Recombinant Lactobacillus plantarum Imidazole glycerol phosphate synthase subunit HisF (hisF)

Basic Research Questions

• What is Imidazole glycerol phosphate synthase (IGPS) and what is the specific role of the HisF subunit in L. plantarum?

Imidazole glycerol phosphate synthase is a heterodimeric enzyme critical in histidine biosynthesis, composed of HisH (glutaminase subunit) and HisF (synthase subunit). The HisF subunit in L. plantarum catalyzes the conversion of PRFAR (N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) to IGP using ammonia produced by HisH . This reaction is essential in histidine biosynthesis and interconnects nitrogen metabolism with purine synthesis . The enzyme plays an important role in cellular metabolism by connecting multiple biochemical pathways.

Methodologically, researchers can study HisF function through gene knockout experiments followed by growth analysis in histidine-deficient media, or by expressing and purifying the recombinant protein for enzymatic assays, using spectrophotometric methods to measure PRFAR consumption or IGP production.

• What expression systems and vectors are most effective for producing recombinant proteins in L. plantarum?

Several expression systems can be used for producing recombinant proteins in L. plantarum, with plasmid-based systems being most common. The pSIP expression system has demonstrated effectiveness for optimizing protein expression in L. plantarum. For proteins that tend to form inclusion bodies, fusion tags can enhance solubility - the pET-43.1a+ vector with its NusA tag has been successfully used to solubilize recombinant proteins in bacterial expression systems .

When designing expression constructs, researchers should consider:

• Codon optimization for L. plantarum

• Selection of appropriate promoters

• Inclusion of secretion signals if extracellular expression is desired

• Optimization of culture conditions

A typical methodology includes:

• Amplification of the target gene using PCR

• Cloning into an appropriate vector

• Transformation into L. plantarum

• Optimization of expression conditions (including temperature, pH, and induction parameters)

• Scale-up production in optimized media

Optimization studies have shown that culture time (66h), starch/water ratio (1.7%), and inoculum concentration (0.02%) can significantly impact protein yield in lactobacilli .

• How is the histidine biosynthesis pathway regulated in L. plantarum, and what factors affect HisF expression?

The histidine biosynthesis pathway in L. plantarum is subject to complex regulation. The IGPS enzyme (including HisF) demonstrates sophisticated allosteric regulation - HisH activity is intrinsically low but becomes activated when PRFAR binds to HisF, inducing conformational changes that propagate across the HisH/HisF interface to synchronize catalysis.

Environmental stress conditions significantly influence the expression of histidine biosynthesis enzymes. The HisH subunit of IGPS has been shown to be upregulated under heat and acid stresses , which likely applies to the HisF subunit as well given their functional interdependence. This upregulation may contribute to stress tolerance in lactobacilli.

The histidine biosynthesis pathway is also affected by other enzymes including hisG (ATP phosphoribosyltransferase) , indicating multiple regulatory points in the pathway. Studies examining differential gene expression under various growth conditions suggest complex regulatory networks controlling these metabolic pathways .

• What purification strategies yield the highest purity and activity of recombinant L. plantarum HisF?

Effective purification of recombinant L. plantarum HisF typically employs a multi-step approach:

• Affinity chromatography: HisPur™ Ni-NTA Resin purification is effective for His-tagged recombinant proteins . For recombinant proteins with specific epitopes, antibody-based affinity purification can also be employed .

• Ion exchange chromatography: As a secondary purification step to remove contaminants with similar affinity characteristics.

• Size exclusion chromatography: For final polishing and to ensure the protein is in the correct oligomeric state.

A typical purification protocol involves:

• Cell lysis using methods appropriate for L. plantarum (sonication or enzymatic treatment)

• Clarification by centrifugation (typically 10,000-15,000 × g for 15-30 minutes)

• Capture using affinity chromatography

• Further purification using ion exchange or size exclusion chromatography

• Quality assessment using SDS-PAGE, Western blotting, and activity assays

The choice of buffer conditions is critical - pH should be maintained near the physiological range of L. plantarum (pH 4.0-6.0), and the inclusion of stabilizing agents such as glycerol (10-20%) can prevent protein aggregation during purification .

• What methods can reliably assess the enzymatic activity of recombinant HisF from L. plantarum?

The assessment of recombinant HisF enzymatic activity requires approaches that account for its role in the heterodimeric IGPS complex:

• Coupled enzyme assays: Since HisF requires ammonia produced by HisH, a complete assay system should include both subunits or provide an alternative ammonia source. The activity can be monitored through the formation of IGP.

• Spectrophotometric methods: The conversion of PRFAR to IGP can be monitored by changes in absorbance at specific wavelengths.

• HPLC or mass spectrometry: For direct quantification of substrate consumption and product formation.

A protocol for assessing HisF activity might include:

• Preparation of reaction buffer (typically at pH 7.5-8.0)

• Addition of purified recombinant HisF (5-20 μg)

• Addition of substrate (PRFAR, 0.1-1.0 mM)

• Provision of ammonia source (either purified HisH with glutamine or direct addition of ammonium salt)

• Incubation at 30-37°C for 10-30 minutes

• Quantification of IGP formation

For studying allosteric regulation, researchers can vary PRFAR concentrations (0.01-1.0 mM) and measure resulting changes in enzymatic activity.

Advanced Research Questions

• How do environmental factors and stress conditions affect the expression and activity of recombinant HisF in L. plantarum?

Environmental conditions significantly impact both expression and activity of recombinant proteins in L. plantarum. pH is particularly important, as "Lactobacilli rapidly modify their surface properties in response to changes in pH" . Lactic acid bacteria naturally produce lactic acid during growth, reducing environmental pH to approximately 4 or below , which affects protein expression and surface presentation.

Cell surface proteins like enolase and glyceraldehyde-3-phosphate dehydrogenase are present on the cell surface at acidic pH but released into the environment at alkaline pH, mediated by lipoteichoic acids that bind these proteins at pH values below their isoelectric point . This pH-dependent localization could affect the expression and activity of recombinant HisF if it is designed as a surface-displayed protein.

The expression of histidine biosynthesis enzymes including IGPS is upregulated under stress conditions, particularly heat and acid stress . This upregulation could potentially be exploited to enhance recombinant HisF production.

For optimizing recombinant protein production, a response surface methodology approach as described in study can be applied:

$Y = \alpha_0 + \alpha_1A + \alpha_2B + \alpha_3C + \alpha_{11}A^2 + \alpha_{22}B^2 + \alpha_{33}C^2 + \alpha_{12}AB + \alpha_{13}AC + \alpha_{23}BC$

Where Y represents protein yield, and A, B, and C represent culture time, starch/water ratio, and inoculum concentration, respectively.

• What are the specific challenges in expressing and maintaining functional heterodimeric enzymes like IGPS in recombinant L. plantarum systems?

Expressing heterodimeric enzymes like IGPS in recombinant L. plantarum presents several specific challenges:

• Maintaining proper stoichiometry: Ensuring balanced expression of both HisH and HisF subunits is critical for optimal enzyme activity. This may require careful design of bicistronic constructs with optimized ribosome binding sites for each subunit.

• Ensuring correct folding and assembly: Heterodimeric enzymes require proper folding of individual subunits and correct assembly. Studies with other recombinant proteins have shown that "misfolded subunits are abundant in uncleaved proteins but very rare in native-like trimer populations" .

• Preserving the allosteric communication between subunits: The HisH-HisF interface is critical for allosteric regulation, where PRFAR binding to HisF activates HisH. This communication can be disrupted by improper folding or mutations.

• Addressing the acidic environment of L. plantarum: The naturally acidic environment created by lactic acid production may affect protein folding and stability, requiring careful optimization of growth conditions.

Strategies to address these challenges include:

• Use of fusion tags to enhance solubility (such as NusA tag)

• Introduction of stabilizing modifications like disulfide bonds (similar to the SOS strategy that increased properly folded proteins to ~20-30%)

• Co-expression of molecular chaperones to assist in proper folding

• Optimization of culture conditions using statistical methods like central composite design

• How can recombinant L. plantarum expressing HisF be utilized as a mucosal vaccine delivery vehicle?

Recombinant L. plantarum has proven effective as a mucosal vaccine delivery system, and this approach could be applied to HisF-based vaccines if HisF from pathogens is being targeted as an antigen. L. plantarum offers several advantages as a vaccine vector:

• Mucosal and systemic immune responses: Oral administration of recombinant L. plantarum can stimulate both mucosal IgA and systemic IgG responses . Studies show that recombinant lactobacilli "could significantly increase the specific anti-HA1 IgA antibody level in the mucosa and the anti-HA1 IgG level in serum, as well as stimulating the splenic lymphocyte proliferative reaction through increased expression of interleukin-4 (IL-4)" .

• T-cell activation: Recombinant L. plantarum can stimulate IFN-γ production in CD4⁺ and CD8⁺ T cells, enhancing cellular immunity.

Comparative immune responses induced by various recombinant L. plantarum strains are shown in Table 1:

For optimal vaccine development, considerations should include:

• Selection of appropriate promoters and secretion signals

• Protection of the antigen from degradation in the gastrointestinal tract

• Dose optimization (studies have used 10¹¹ cells per dose)

• Evaluation of immune responses in appropriate animal models before human trials

• How do mutations in key residues of HisF affect the allosteric regulation and catalytic efficiency of the IGPS complex?

While specific data on L. plantarum HisF mutations is limited in the search results, general principles of enzyme allostery suggest that mutations could affect:

• PRFAR binding site: Mutations in residues that directly interact with PRFAR would alter binding affinity and subsequently affect allosteric signaling.

• HisH-HisF interface: Mutations at the interface could disrupt the transmission of conformational changes between subunits. Research indicates that "molecular docking studies identify small molecules that disrupt HisH/HisF interactions", suggesting the importance of this interface.

• Catalytic residues: Mutations in the catalytic site of HisF would directly impact its ability to convert PRFAR to IGP, regardless of allosteric signaling.

Methodological approaches to study mutation effects include:

• Site-directed mutagenesis targeting conserved residues

• Steady-state kinetic analysis to determine changes in catalytic parameters (Km, kcat)

• Binding assays to measure PRFAR affinity

• Structural studies (X-ray crystallography, NMR) to observe conformational changes

• Molecular dynamics simulations to visualize allosteric communication pathways

These studies are particularly relevant as HisF is "absent in mammals but essential in pathogens", making it a potential antimicrobial target.

• What experimental approaches can be used to study the HisH-HisF interface and interaction dynamics in the IGPS complex?

Understanding the HisH-HisF interface is crucial for elucidating the allosteric regulation of IGPS. Several experimental approaches can be employed:

• Biophysical methods:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics between purified subunits

  • Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters

  • Analytical ultracentrifugation to study complex formation

  • Förster Resonance Energy Transfer (FRET) with fluorescently labeled subunits to monitor interaction dynamics in real-time

• Structural biology approaches:

  • X-ray crystallography to determine atomic resolution structures

  • Cryo-electron microscopy for visualization of different conformational states

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered solvent accessibility upon complex formation

• Computational methods:

  • Molecular docking to predict interaction sites

  • Molecular dynamics simulations to study conformational changes during allosteric regulation

  • Normal mode analysis to identify cooperative motions between subunits

• Biochemical techniques:

  • Site-directed mutagenesis of interface residues followed by activity assays

  • Chemical cross-linking combined with mass spectrometry to identify interacting regions

  • Limited proteolysis to probe structural changes upon complex formation

These approaches can be combined to develop a comprehensive understanding of how PRFAR binding to HisF induces conformational changes that propagate to HisH, activating its glutaminase activity.

• How can transcriptomic and metabolomic analyses provide insights into the impact of recombinant HisF expression on L. plantarum metabolism?

Transcriptomic and metabolomic approaches offer powerful tools to understand how recombinant HisF expression affects global metabolism in L. plantarum:

• Transcriptomic analysis:

  • RNA sequencing can identify differentially expressed genes in response to recombinant protein production

  • Studies have shown that L. plantarum modifies gene expression under different growth conditions, affecting pathways related to cell-cell adhesion and stress responses

  • The analysis could reveal metabolic adaptations, stress responses, and regulatory networks activated during recombinant protein expression

  • Example approach: Compare transcriptomes of wild-type L. plantarum versus strains expressing recombinant HisF under various conditions

• Metabolomic analysis:

  • Targeted and untargeted metabolomics can detect changes in metabolite levels

  • Particularly relevant would be changes in histidine pathway intermediates, purine metabolites, and nitrogen metabolism

  • Metabolic flux analysis using isotope-labeled substrates can trace carbon and nitrogen flow through relevant pathways

  • L. plantarum's facultative hetero-fermentative metabolism involves multiple pyruvate-dissipating routes , which might be affected by recombinant protein production

Integrated analysis:

• Correlating transcriptomic changes with metabolite profiles

• Pathway analysis to identify metabolic bottlenecks or compensatory mechanisms

• Construction of genome-scale metabolic models to predict the impact of recombinant protein expression

These approaches could help optimize expression conditions and identify potential metabolic engineering targets to enhance recombinant protein production.

• How can recombinant L. plantarum HisF be exploited for developing novel antimicrobials against pathogenic fungi and bacteria?

The potential of recombinant L. plantarum HisF as a tool for antimicrobial development is rooted in the fact that "HisH is absent in mammals but essential in pathogens like Cryptococcus and Candida, making IGPS a target for antifungal/antibiotic development". This selective essentiality makes IGPS an attractive target for antimicrobial development.

Strategies for exploiting recombinant L. plantarum HisF include:

• High-throughput screening platform:

  • Expression of recombinant HisF in L. plantarum

  • Development of activity-based assays for screening compound libraries

  • Identification of molecules that specifically inhibit HisF activity

• Structure-based drug design:

  • Determination of L. plantarum HisF crystal structure

  • In silico docking studies to identify potential binding sites

  • Design of small molecules that disrupt HisH/HisF interactions

  • Optimization of lead compounds through medicinal chemistry

• Immunological approaches:

  • Use of recombinant L. plantarum expressing pathogen-derived HisF as a vaccine

  • Induction of mucosal and systemic immune responses against HisF from pathogens

  • Evaluation of protective efficacy in appropriate animal models

• Combination approaches:

  • Integration of HisF inhibitors with existing antimicrobials

  • Exploiting L. plantarum's natural antimicrobial properties alongside HisF-targeting compounds

Development pipeline:

• Target validation using genetic approaches (e.g., CRISPR-Cas9 to confirm essentiality)

• Screening of compound libraries against recombinant HisF

• Validation in pathogen-specific models

• Optimization of lead compounds for improved pharmacokinetics and reduced toxicity

• Evaluation in animal models of infection

This approach leverages both the unique properties of IGPS as an antimicrobial target and the safety profile of L. plantarum as a generally recognized as safe (GRAS) organism.