Recombinant Lactobacillus plantarum Imidazoleglycerol-phosphate dehydratase (hisB)

What is imidazoleglycerol-phosphate dehydratase (hisB) and what is its role in Lactobacillus plantarum?

Imidazoleglycerol-phosphate dehydratase (IGPD) is a critical enzyme in the histidine biosynthetic pathway (HBP) that catalyzes the sixth step, specifically the dehydration of imidazole-glycerol phosphate (IGP) to form imidazole-acetol phosphate (IAP). In bacteria, this enzyme is typically encoded by the hisB gene, whereas it is referred to as HISN5 in plants and HIS3 in yeast .

In Lactobacillus plantarum, hisB functions as part of the essential histidine biosynthetic pathway. It is worth noting that in some bacterial species, IGPD exists as a bifunctional enzyme resulting from gene fusion between genes encoding IGPD and histidinol-phosphate phosphatase (HPP, EC 3.1.3.15) . The enzyme contains a bi-manganese (Mn²⁺) cluster in its active site that has high affinity for carboxylate groups and imidazole structures, which are important for its catalytic function .

What are the standard methods for constructing recombinant L. plantarum expressing hisB?

Standard methodologies for creating recombinant L. plantarum expressing hisB typically involve:

• Vector Selection: Researchers often use specialized vectors designed for lactic acid bacteria. For example, a vector system similar to that described in the literature can be employed, which combines a plasmid backbone with an erythromycin resistance gene as a selection marker .

• Promoter Selection: Selection of an appropriate promoter is crucial for efficient expression. Inducible promoters like the bile-inducible promoter of the lactate dehydrogenase 1 gene have been successfully used in L. plantarum .

• Cloning Strategy: The hisB gene can be amplified from genomic DNA using PCR with appropriate restriction sites incorporated into primers. The gene is then inserted into the expression vector using restriction enzymes and ligation .

• Transformation: Electroporation is commonly used to introduce the recombinant plasmid into L. plantarum. The transformation protocol typically involves:

  • Growing L. plantarum to mid-logarithmic phase

  • Washing cells with an electroporation buffer

  • Mixing cells with the recombinant plasmid

  • Applying an electric pulse

  • Recovering cells in growth medium before plating on selective media

• Verification: Successful transformants are verified by PCR, restriction digestion, and sequencing. Functional expression is confirmed by enzyme activity assays or immunodetection methods .

How can researchers evaluate the immunogenic properties of recombinant L. plantarum expressing hisB?

Evaluation of immunogenic properties involves several established methodologies:

In vitro assessment:

• Human PBMC Proliferation Assay: Peripheral blood mononuclear cells (PBMCs) from donors can be isolated and cultured with UV-inactivated recombinant L. plantarum. Cell proliferation can be measured using [³H]thymidine incorporation. Briefly:

  • 1×10⁵ PBMCs are seeded in triplicate in 96-well plates

  • 1×10⁷ CFU of UV-inactivated L. plantarum strains are added

  • Cells are incubated for 8-10 days

  • [³H]thymidine is added 24 hours before harvesting

  • Proliferation is measured using a scintillation counter

• Cytokine Production Analysis: Production of cytokines such as IFN-γ, TNF-α, IL-6, and IL-10 can be measured by ELISA or multiplex cytokine assays following stimulation of immune cells with recombinant L. plantarum .

In vivo assessment:

• Mouse Immunization Models: Oral or intranasal administration of recombinant L. plantarum to mice, followed by:

  • Measurement of antigen-specific IFN-γ production by splenocytes after in vitro restimulation

  • Assessment of PBMC proliferative responses

  • Evaluation of antigen-specific IgA secretion in mucosal sites (e.g., fecal samples)

• Sampling Schedule: Typical protocols involve:

  • Primary immunization followed by booster doses

  • Collection of samples at various time points (e.g., 21, 35 days post-immunization)

  • Analysis of serum IgG and mucosal IgA levels

What factors influence the expression efficiency of recombinant proteins in L. plantarum?

Several key factors affect expression efficiency:

Research indicates that surface-displayed proteins in L. plantarum can effectively stimulate immune responses, suggesting efficient expression and localization of recombinant proteins . For instance, L. plantarum strains expressing surface-displayed epitopes from SARS-CoV-2 induced significant cytokine responses and antibody production in animal models .

How does the structural conformation of recombinant hisB impact its enzymatic activity when expressed in L. plantarum?

The structural conformation of hisB is critical to its enzymatic function. Research findings on imidazole-glycerol phosphate dehydratase provide insights into structure-function relationships:

• Active Site Architecture: The enzyme contains a bi-manganese (Mn²⁺) cluster in its active site. High-resolution structural studies have shown that these metal ions are crucial for catalytic activity, coordinating with the substrate during the reaction .

• Substrate Binding: The active site has high affinity for carboxylate groups and imidazole structures. Crystallographic studies have revealed that formiate (FMT) binds between Mn²⁺ ions while imidazole can bind in different positions, suggesting flexibility in substrate recognition .

• Expression Location Effects: When expressed in recombinant systems, the localization of hisB can affect its conformation and activity:

  • Cytoplasmic expression generally preserves native folding

  • Surface display may alter protein conformation due to interactions with the cell wall

  • Secreted forms might encounter different folding environments affecting structure

• Conformational Stability: The stability of hisB in L. plantarum depends on factors such as pH, temperature, and ionic strength of the cellular environment. Maintaining proper conformation is essential for preserving catalytic activity.

Researchers should consider these structural aspects when designing recombinant L. plantarum expressing hisB, particularly if the goal is to maintain enzymatic activity.

What immunological mechanisms are activated by recombinant L. plantarum expressing foreign antigens?

Recombinant L. plantarum activates multiple immunological pathways:

• Cellular Immune Responses:

  • Studies with recombinant L. plantarum expressing M. tuberculosis antigens demonstrated significant induction of antigen-specific T-cell proliferation in human PBMCs

  • IFN-γ production by splenocytes from immunized mice indicates activation of Th1 responses

  • The strain expressing cytoplasmic antigens (Lp_1261AgE6-DC) induced stronger T-cell responses than the surface-displayed variant in some experimental settings

• Humoral Immune Responses:

  • Recombinant L. plantarum induces antigen-specific IgA secretion at mucosal sites, critical for mucosal immunity

  • Significant increases in serum IgG have been observed in mice immunized with recombinant L. plantarum expressing viral antigens

  • Time-dependent antibody responses show that booster immunizations enhance mucosal IgA levels with peak responses typically after 35 days

• Cytokine Profiles:

  • Recombinant L. plantarum strains can induce both pro-inflammatory (TNF-α, IL-6) and anti-inflammatory (IL-10) cytokines

  • The balance between these cytokines influences the type of immune response generated

  • Certain antigen constructs demonstrate higher anti-inflammatory to pro-inflammatory cytokine ratios

• Dendritic Cell Maturation:

  • Recombinant L. plantarum has been shown to induce maturation of dendritic cells, crucial for initiating adaptive immune responses

  • This effect contributes to enhanced antigen presentation and subsequent T-cell activation

How do different antigen localization strategies in L. plantarum affect immune response profiles?

Research findings demonstrate that antigen localization significantly impacts immune response profiles:

Experimental evidence suggests that:

• After oral vaccination, the cytoplasmic antigen-expressing strain (Lp_1261AgE6-DC) elicited significantly higher IFN-γ responses compared to the surface-displayed variant

• Intranasal immunization with the cytoplasmic antigen-expressing strain also resulted in significantly higher PBMC proliferative responses compared to all other groups, indicating that this localization strategy may be particularly effective for certain administration routes

• Surface-displayed antigens, while sometimes generating lower cellular responses, effectively stimulate mucosal IgA production, which is critical for protection at mucosal surfaces

What are the comparative advantages of using L. plantarum versus other lactic acid bacteria as expression hosts for recombinant proteins?

L. plantarum offers several distinct advantages compared to other lactic acid bacteria:

• Enhanced Immunogenicity: Research indicates that L. plantarum-based vaccines demonstrate higher immunogenicity than Lactococcus lactis when orally administered to mouse models

• Gastrointestinal Survival: Certain L. plantarum strains show superior ability to survive gastrointestinal conditions, making them particularly suitable for oral vaccine delivery

• Immune Response Modulation: L. plantarum can improve both local and distal immune responses in vivo, enhancing vaccine efficacy

• Expression System Flexibility: L. plantarum offers various expression systems, including:

  • Bile-responsive expression systems that enable intestine-targeted antigen delivery

  • Strong constitutive promoters for consistent expression

  • Inducible systems for controlled expression

• Safety Profile: L. plantarum has Generally Recognized as Safe (GRAS) status and has been safely consumed in fermented foods for centuries, reducing regulatory hurdles

• Adjuvant Properties: The intrinsic immunomodulatory properties of L. plantarum can serve as natural adjuvants, potentially eliminating the need for additional adjuvants

• Antigen Delivery Efficiency: Studies demonstrate that L. plantarum effectively delivers antigens to mucosal sites, as evidenced by successful induction of antigen-specific immune responses against tuberculosis antigens and SARS-CoV-2 epitopes

What methodological approaches can optimize mucosal immune responses to antigens delivered by recombinant L. plantarum?

Optimizing mucosal immune responses requires strategic methodological approaches:

• Administration Route Selection:

  • Intranasal administration has been shown to increase the numbers of antigen-specific cytokine-producing splenocytes for both cytoplasmic and surface-displayed antigens

  • Oral administration may be more effective for certain antigen localizations, particularly cytoplasmic expression (Lp_1261AgE6-DC)

  • Route selection should be based on the target pathogen and desired immune response

• Immunization Protocol Design:

  • Multiple doses significantly enhance immune responses

  • Typical protocols involve primary immunization followed by booster doses at 3-4 week intervals

  • Measurements at 21 and 35 days post-immunization can track response development

• Antigen Design Strategies:

  • Epitope selection: Identifying highly immunogenic epitopes through computational prediction and validation

  • Surface anchoring: Using appropriate anchoring domains for effective surface display

  • Expression level optimization: Selecting promoters and signal sequences for optimal expression

• Adjuvant Co-expression:

  • Co-expression of cytokines or other immune modulators can enhance responses

  • Research suggests potential for "co-expression and co-administration of adjuvants" to improve the L. plantarum vaccine delivery system

• Strain Selection:

  • Different L. plantarum strains have varying abilities to survive gastrointestinal transit and stimulate immune responses

  • For example, L. plantarum SK156 has been identified as an effective host for expressing bioactive substances in the intestine due to its bile-responsive expression system

• Formulation Considerations:

  • Protection of the bacterial vector from harsh environmental conditions

  • Ensuring viability until reaching target mucosal sites

  • Controlled release of antigen at mucosal surfaces