Recombinant Lactobacillus plantarum 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ispD)

Basic Research Questions

• What is 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ispD) and what is its role in bacterial metabolism?

  2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) is a critical enzyme (EC 2.7.7.60) that catalyzes the third step in the mevalonate-independent (MEP) pathway of isoprenoid biosynthesis. This enzyme specifically mediates the formation of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) from 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP), releasing inorganic pyrophosphate (PPi) as a byproduct .

  The MEP pathway consists of seven enzymatic steps that ultimately lead to the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which serve as fundamental building blocks for all isoprenoids. After IspD catalyzes the formation of CDP-ME, the pathway continues with CDPME kinase (IspE) mediating the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P), followed by MEcDP synthase (IspF) forming 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP). The final steps involve conversion to 4-hydroxy-3-mehtyl-butenyl 1-diphosphate (HMBPP) by IspG, then to IPP and DMAPP by IspH .

  The importance of IspD as a research target stems from its presence in many bacteria but absence in mammals, making it an attractive target for anti-infective drug development with potentially minimal effects on human cells .

• Why is Lactobacillus plantarum used as a host for recombinant protein expression?

  Lactobacillus plantarum (also known as Lactiplantibacillus plantarum in updated taxonomy) offers several advantages as a host for heterologous protein expression, particularly for immunological applications:

  Characteristic	Research Advantage
  GRAS status (Generally Recognized As Safe)	Enables safe oral administration of recombinant constructs
  Inherent adjuvanticity	Enhances immune responses to expressed antigens
  Robust secretion systems	Allows efficient protein production and secretion
  Survival through GI tract	Facilitates delivery to intestinal immune tissues
  Adaptable expression systems	Enables controlled expression with systems like pSIP

  L. plantarum WCFS1 has been successfully exploited for intracellular expression, secretion, and cell-surface display of heterologous proteins using the pSIP expression systems. These systems can be optimized through selection of appropriate signal peptides and culture conditions to maximize protein yields .

  The bacterium's ability to stimulate dendritic cells and influence both innate and adaptive immune responses makes it particularly valuable for vaccine development and immunomodulatory applications .

• How does recombinant L. plantarum affect host immune responses?

  Recombinant L. plantarum can significantly modulate host immunity through multiple immunological pathways. Research demonstrates that recombinant L. plantarum strains can:

  • Activate dendritic cells in Peyer's patches, increasing antigen presentation capacity

  • Enhance CD4+IFN-γ+ and CD8+IFN-γ+ T cell populations in the spleen and mesenteric lymph nodes

  • Promote T cell proliferation in response to specific antigens

  • Increase B220+IgA+ cells in Peyer's patches, enhancing mucosal immunity

  • Induce high levels of specific IgG, IgG1, and IgG2a antibodies in serum

  • Elevate secretory IgA (sIgA) levels in the lungs and various intestinal segments

  A meta-analysis of clinical trials demonstrated that L. plantarum significantly modulates cytokine profiles, increasing anti-inflammatory IL-10 (mean difference: 9.88 pg/mL; 95% CI: 6.52 to 13.2; p < 0.05) while reducing pro-inflammatory cytokines including IL-4 (-0.48 pg/mL; 95% CI: -0.79 to -0.17; p < 0.05), TNF-α (-2.34 pg/mL; 95% CI: -3.5 to -1.19; p < 0.05), and IFN-γ (-0.99 pg/mL; 95% CI: -1.56 to -0.41; p < 0.05) .

  Interestingly, L. plantarum-matured dendritic cells can also upregulate immunosuppressive factors like PD-L1 and IDO, suggesting complex immunoregulatory properties that might be beneficial in controlling excessive inflammation .

• What are the critical factors affecting expression efficiency of recombinant proteins in L. plantarum?

  Several key factors influence the expression efficiency of recombinant proteins in L. plantarum:

  Factor	Impact on Expression
  Signal peptide selection	Determines secretion efficiency and sometimes growth rate
  Promoter strength	Controls transcription levels of the target gene
  Codon optimization	Enhances translation efficiency for heterologous proteins
  Culture conditions	Temperature, pH, and media composition affect protein yields
  Plasmid stability	Influences long-term expression in the absence of selection

  Research has demonstrated that signal peptide selection can dramatically impact protein yields. For instance, constructs containing signal peptides Lp_2145, Lp_0373, and Lp_AmyA showed significantly higher recombinant protein yields compared to those with Lp_3050 and SP_AmyL signal peptides .

  The secretion pathway is also important to consider. In L. plantarum, most secreted proteins are translocated via the Sec secretion machinery as unfolded polypeptides, with the Twin-arginine translocation (Tat) pathway being absent. During translocation, signal peptidase type I cleaves the N-terminal signal peptide sequences before proteins are released into the medium .

  Growth rates of recombinant strains can vary significantly based on the expression construct. For example, strains containing constructs with the signal peptide Lp_3050 demonstrated noticeably slower growth rates compared to other signal peptide variants .

• What methods are used to confirm successful expression of recombinant proteins in L. plantarum?

  Multiple complementary methods are employed to verify successful expression of recombinant proteins in L. plantarum:

  Protein Detection and Quantification:

  • SDS-PAGE analysis: Separates proteins by molecular weight to visualize target protein bands in cell lysates and supernatants

  • Western blot: Confirms protein identity using specific antibodies (as demonstrated with rTsPPase expression)

  • Immunofluorescence assay (IFA): Verifies surface expression of recombinant proteins on bacterial cells

  Functional Assessment:

  • Enzymatic activity assays: Measures specific activity of expressed enzymes (e.g., amylase activity measured in kU/L of culture medium)

  • Hemagglutination inhibition (HI) assays: Evaluates functional activity of expressed viral antigens

  Growth and Stability Testing:

  • Growth curve analysis: Assesses impact of recombinant constructs on bacterial growth rates

  • Plasmid stability testing: Determines maintenance of expression plasmids over multiple generations

  For example, in a study expressing α-amylase in L. plantarum, researchers confirmed expression through SDS-PAGE visualization of ~95 kDa (AmyL) and ~49 kDa (AmyA) protein bands in both supernatant and cell lysates, complemented by enzymatic activity measurements showing up to 8.1 kU/L of AmyL activity and higher activities for AmyA .

Advanced Research Questions

• What strategies can optimize recombinant IspD expression and secretion in L. plantarum?

  Optimizing recombinant IspD expression and secretion in L. plantarum requires a multi-faceted approach addressing gene design, expression systems, and growth parameters:

  Signal Peptide Engineering:

  Selection of an appropriate signal peptide is critical for efficient secretion. Comparative studies have shown that signal peptides like Lp_2145 can yield significantly higher secretion efficiency than others such as Lp_3050 . For IspD expression, researchers should test multiple signal peptides, prioritizing those that have demonstrated high secretion efficiency with proteins of similar size and properties.

  Expression System Selection:

  The pSIP expression systems have proven effective for controlled expression in L. plantarum. These systems allow inducible expression and can be optimized for specific proteins. For example, pSIP409 has been successfully used for high-level protein secretion . When expressing IspD, the vector backbone should be selected based on copy number, promoter strength, and compatibility with the signal peptide.

  Codon Optimization:

  Since IspD derives from different bacterial species, codon optimization for L. plantarum can significantly improve translation efficiency. This involves adjusting the coding sequence to use preferred codons without altering the amino acid sequence, potentially increasing protein yields by 2-5 fold.

  Culture Optimization:

  Fine-tuning of growth conditions can dramatically affect recombinant protein yields:

  • Temperature: Lowering cultivation temperature from 37°C to 25-30°C after induction can reduce protein aggregation

  • Media composition: MRS medium supplemented with specific carbon sources can enhance secretion

  • pH control: Maintaining pH between 6.0-6.5 can improve protein stability

  • Induction timing: Inducing expression at mid-log phase (OD600 ~0.8) rather than early growth phases

  Protease Inhibition:

  L. plantarum secretes various proteases that may degrade recombinant proteins. Adding protease inhibitors to the culture medium or engineering protease-deficient strains can increase protein recovery and stability, particularly important for sensitive proteins like IspD.

• How can recombinant L. plantarum expressing IspD be developed as a vaccine vector?

  Developing recombinant L. plantarum expressing IspD as a vaccine vector involves a comprehensive strategy addressing antigen design, immunization protocols, and efficacy evaluation:

  Antigen Design Considerations:

  • IspD-antigen fusion constructs: Engineering IspD fused with immunogenic epitopes from target pathogens

  • Addition of molecular adjuvants: Incorporating dendritic cell-targeting peptides (DCpep) significantly enhances immune responses, as demonstrated in avian influenza studies where HA1-DCpep constructs induced higher immune responses than HA1 alone

  • Surface display vs. secretion: Determining optimal localization based on desired immune response type (surface display may enhance B-cell responses while secretion may favor T-cell responses)

  Immunization Protocol Development:

  Effective oral immunization protocols typically involve:

  • Primary immunization: Initial dose of recombinant bacteria (10^9-10^10 CFU)

  • Booster immunization: Secondary dose 2-4 weeks after primary immunization

  • Dosage optimization: Testing multiple concentrations to identify minimum effective dose

  Immune Response Assessment:

  Comprehensive evaluation of:

  • Humoral immunity: Measuring specific serum IgG, IgG1, IgG2a, and mucosal IgA antibodies by ELISA

  • Cellular immunity: Flow cytometry analysis of CD4+IFN-γ+ and CD8+IFN-γ+ T cells in spleen and mesenteric lymph nodes

  • B cell activation: Quantifying B220+IgA+ cells in Peyer's patches

  • Cytokine profiles: Measuring IL-4, IL-10, TNF-α, and IFN-γ levels

  Previous studies with recombinant L. plantarum vaccines have demonstrated protection efficacy up to 67.18% reduction in pathogens after challenge, suggesting similar approaches could be effective with IspD-based constructs .

  Challenge Studies:

  Ultimate validation requires challenge studies showing protection against relevant pathogens, with careful monitoring of:

  • Pathogen burden: Quantification in relevant tissues

  • Clinical symptoms: Monitoring disease progression

  • Survival rates: Comparing vaccinated versus control groups

• What methodologies are most effective for studying IspD enzymatic activity in recombinant L. plantarum?

  Studying IspD enzymatic activity in recombinant L. plantarum requires specialized assays to measure both enzyme production and catalytic function:

  Spectrophotometric Coupled Assays:

  The most widely used approach for measuring IspD activity is a coupled enzyme assay that tracks the release of pyrophosphate (PPi) during the reaction:

  1. IspD catalyzes: MEP + CTP → CDP-ME + PPi

  2. Inorganic pyrophosphatase converts PPi to 2Pi

  3. Formation of Pi is detected using malachite green or other colorimetric methods

  This assay can be performed with cell lysates or purified enzyme, with activity expressed as μmol product formed per minute per mg protein.

  Radiometric Assays:

  For higher sensitivity, radiometric assays using [14C]-labeled CTP can track the formation of [14C]-CDP-ME:

  1. Incubate recombinant IspD with [14C]-CTP and MEP

  2. Separate reaction products by thin-layer chromatography

  3. Quantify radioactivity in CDP-ME spot by scintillation counting

  LC-MS/MS Analysis:

  Liquid chromatography coupled with tandem mass spectrometry provides direct quantification of reaction products:

  1. Extract metabolites from reaction mixture

  2. Separate compounds by HPLC

  3. Detect and quantify CDP-ME using mass spectrometry

  4. Compare with authentic standards

  In Vivo Metabolic Flux Analysis:

  To understand IspD function within bacterial metabolism:

  1. Grow recombinant L. plantarum with 13C-labeled glucose

  2. Extract and analyze labeled intermediates in the MEP pathway

  3. Calculate flux through the pathway based on labeling patterns

  Inhibition Studies:

  To validate IspD as a drug target:

  1. Test potential inhibitors against recombinant IspD

  2. Determine IC50 values using standard enzyme kinetics

  3. Correlate enzyme inhibition with growth inhibition of recombinant L. plantarum

  These methodologies can be combined with structural studies and molecular modeling to gain comprehensive insights into IspD function and regulation in the bacterial system.

• How do the structural characteristics of IspD influence its function and potential as a drug target?

  The structural characteristics of IspD play a critical role in both its enzymatic function and its attractiveness as a drug target:

  Core Structural Features:

  2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) belongs to the nucleotidyltransferase family . The crystal structure of E. coli IspD (PDB: 1I52, 1INI, 1INJ) revealed several key structural elements:

  • Homodimeric quaternary structure with two active sites

  • Nucleotide-binding fold similar to other cytidylyltransferases

  • Distinct CTP binding pocket with conserved residues across bacterial species

  • MEP binding site that accommodates the unique structure of 2-C-methyl-D-erythritol 4-phosphate

  • Catalytic magnesium ion coordinated by conserved aspartate residues

  Structure-Function Relationships:

  The enzyme catalyzes the reaction between MEP and CTP through a sequential ordered mechanism:

  1. CTP binds first, inducing conformational changes

  2. MEP then binds in the correct orientation for nucleophilic attack

  3. The reaction proceeds via an SN2-like mechanism

  4. Products (CDP-ME and PPi) are released sequentially

  This ordered binding mechanism creates opportunities for designing inhibitors that target either substrate binding site or capture transition state conformations.

  Drug Target Potential:

  IspD presents several advantages as an antimicrobial target:

  • Structural uniqueness: The MEP pathway enzymes show no homology to mammalian proteins

  • Essential function: IspD catalyzes a critical step in isoprenoid biosynthesis

  • Conservation: The active site is highly conserved across bacterial species, allowing broad-spectrum targeting

  • Druggability: The binding pockets have favorable properties for small molecule binding

  Inhibitor Design Strategies:

  Based on structural knowledge, several approaches can be pursued for inhibitor development:

  • Substrate analogs: Compounds that mimic CTP or MEP but cannot undergo reaction

  • Transition state mimics: Molecules that resemble the high-energy intermediate

  • Allosteric inhibitors: Compounds binding outside the active site that disrupt enzyme function

  • Covalent inhibitors: Molecules that form irreversible bonds with active site residues

  Expressing recombinant IspD in L. plantarum provides a valuable tool for screening potential inhibitors, as growth inhibition can serve as a preliminary indicator of target engagement before proceeding to more complex assays.

• What mechanisms underlie the immunomodulatory effects of recombinant L. plantarum, and how can they be optimized for therapeutic applications?

  The immunomodulatory effects of recombinant L. plantarum involve complex interactions with the host immune system that can be strategically optimized for therapeutic applications:

  Molecular Mechanisms of Immunomodulation:

  Pattern Recognition Receptor (PRR) Activation:

  • L. plantarum cell wall components (peptidoglycan, lipoteichoic acids) engage TLR2 and NOD receptors on dendritic cells and macrophages

  • This activation initiates signaling cascades leading to cytokine production and antigen presentation

  Dendritic Cell Modulation:

  • Recombinant L. plantarum activates dendritic cells in Peyer's patches, enhancing their ability to present antigens

  • L. plantarum-matured dendritic cells express immunoregulatory molecules including PD-L1 and indoleamine 2,3-dioxygenase (IDO)

  • These mature DCs can effectively drive specific T cell responses depending on the cytokine environment

  Cytokine Regulation:
  Meta-analysis of clinical trials has established that L. plantarum significantly modulates cytokine profiles by:

  • Increasing anti-inflammatory IL-10 (mean difference: 9.88 pg/mL; 95% CI: 6.52-13.2; p<0.05)

  • Decreasing pro-inflammatory cytokines:

    • IL-4: -0.48 pg/mL (95% CI: -0.79 to -0.17; p<0.05)

    • TNF-α: -2.34 pg/mL (95% CI: -3.5 to -1.19; p<0.05)

    • IFN-γ: -0.99 pg/mL (95% CI: -1.56 to -0.41; p<0.05)

  Mucosal Antibody Induction:

  • Recombinant L. plantarum increases B220+IgA+ cells in Peyer's patches

  • Enhances IgA secretion in mucosal tissues (lungs, intestinal segments)

  • Induces specific systemic antibodies (IgG, IgG1, IgG2a)

  Optimization Strategies for Therapeutic Applications:

  Targeting Specific Immune Pathways:

  • Co-expression of immunomodulatory molecules with IspD

  • Addition of dendritic cell-targeting peptides (DCpep) to enhance antigen delivery and presentation

  • Engineering strains to express cytokines that can enhance or suppress specific immune responses

  Dosage and Administration Optimization:
  Clinical studies have demonstrated dose-dependent effects, with significant differences between:

  • Low dose: 1 × 10^9 CFU/day

  • High dose: 1 × 10^10 CFU/day

  For IBS-D treatment, higher doses showed greater improvement in IBS severity scoring system (IBS-SSS) total scores, abdominal pain severity, quality of life, and normalization of diarrheal stool type .

  Adjuvant Selection:
  For vaccine applications, combining recombinant L. plantarum with specific adjuvants can enhance responses:

  • Mucosal adjuvants (e.g., cholera toxin B subunit) for enhanced secretory IgA

  • TLR agonists for stronger Th1-biased responses

  • Alum-based adjuvants for Th2-biased responses

  Genetically Modified Strains:
  Engineering L. plantarum to modulate specific immunological parameters:

  • Knockout of genes involved in immunosuppressive factor production

  • Enhancement of immunostimulatory component expression

  • Addition of surface proteins that target specific immune cell subsets

  These mechanistic insights and optimization strategies provide a foundation for developing recombinant L. plantarum expressing IspD for various therapeutic applications, from vaccines to treatment of inflammatory conditions.