressing lymphocytic choriomeningitis virus (LCMV) nucleoprotein protected mice against challenge with virulent LCMV strains .

What methodological approaches are optimal for constructing recombinant L. monocytogenes expressing menD?

The construction of recombinant L. monocytogenes expressing menD typically involves:

• Gene amplification: PCR amplification of the menD gene (complete or partial) from the source organism

• Vector construction: Cloning into a suitable shuttle vector containing:

  • A promoter active in L. monocytogenes (e.g., hly promoter)

  • Signal sequence for protein secretion if desired

  • Antibiotic resistance markers for selection

• Transformation: Electroporation of the construct into competent L. monocytogenes cells

• Selection and verification: Antibiotic selection followed by PCR, sequencing, and expression analysis

The genetic system established for L. monocytogenes allows for stable site-specific integration of expression cassettes into the bacterial genome, ensuring maintained expression over multiple generations .

How can menD enzyme activity be accurately measured in recombinant L. monocytogenes?

Two complementary bioanalytical techniques have proven particularly useful for measuring menD activity:

• Intrinsic protein fluorescence: Monitors conformational changes upon substrate binding

• Differential scanning fluorimetry (DSF): Measures thermal stability shifts upon ligand binding

DSF is especially valuable due to the small sample volumes required, ability to perform replicates, and flexibility in testing various ligand conditions. The technique can reveal interactions with both natural ligands and potential inhibitors .

How does menD from different bacterial species compare in structure and function when expressed in L. monocytogenes?

Expression of menD from different bacterial sources in L. monocytogenes allows for comparative studies of enzyme properties. Research with menD from M. tuberculosis, E. coli, and S. aureus has revealed:

• Conservation of core catalytic function across species

• Presence of potential allosteric regulatory sites in all examined menD enzymes

• Differential responses to inhibitors, with some compounds showing species-specific effects

The allosteric binding site for dihydroxynaphthoic acid (DHNA) in M. tuberculosis menD appears to have counterparts in menD from other species, suggesting evolutionary conservation of regulatory mechanisms .

What approaches can be used to optimize menD expression and activity in recombinant L. monocytogenes?

Optimization strategies include:

• Promoter selection: Testing constitutive vs. inducible promoters for optimal expression levels

• Codon optimization: Adjusting the menD coding sequence to match L. monocytogenes codon usage preferences

• Fusion tags: Incorporating tags that can:

  • Enhance protein solubility

  • Facilitate purification (e.g., His-tag)

  • Be cleaved post-purification for native protein studies

• Growth conditions: Optimizing temperature, media composition, and induction timing

• Co-expression of chaperones: To assist proper protein folding if needed

How can tumor microenvironment (TME) remodeling by recombinant L. monocytogenes be measured and exploited in cancer immunotherapy?

When developing L. monocytogenes-based cancer vaccines, understanding TME remodeling is crucial. Research has shown that live-attenuated double-deleted L. monocytogenes expressing tumor antigens (LADD-Ag) can significantly alter the TME through:

• Recruiting tumor-specific KLRG1+PD1loCD62L−CD8+ T cells that produce IFNγ

• Decreasing regulatory T cell (Treg) levels

• Creating a proinflammatory cytokine milieu

• Shifting macrophage polarization from M2 to M1 (iNOS+CD206−) phenotype

These changes convert the tumor from an immunosuppressive to an inflamed microenvironment, enabling tumor rejection . Key parameters to measure include:

What are the common challenges in purifying active menD enzyme from recombinant sources?

Purification of active menD enzyme presents several challenges:

• Maintaining enzyme stability throughout purification

• Obtaining sufficient quantities of soluble protein

• Removing fusion tags without compromising activity

• Ensuring proper cofactor association

Successful approaches have included the development of constructs that allow for His-tag cleavage post-purification, as implemented for Smeg-MenD . Differential scanning fluorimetry can be used to monitor protein stability throughout the purification process and in the presence of various buffer conditions .

How can researchers address safety concerns when working with recombinant L. monocytogenes?

Safety considerations include:

• Use of attenuated strains: Live-attenuated double-deleted L. monocytogenes (LADD) strains have been developed for clinical applications with reduced pathogenicity

• Containment protocols: Proper biosafety level facilities and procedures

• Antibiotic sensitivity: Ensuring recombinant strains maintain sensitivity to clinically relevant antibiotics

• Genetic stability: Monitoring for genetic rearrangements or loss of attenuating mutations

What methods can detect unexpected interactions between menD and host cellular components?

Unexpected interactions can be identified through:

• Pull-down assays coupled with mass spectrometry to identify binding partners

• Transcriptomic analysis to detect host cell responses

• Metabolomic profiling to identify alterations in metabolic pathways

• Cellular localization studies using fluorescently tagged menD protein

How can researchers troubleshoot poor expression of recombinant menD in L. monocytogenes?

Troubleshooting approaches include:

• Verifying construct integrity by sequencing

• Testing alternate promoters or ribosome binding sites

• Evaluating protein toxicity effects on the host

• Optimizing growth and induction conditions

• Considering codon optimization or fusion partners to enhance expression

• Examining potential proteolytic degradation

What statistical approaches are recommended for analyzing menD enzyme kinetics data?

Recommended statistical approaches include:

• Non-linear regression for determining Michaelis-Menten parameters (Km, Vmax)

• Global fitting for inhibition studies to determine Ki values

• Statistical comparison of enzyme variants using ANOVA with post-hoc tests

• Bootstrap resampling for robust confidence interval estimation

How should researchers interpret thermal shift assay data for menD-ligand interactions?

Interpretation of differential scanning fluorimetry data for menD should consider:

• Magnitude of ΔTm: Larger shifts typically indicate stronger binding

• Shape of melting curves: Multiple transitions may indicate domain-specific effects

• Concentration-dependence: Testing various ligand concentrations to determine saturation

• Comparison across species: Differential effects on menD from different sources may reveal species-specific binding pockets

Research has shown that menD from multiple bacterial species exhibits thermal stability shifts in the presence of DHNA, suggesting conservation of allosteric regulatory mechanisms across species .

What bioinformatic approaches can identify potential inhibitor binding sites on menD?

Effective computational approaches include:

• Molecular docking of potential inhibitors to crystal or homology models

• Molecular dynamics simulations to identify transient binding pockets

• Sequence conservation analysis to identify functionally important residues

• Fragment-based virtual screening to identify novel chemical scaffolds

• Quantitative structure-activity relationship (QSAR) modeling for rational inhibitor design

How might recombinant L. monocytogenes expressing menD contribute to vaccine development against multidrug-resistant pathogens?

This approach offers several advantages:

• Potential dual action: Targeting both pathogen-specific antigens and disrupting menaquinone biosynthesis

• Strong CD8+ T cell induction: Critical for intracellular pathogen clearance

• Adjuvant-free formulation: L. monocytogenes itself serves as a natural adjuvant

• Mucosal immunity: Potential for oral administration inducing mucosal immune responses

What innovative approaches could enhance the specificity and efficacy of menD-targeting antimicrobials?

Promising approaches include:

• Structure-based drug design targeting species-specific features of menD

• Development of prodrugs activated by pathogen-specific enzymes

• Dual-targeting compounds affecting both menD and other essential pathways

• Allosteric modulation: Exploiting regulatory sites like the DHNA binding pocket

• Nanoparticle delivery systems for enhanced bioavailability

Research with potential inhibitors has identified compounds that display differential effects on menD from different bacterial species, suggesting the possibility of developing species-selective inhibitors .

How might combining recombinant L. monocytogenes with immune checkpoint inhibitors enhance cancer immunotherapy?

This combination approach could provide synergistic benefits through:

• LADD-Ag-induced tumor-specific T cells persisting for extended periods (>2 months) and maintaining capacity to control secondary tumor challenges

• Conversion of "cold" tumors to "hot" immunogenic environments more susceptible to checkpoint inhibition

• Decreased regulatory T cell levels potentially enhancing effects of checkpoint inhibitors

• Shift toward M1 macrophage polarization supporting sustained anti-tumor responses

Research indicates that while non-recombinant LADD induces some changes in the tumor microenvironment, antitumor efficacy requires the expression of tumor antigens by the recombinant bacteria, even when combined with immune checkpoint blockade .