Recombinant Lactobacillus plantarum Glutathione biosynthesis bifunctional protein gshAB (gshAB), partial

Basic Research Questions

• What is Lactiplantibacillus plantarum and why is it significant for recombinant protein expression?

  Lactiplantibacillus plantarum (formerly known as Lactobacillus plantarum) is a versatile gram-positive lactic acid bacterium found across diverse ecological niches, including fermented foods and the human gastrointestinal tract. Its significance for recombinant protein expression stems from several key attributes:

  • It maintains high survival rates in the human gastrointestinal tract (7±2% survival in the human ileum compared to 1±0.8% for Lactococcus lactis)

  • It has a remarkable plasticity of its genome, making it amenable to genetic engineering

  • It is recognized as safe for human consumption (GRAS status)

  • It elicits beneficial immunomodulatory effects

  • It can be engineered to express heterologous proteins on its surface or intracellularly

  L. plantarum NC8 strain is particularly valued in recombinant expression systems due to its well-characterized genetics and robust performance in laboratory conditions.

• What is the glutathione biosynthesis bifunctional protein gshAB in L. plantarum?

  The glutathione biosynthesis bifunctional protein gshAB in L. plantarum is an enzyme that catalyzes key steps in glutathione synthesis, an important antioxidant tripeptide (γ-L-glutamyl-L-cysteinylglycine). The bifunctional nature of gshAB refers to its dual enzymatic activities:

  • γ-glutamylcysteine synthetase activity (first step in glutathione synthesis)

  • Glutathione synthetase activity (second step in glutathione synthesis)

  This bifunctionality differs from many other organisms where these activities are performed by separate proteins, making the L. plantarum gshAB protein particularly interesting for research . The protein contributes to L. plantarum's oxidative stress resistance and potentially to its probiotic properties.

Advanced Research Questions

• What are the most effective methods for optimizing surface display of recombinant proteins on L. plantarum?

  Optimizing surface display of recombinant proteins on L. plantarum involves several critical methodological considerations:

  a) Selection of anchoring motifs: The pgsA' anchoring system has proven highly effective for surface display. Studies show that proteins anchored to the cell wall using pgsA' demonstrate higher surface exposure than membrane-anchored proteins, as confirmed by flow cytometry . Other effective anchoring systems include:

  Anchoring System	Location	Exposure Level	Stability
  pgsA'	Cell membrane	Moderate-High	High
  LysM domain	Cell wall	Very High	High
  LPXTG motif	Cell wall	High	Moderate
  Transmembrane domains	Cell membrane	Moderate	Variable

  b) Codon optimization: Codon optimization significantly improves expression levels. For example, the spike protein of SARS-CoV-2 was efficiently expressed on L. plantarum after codon optimization for Lactobacillus usage .

  c) Induction parameters: Optimal expression conditions include:

  • Induction with 50 ng/mL SppIP at 37°C for 6-10 hours

  • Culture in MRS broth supplemented with appropriate selective markers

  • Induction at OD600nm of 0.3 followed by continued cultivation

  d) Structural considerations: The insertion of linker sequences between the anchoring motif and the target protein has been shown to improve surface accessibility and proper folding of the expressed protein .

• How can the stability and inheritance of recombinant plasmids in L. plantarum be assessed and improved?

  The stability and inheritance of recombinant plasmids in L. plantarum are critical for consistent protein expression. Methodological approaches include:

  a) Stability assessment protocols:

  • PCR detection of the recombinant plasmid in successive generations (typically through 20 generations)

  • Growth curve analysis comparing recombinant strains with wild-type strains

  • Spectrophotometric monitoring at OD600nm every 2 hours during cultivation

  b) Stability improvement strategies:

  • Development of antibiotic-free selection systems using auxotrophic markers (e.g., asd and alr genes)

  • Integration of expression cassettes into the chromosome

  • Use of balanced promoter systems that minimize metabolic burden

  • Construction of food-grade selection systems

  Research on the TsPPase/pSIP409-pgsA' plasmid showed stable inheritance through 20 generations of L. plantarum NC8, with no significant differences in growth curves between recombinant and wild-type strains (t = 6.062, P = 0.116) .

• What are the key parameters for evaluating recombinant L. plantarum survival under gastrointestinal conditions?

  Evaluating the survival of recombinant L. plantarum under gastrointestinal conditions is essential for applications targeting intestinal delivery. Robust methodological approaches include:

  a) In vitro simulated gastric conditions assessment:

  • pH resistance testing: Expose bacteria to pH ranges from 1.0 to 6.4 for defined time periods

  • Bile salt tolerance: Test survival in 0.1-0.5% bile salt concentrations

  • Pancreatic enzyme resistance: Evaluate survival after exposure to pancreatic enzymes

  b) Quantification methods:

  • Colony counting on MRS plates after exposure to simulated conditions

  • Logarithmic representation of survival data

  • Flow cytometric analysis with viability dyes

  Research demonstrates that recombinant L. plantarum can survive for 2-3 hours in highly acidic environments (pH 1.0-2.0) and longer in less acidic conditions (pH 3.0-4.0) . Survival rates are significantly lower in acidic environments compared to neutral pH 6.4 (F = 243.031, P < 0.05) .

  c) In vivo transit assessment:

  • Recovery from fecal samples

  • Intestinal intubation studies

  • PCR-based detection methods

  Studies show that 7±2% of L. plantarum NCIMB 8826 survives passage to the human ileum, compared to 1±0.8% for Lactococcus lactis .

• How can the immunogenicity of recombinant proteins displayed on L. plantarum be comprehensively evaluated?

  Comprehensive evaluation of immunogenicity for surface-displayed recombinant proteins involves multi-parameter assessment:

  a) Humoral immune response evaluation:

  • Measurement of serum IgG, IgG1, IgG2a titers via ELISA

  • Detection of mucosal secretory IgA (sIgA) in bile, duodenal fluids, and feces

  • Hemagglutination inhibition (HI) assays for viral antigens

  b) Cellular immune response assessment:

  • Quantification of CD4+IFN-γ+ and CD8+IFN-γ+ T cells in spleen and mesenteric lymph nodes

  • T cell proliferation assays using CFSE labeling

  • Evaluation of B220+IgA+ cells in Peyer's patches

  • Cytokine profile analysis (typically IFN-γ and IL-4)

  c) Mucosal immunity metrics:

  • sIgA levels in intestinal segments and lungs

  • Activation status of dendritic cells in Peyer's patches

  Research has shown that oral vaccination with recombinant L. plantarum (e.g., expressing TsPPase) induces significantly higher levels of specific serum IgG, IgG1, IgG2a, and mucosal sIgA compared to control groups .

• What are the critical factors in experimental design for evaluating protective efficacy of recombinant L. plantarum vaccines?

  Designing robust experiments to evaluate protective efficacy of recombinant L. plantarum vaccines requires attention to several key factors:

  a) Challenge model selection:

  • Appropriate pathogen challenge strain/dose determination

  • Timing of challenge post-immunization

  • Route of challenge appropriate to the natural infection

  b) Immunization protocol optimization:

  • Dose determination (typically 5 × 10^9 CFU/mL)

  • Immunization schedule (primary + booster doses)

  • Route of administration (typically oral, but also intranasal for respiratory pathogens)

  c) Protection assessment parameters:

  • Reduction in pathogen burden (e.g., viremia detection)

  • Reduction in clinical symptoms

  • Survival rates

  • Quantification of specific immune correlates of protection

  d) Statistical analysis considerations:

  • Adequate group sizes for statistical power

  • Appropriate control groups (PBS, non-recombinant L. plantarum)

  • Statistical methods appropriate for data type

  Research using recombinant L. plantarum expressing TsPPase demonstrated 67.18%, 54.78%, and 51.91% reduction of Trichinella spiralis intestinal infective larvae, adult worms, and muscle larvae, respectively, compared to control groups (P < 0.05) .

• How can recombinant L. plantarum strains be effectively characterized to confirm surface expression of target proteins?

  Comprehensive characterization of surface expression requires multiple complementary techniques:

  a) Protein expression verification:

  • SDS-PAGE analysis of cell lysates

  • Western blotting using target protein-specific antibodies

  • Mass spectrometry identification

  b) Surface localization confirmation:

  • Immunofluorescence assay (IFA) using target protein-specific antibodies

  • Flow cytometry to quantify surface expression levels

  • Confocal microscopy for visual confirmation

  • Cell fractionation and compartment-specific analysis

  c) Functionality assessment:

  • Antigen-specific binding assays

  • Enzymatic activity assays (if applicable)

  • Receptor-ligand interaction studies

  When characterizing recombinant L. plantarum expressing spike protein, flow cytometry revealed significantly greater intensity of fluorescence signals in cells displaying the fusion protein compared to control cells . Similarly, western blotting and IFA confirmed surface expression of TsPPase in recombinant L. plantarum NC8 .

• What are the molecular mechanisms through which L. plantarum modulates mucosal immune responses, and how can these be leveraged in recombinant protein design?

  The molecular mechanisms of L. plantarum-mediated mucosal immune modulation are complex and can be leveraged in recombinant protein design:

  a) Pattern recognition receptor (PRR) engagement:

  • L. plantarum cell wall components interact with TLR2 and NOD2 receptors

  • These interactions can be enhanced by strategic design of recombinant proteins

  b) Dendritic cell activation:

  • L. plantarum activates dendritic cells in Peyer's patches

  • Fusion of dendritic cell-targeting peptide (DCpep) to recombinant antigens enhances immune responses

  c) Induction of regulatory vs. inflammatory responses:

  • L. plantarum strains can differentially modulate IL-10/IL-12 ratios

  • This property can be exploited for targeted immune outcomes

  d) Mucosal adherence mechanisms:

  • Mannose-specific adherence to intestinal epithelial cells

  • Potential to induce human mucin genes

  e) Leveraging strategies in recombinant design:

  • Co-expression of immunomodulatory molecules (e.g., cytokines, TLR ligands)

  • Fusion of antigens with DC-targeting moieties

  • Optimization of cellular localization based on immune response goals

  Research demonstrates that recombinant L. plantarum expressing HA1-DCpep showed superior induction of B220+IgA+ cells in Peyer's patches compared to those expressing HA1 alone (P < 0.05) , illustrating the value of targeting dendritic cells in recombinant design.

Advanced Technical Questions

• What are the optimal electroporation parameters for transforming L. plantarum with recombinant plasmids, and how can transformation efficiency be improved?

  Successful transformation of L. plantarum with recombinant plasmids requires optimized electroporation protocols:

  a) Standard electroporation parameters:

  • Electric pulse: 2.0 kV/cm, 200 Ω, 25 μF using a 0.2 cm cuvette

  • Temperature: typically performed at 4°C

  • Recovery medium: MRS broth supplemented with appropriate osmotic stabilizers

  b) Critical pre-electroporation steps:

  • Growth phase: cells harvested in early exponential phase (OD600 0.4-0.6)

  • Cell wall weakening: glycine (1-2%) treatment during growth

  • Washing steps: multiple washes with ice-cold electroporation buffer

  c) Post-electroporation optimization:

  • Recovery time: 2-3 hours at optimal temperature before selective plating

  • Selection methodology: appropriate antibiotic concentration (e.g., 5 μg/ml erythromycin)

  • Incubation conditions: anaerobic at 37°C for 36-48 hours

  d) Efficiency improvement strategies:

  • Cell wall modification with glycine or lysozyme treatment

  • Optimization of DNA concentration and purity

  • Use of methylation-deficient plasmid DNA

  • Selection of highly competent L. plantarum strains

  Improved protocols have achieved transformation efficiencies of 10^5-10^6 transformants per μg DNA, representing significant improvement over earlier methods.

• How can protein expression be quantitatively monitored and optimized in recombinant L. plantarum systems?

  Quantitative monitoring and optimization of protein expression in recombinant L. plantarum systems involves several sophisticated approaches:

  a) Quantitative monitoring techniques:

  • Western blotting with densitometric analysis

  • Flow cytometry with fluorescently labeled antibodies

  • ELISA-based quantification

  • Fluorescence microscopy with image analysis

  • Mass spectrometry-based proteomics

  b) Expression optimization strategies:

  • Promoter strength optimization: constitutive vs. inducible systems

  • Codon optimization for L. plantarum (shown to significantly improve yields)

  • Signal peptide selection for secreted proteins

  • Optimization of ribosome binding site (RBS) strength

  • Modulation of growth conditions (temperature, pH, media composition)

  c) Induction parameter optimization for inducible systems:

  • Inducer concentration (e.g., 50 ng/mL SppIP for sakacin P-based systems)

  • Induction timing (typically at OD600 0.3-0.6)

  • Induction duration (optimization between 6-10 hours)

  • Temperature during induction (typically 30-37°C)

  Studies with the SARS-CoV-2 spike protein demonstrated highest yields when recombinant L. plantarum was induced with 50 ng/mL SppIP at 37°C for 6-10 hours . Stability testing revealed the recombinant protein remained stable under normal conditions as well as at elevated temperatures (50°C), acidic pH (1.5), and high salt concentrations .

• What advanced genomic engineering approaches are being developed for stable chromosomal integration of expression cassettes in L. plantarum?

  Advanced genomic engineering techniques for stable chromosomal integration in L. plantarum include:

  a) CRISPR-Cas9 based approaches:

  • Precise genome editing with reduced off-target effects

  • Development of L. plantarum-specific guide RNA design tools

  • Optimization of cas9 expression for L. plantarum

  b) Recombineering systems:

  • Adaptation of lambda Red-like systems for L. plantarum

  • Development of ssDNA-mediated genome editing

  • Temperature-sensitive plasmid-based approaches

  c) Site-specific recombination systems:

  • Integration at defined chromosomal loci (attB sites)

  • Use of phage integrases (e.g., ΦC31) for stable integration

  • Development of serine recombinase-based tools

  d) Selection strategies for integration events:

  • Counter-selection markers (e.g., sacB)

  • Food-grade selection systems

  • Development of auxotrophic complementation markers

  e) Neutral integration sites identification:

  • Genome-wide analysis to identify regions with minimal impact on fitness

  • Characterization of expression levels at different chromosomal positions

  • Development of standardized integration modules

  Recent advances include the development of antibiotic-free selection systems using the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene as markers, combined with asd gene-deficient E. coli (χ6212) as the plasmid donor and alr gene deletion L. plantarum NC8Δ as the host strain .

• What analytical methods are most effective for studying the impact of recombinant L. plantarum on host immune cell populations and activation status?

  Comprehensive analysis of immune responses to recombinant L. plantarum requires sophisticated immunological techniques:

  a) Flow cytometry-based immune profiling:

  • Multiparameter analysis of cell surface markers

  • Intracellular cytokine staining

  • Proliferation assays using CFSE labeling

  • Identification of specific cell populations (e.g., CD4+IFN-γ+, CD8+IFN-γ+, B220+IgA+)

  b) Tissue-specific immune response assessment:

  • Isolation and analysis of cells from spleen, mesenteric lymph nodes, and Peyer's patches

  • Evaluation of lung and intestinal mucosal immunity

  • Trafficking studies of immune cells after vaccination

  c) Functional assays:

  • T cell proliferation in response to specific antigens

  • Antigen-specific cytokine production (ELISPOT, ELISA)

  • B cell activation and antibody secretion

  • Neutrophil and macrophage activation status

  d) Advanced imaging techniques:

  • Immunohistochemistry of relevant tissues

  • Intravital microscopy to track cellular interactions

  • Confocal microscopy for detailed cellular localization

  Research with recombinant L. plantarum expressing HA1 and HA1-DCpep demonstrated activation of dendritic cells in Peyer's patches, increased numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes, and enhanced CD4+ and CD8+ cell proliferation . These findings were established using flow cytometry with appropriate cell surface and intracellular markers.

• How can transcriptomic and proteomic approaches be integrated to optimize recombinant protein expression in L. plantarum?

  Integration of transcriptomic and proteomic approaches offers powerful insights for optimizing recombinant protein expression:

  a) Transcriptomic analysis approaches:

  • RNA-Seq to identify global gene expression patterns under different conditions

  • Targeted RT-qPCR for key pathway components

  • Transcriptional start site mapping to optimize promoter design

  • Identification of regulatory RNAs affecting expression

  b) Proteomic analysis techniques:

  • Mass spectrometry-based quantitative proteomics

  • 2D gel electrophoresis for protein profiling

  • Pulse-chase experiments to assess protein stability

  • Post-translational modification analysis

  c) Integration strategies:

  • Correlation of transcript and protein levels for key genes

  • Identification of rate-limiting steps in expression

  • Analysis of stress responses to recombinant protein production

  • Pathway analysis to identify metabolic bottlenecks

  d) Systems biology approaches:

  • Metabolic flux analysis

  • Genome-scale models to predict expression outcomes

  • Network analysis of regulatory interactions

  Studies of L. plantarum in the gastrointestinal tract have revealed that passage through the GI tract affects expression of genes involved in nutrient acquisition and synthesis, stress responses, and extracellular functions . Similar approaches can be applied to optimize recombinant protein expression systems.