Recombinant Lactobacillus plantarum Diaminopimelate epimerase (dapF)

What are the key advantages of using L. plantarum as an expression system for recombinant proteins?

Lactobacillus plantarum offers numerous advantages as an expression system, particularly for vaccine development and therapeutic applications. As demonstrated in multiple studies, L. plantarum can effectively resist damage from extreme gastrointestinal environments, making it ideal for oral administration routes. The bacterium serves dual roles as both an adjuvant and vector for recombinant vaccines, enhancing the immunogenicity of expressed antigens. Additionally, L. plantarum is generally regarded as safe (GRAS), can maintain intestinal flora, enhance immunity, and promote nutrient absorption, making it a particularly versatile expression platform .

How does surface display technology function in L. plantarum expression systems?

Surface display technology in L. plantarum involves the expression of target proteins on the bacterial surface using specific anchoring motifs. In reported studies, the surface-display motif pgsA (derived from the poly-γ-glutamic synthetase complex) has been successfully employed to display proteins on the L. plantarum surface. This approach significantly enhances antigen immunogenicity compared to conventional expression methods. Surface localization can be verified through techniques such as western blotting and flow cytometry, with displayed proteins showing significantly greater fluorescence intensity compared to control cells .

What immune responses can be induced by recombinant L. plantarum vaccines?

Recombinant L. plantarum vaccines can induce both mucosal and systemic immune responses. Orally administered recombinant L. plantarum has been shown to stimulate the production of secretory IgA (sIgA) in bile, duodenal lavages, and sera, reaching peak levels after multiple immunizations. This robust mucosal immune response represents an important defense mechanism, as sIgA is the predominant antibody on mucosal surfaces. Additionally, recombinant L. plantarum vaccines can trigger specific serum IgG production, alongside cellular immune responses involving dendritic cell activation in Peyer's patches and increased numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes .

How does the metabolic integration of dapF affect the growth characteristics of recombinant L. plantarum strains?

Diaminopimelate epimerase (dapF) plays a critical role in bacterial cell wall biosynthesis by catalyzing the conversion of L,L-diaminopimelate to meso-diaminopimelate. When expressed in recombinant L. plantarum, researchers should evaluate how this enzyme affects growth characteristics compared to wild-type strains. Studies of recombinant L. plantarum expressing other proteins have shown significant differences in body weight and growth parameters between vaccinated and control groups, suggesting metabolic impacts from recombinant protein expression. For dapF specifically, researchers should monitor growth curves, cell morphology, and peptidoglycan integrity to fully characterize the metabolic burden or potential benefits of dapF integration .

What are the optimal promoter and signal sequence combinations for maximizing dapF expression in L. plantarum?

Promoter selection significantly impacts recombinant protein expression levels in L. plantarum. While the search results don't specifically address dapF expression, studies with other recombinant proteins have utilized shuttle vectors such as pMG36e with considerable success. When designing expression systems for dapF, researchers should consider constitutive promoters (like P32 or Ppgm) versus inducible systems (like PnisA), depending on whether continuous or controlled expression is desired. Signal sequence selection should be optimized based on whether cytoplasmic, membrane-anchored, or secreted dapF is required. Methodological approaches should include comparative assessment of multiple promoter-signal sequence combinations using quantitative protein analysis techniques .

What protocols are most effective for verifying surface display of dapF in recombinant L. plantarum?

Verification of dapF surface display in recombinant L. plantarum requires multiple complementary techniques. Based on successful protocols with other recombinant proteins, researchers should employ the following methodology:

• SDS-PAGE analysis: Separate bacterial proteins and look for bands at the expected molecular weight of the fusion protein (dapF plus anchoring motif).

• Western blotting: Use dapF-specific antibodies to confirm the identity of the expressed protein.

• Flow cytometry: Employ primary antibodies against dapF and fluorescently-labeled secondary antibodies to quantify surface expression levels.

• Immunofluorescence microscopy: Visualize the cellular localization of dapF on the bacterial surface.

For flow cytometry specifically, researchers should compare fluorescence intensity between recombinant and control strains, with significantly higher fluorescence indicating successful surface display of dapF, as demonstrated in previous studies with other recombinant proteins in L. plantarum .

What experimental design best assesses the enzymatic activity of recombinant dapF expressed in L. plantarum?

To assess enzymatic activity of recombinant dapF expressed in L. plantarum, researchers should implement a comprehensive experimental design:

• Enzyme extraction: Prepare separate fractions (cytoplasmic, membrane-bound, and surface-displayed) depending on the expression system design.

• Spectrophotometric assay: Measure dapF activity by following the conversion of L,L-diaminopimelate to meso-diaminopimelate using HPLC or specialized enzymatic assays.

• Kinetic parameters: Determine Km, Vmax, and catalytic efficiency under various pH and temperature conditions.

• Comparative analysis: Benchmark against purified native dapF from original bacterial sources.

• Inhibition studies: Assess sensitivity to known inhibitors to confirm enzymatic mechanism.

This multi-parameter approach allows for thorough characterization of recombinant dapF functionality and provides insights into any structural or functional alterations resulting from expression in L. plantarum.

How should immunological responses to dapF-expressing L. plantarum be evaluated in experimental models?

Based on successful immunological evaluation protocols with other recombinant L. plantarum strains, assessment of dapF-expressing strains should include:

• Antibody response analysis:

  • Measure serum IgG levels using ELISA with S/P ratios (sample-to-positive ratios)

  • Assess secretory IgA in bile, intestinal lavages, and feces

  • Monitor antibody kinetics at regular intervals post-immunization

• Cellular immune response evaluation:

  • Quantify CD4+IFN-γ+ and CD8+IFN-γ+ cells in spleen and mesenteric lymph nodes

  • Assess dendritic cell activation in Peyer's patches

  • Measure T and B cell proliferation in response to stimulation

• Protection studies:

  • Challenge with relevant pathogens if applicable

  • Monitor physiological parameters (weight gain, temperature)

  • Assess pathogen loads and viremia

This comprehensive immunological assessment provides insight into both mucosal and systemic immune responses, which is critical for evaluating the efficacy of dapF-expressing L. plantarum strains .

What control groups are essential when testing recombinant L. plantarum expressing dapF?

Proper experimental design for testing recombinant L. plantarum expressing dapF should include the following control groups:

• Wild-type L. plantarum without any genetic modification: Establishes baseline probiotic effects

• L. plantarum containing empty vector*: Controls for vector effects independent of dapF expression

• L. plantarum expressing non-functional dapF mutant*: Distinguishes between enzymatic activity and protein presence

• PBS or buffer control: Provides absolute negative control

• Purified dapF enzyme (if applicable): Serves as positive control for enzymatic activity

These control groups help differentiate the specific effects of recombinant dapF expression from other variables. In previous studies with recombinant L. plantarum, significant differences were observed between recombinant strains and both wild-type L. plantarum and PBS control groups, highlighting the importance of comprehensive controls .

What parameters should be measured when optimizing culture conditions for maximum dapF expression in L. plantarum?

Optimization of culture conditions for maximum dapF expression in L. plantarum should involve systematic assessment of:

• Growth media composition:

  • Base media type (MRS, M17, defined media)

  • Carbon source type and concentration

  • Nitrogen source composition

  • Micronutrient requirements

• Physical parameters:

  • Temperature profiles (25-40°C range)

  • pH optimization (4.5-7.0)

  • Oxygen levels (aerobic, microaerophilic, anaerobic)

  • Agitation rate

• Induction conditions (if using inducible promoters):

  • Inducer concentration

  • Induction timing

  • Duration of expression period

• Harvest timing:

  • Growth phase (early log, mid-log, late log, stationary)

  • Cell density at harvest

Each parameter should be optimized using factorial experimental design, with dapF expression levels quantified through protein assays, western blotting, and enzymatic activity measurements to determine optimal conditions.

How do different administration routes affect the immunological response to dapF-expressing L. plantarum?

Different administration routes can significantly impact immunological responses to recombinant L. plantarum. Based on studies with other recombinant antigens, researchers should compare:

• Oral administration:

  • Induces strong mucosal immune responses with significant sIgA production

  • May require higher doses due to gastric degradation

  • Typically requires multiple doses to achieve optimal response

• Intranasal administration:

  • Generates robust respiratory mucosal immunity

  • Often produces both mucosal and systemic antibody responses

  • May require different formulation than oral delivery

• Parenteral administration (subcutaneous/intramuscular):

  • Primarily generates systemic IgG responses

  • May not induce strong mucosal immunity

  • Different dose requirements than mucosal routes

Comparative studies should measure route-specific immune parameters, including mucosal sIgA levels, serum IgG titers, and cellular responses in relevant tissues. Previous research has shown oral administration of recombinant L. plantarum generates significant mucosal immunity with high levels of sIgA in the intestine and bile, as well as serum IgG responses .

How can protein degradation of dapF be minimized during expression in L. plantarum?

Minimizing dapF degradation in L. plantarum expression systems requires multiple strategic approaches:

• Protease deficient strains: Consider using or developing L. plantarum strains with reduced protease activity.

• Fusion partners:

  • Use stability-enhancing fusion partners like thioredoxin or SUMO

  • Incorporate the pgsA surface display system shown to effectively present proteins on the bacterial surface

• Expression conditions:

  • Lower incubation temperature (25-30°C instead of 37°C)

  • Adjust media composition to reduce protease production

  • Optimize induction parameters to balance expression rate with folding capacity

• Protease inhibitors:

  • Add appropriate inhibitors during extraction if conducting in vitro studies

  • Consider co-expression of protease inhibitors for in vivo stability

• Codon optimization:

  • Adapt the dapF coding sequence to L. plantarum codon usage for efficient translation

This multi-faceted approach addresses the various mechanisms of protein degradation that might affect dapF stability in recombinant L. plantarum systems .

What methods are most effective for resolving poor transformation efficiency when introducing dapF constructs into L. plantarum?

Poor transformation efficiency is a common challenge when introducing recombinant constructs into L. plantarum. Researchers should consider these methodological improvements:

• Electroporation optimization:

  • Cell wall weakening treatments (glycine, lysozyme)

  • Buffer composition adjustment (reduced salt concentration)

  • Field strength optimization (typically 1.5-2.5 kV/cm)

  • Cuvette gap width selection (1 mm or 2 mm)

• Plasmid considerations:

  • Minimize plasmid size where possible

  • Ensure plasmid is demethylated if L. plantarum has restriction systems

  • Use shuttle vectors with proven efficiency in L. plantarum (like pMG36e used successfully in previous studies)

• Growth phase optimization:

  • Harvest cells in early-mid log phase

  • Growth in media containing glycine (0.5-2%) to weaken cell walls

• Recovery conditions:

  • Extended recovery period (3-5 hours)

  • Recovery at optimal growth temperature

  • Rich recovery media containing optimal osmolytes

Implementing these optimizations can significantly improve transformation efficiency for recombinant dapF constructs in L. plantarum .

How can specific immune responses to dapF be distinguished from general immune stimulation by L. plantarum?

Distinguishing specific immune responses to dapF from the general immunostimulatory effects of L. plantarum requires carefully designed experiments:

• Comprehensive control groups:

  • Wild-type L. plantarum (general immune effects)

  • L. plantarum with empty vector (vector effects)

  • L. plantarum expressing an irrelevant protein (protein expression effects)

• Antigen-specific assays:

  • ELISA using purified dapF protein as coating antigen

  • Antigen-specific B-cell ELISpot

  • T-cell proliferation assays with dapF stimulation

  • Cytokine profiling following dapF restimulation

• Epitope mapping:

  • Identify specific epitopes within dapF that generate immune responses

  • Develop peptide arrays to pinpoint immunodominant regions

• Adoptive transfer experiments:

  • Transfer T or B cells from immunized animals to naïve recipients

  • Challenge with purified dapF to confirm antigen specificity

Previous studies with recombinant L. plantarum have shown significant differences in immune responses between groups receiving recombinant bacteria versus wild-type L. plantarum or PBS, demonstrating the ability to detect antigen-specific responses above background stimulation .

What statistical approaches are most appropriate for analyzing immune response data from dapF-expressing L. plantarum studies?

Appropriate statistical analysis of immune response data from dapF-expressing L. plantarum studies should include:

• Descriptive statistics:

  • Mean, median, standard deviation, standard error

  • Box plots or violin plots for data distribution visualization

• Hypothesis testing:

  • One-way ANOVA with appropriate post-hoc tests (Tukey, Bonferroni, or Dunnett's) for comparing multiple groups

  • Student's t-test (paired or unpaired) for two-group comparisons

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normally distributed data

• Longitudinal data analysis:

  • Repeated measures ANOVA

  • Mixed models for handling missing data points

• Correlation analysis:

  • Pearson or Spearman correlation between antibody levels and protection

  • Multivariate analysis to identify immune correlates of protection

• Significance thresholds:

  • P-values < 0.05 considered statistically significant

  • Multiple comparison correction when appropriate (FDR, Bonferroni)

The statistical significance threshold should be clearly defined (typically P < 0.05 or P < 0.01), as demonstrated in previous recombinant L. plantarum studies .

How can dapF-expressing L. plantarum systems be applied to develop novel vaccines?

dapF-expressing L. plantarum systems can be leveraged for novel vaccine development through several strategic applications:

• Adjuvant properties:

  • dapF may enhance immunogenicity of co-expressed antigens

  • The system could serve as a molecular adjuvant platform for mucosal vaccines

• Multi-antigen delivery:

  • Co-express dapF with pathogen antigens to enhance immune response

  • Develop bivalent or multivalent mucosal vaccines

• Targeted immunity:

  • Engineer strains for tissue-specific colonization and immune induction

  • Optimize for particular immune response profiles (Th1, Th2, or Th17)

• Prime-boost strategies:

  • Use as oral primer followed by parenteral boost

  • Develop complementary vaccine strategies with other platforms

• Modulation of immune tolerance:

  • Potential applications in autoimmune disease therapy

  • Development of tolerance-inducing vaccines

Previous studies have demonstrated that recombinant L. plantarum expressing viral antigens can provide effective protection against viral challenges, suggesting similar approaches could be applied with dapF-based systems for relevant applications .