Recombinant Lactobacillus plantarum UPF0223 protein lp_2149 (lp_2149)

What is Lactobacillus plantarum UPF0223 protein lp_2149 and how does it function in immunomodulation?

Lactobacillus plantarum UPF0223 protein lp_2149 belongs to a family of proteins with partially characterized functions involved in bacterial cellular processes. The immunomodulatory properties of L. plantarum strains are largely mediated through TLR signaling pathways. Studies indicate that L. plantarum stimulates innate immunity primarily through TLR2 and TLR9, leading to the activation of NF-κB and MAPK pathways . These pathways are crucial for the production of cytokines involved in immunomodulation.

The mechanism of action typically involves:

• Engagement with pattern recognition receptors on immune cells

• Activation of dendritic cells (DCs) in Peyer's patches

• Modulation of cytokine production profiles

• Stimulation of T cell responses

Experimental data shows that L. plantarum strains can significantly alter cytokine production, with meta-analysis results demonstrating the following mean differences (95% confidence interval):

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

• IL-10: 9.88 pg/mL (6.52 to 13.2; p < 0.05)

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

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

How can I distinguish between native and recombinant forms of lp_2149 protein during analysis?

Distinguishing between native and recombinant forms of lp_2149 requires multiple analytical approaches:

• Western blotting: Use antibodies specific to unique tags (e.g., His-tag, HA-tag) incorporated into the recombinant protein. Lysates from both wild-type and recombinant strains should be compared side-by-side.

• Mass spectrometry: The recombinant protein will show mass shifts corresponding to any fusion tags or modifications. High-resolution MS can detect even small differences.

• Immunofluorescence analysis: This can be performed using:

  • Anti-tag antibodies (for recombinant protein)

  • Secondary antibodies conjugated with fluorescent markers like FITC

• Flow cytometry: As demonstrated in studies with other L. plantarum recombinant proteins, flow cytometry with specific antibodies can effectively differentiate expression levels between wild-type and recombinant strains .

For cell surface-expressed recombinant proteins, indirect immunofluorescence followed by analysis with FITC-conjugated anti-mouse IgG antibodies has been successfully employed, as demonstrated in comparable recombinant L. plantarum studies .

What are the optimal expression conditions for recombinant lp_2149 in L. plantarum?

Optimizing expression of recombinant lp_2149 in L. plantarum requires careful consideration of several parameters:

Medium Composition: Based on studies optimizing L. plantarum growth, the following medium components have shown significant effects on biomass production:

Induction Parameters: For inducible expression systems, optimal conditions based on analogous recombinant L. plantarum studies are:

• Inducer concentration: 50 ng/mL (if using SppIP induction system)

• Temperature: 37°C

• Induction time: 6-10 hours

Growth Phase: Expression yield is highly dependent on growth phase. For maximum protein production, harvest cells during late log phase to early stationary phase. Recent findings suggest that encapsulation of L. plantarum in alginate can trap cells in a growth phase that maximizes switch performance and protein expression .

What molecular cloning strategies are most effective for creating recombinant L. plantarum expressing lp_2149?

Based on successful approaches with other recombinant L. plantarum proteins, the following molecular cloning strategies are recommended:

• Vector Selection:

  • pSIP411-based vectors show high efficiency for L. plantarum

  • pLP-based vectors for surface display applications

  • pWCF series for protein secretion

• Codon Optimization:

  • Essential for high-level expression

  • Adjust codons according to L. plantarum codon usage bias

  • This strategy significantly improves translation efficiency

• Signal Sequence Selection:

  • For surface display: Use L. plantarum endogenous signal peptides (e.g., SP1320)

  • For secretion: Consider the Usp45 signal sequence

• Cloning Methods:

  • Gibson Assembly has shown excellent results for complex constructs

  • Traditional restriction-ligation for simpler constructs

  • Homologous recombination for chromosomal integration

• Verification Protocol:

  • Colony PCR for initial screening

  • Restriction digestion to confirm insert

  • Sequencing to verify the entire construct

  • Western blot to confirm protein expression

A typical workflow includes:

• Gene synthesis with optimized codons

• PCR amplification with primers containing appropriate restriction sites

• Digestion of both vector and insert

• Ligation and transformation into an intermediate host (E. coli)

• Verification by sequencing

• Electrotransformation into L. plantarum

• Confirmation of expression by Western blot and functional assays

How can I evaluate the immunomodulatory effects of recombinant L. plantarum expressing lp_2149?

Comprehensive evaluation of immunomodulatory effects requires both in vitro and in vivo approaches:

In Vitro Assays:

• Dendritic Cell Activation:

  • Culture bone marrow-derived dendritic cells (BMDCs) with the recombinant strain

  • Measure maturation markers (CD80, CD86, CCR7) by flow cytometry

  • Quantify cytokine production (IL-6, IL-10, IL-12) by ELISA

  • Analyze mRNA levels using qRT-PCR

• TLR Signaling Assessment:

  • Use TLR-deficient mouse models (TLR2-/-, TLR9-/-, MyD88-/-)

  • Compare cytokine production in wild-type vs. deficient BMDCs

  • Employ HEK293 cells expressing specific TLRs with NF-κB reporter plasmids

  • Measure NF-κB activation and IL-8 secretion

In Vivo Studies:

• Immune Cell Analysis:

  • After oral administration, collect tissues (spleen, mesenteric lymph nodes, Peyer's patches)

  • Analyze T cell populations (CD4+IFN-γ+, CD8+IFN-γ+) by flow cytometry

  • Measure T cell proliferation using CFSE staining

  • Evaluate B cell activation (B220+IgA+) in Peyer's patches

• Antibody Production:

  • Collect serum at different time points post-immunization

  • Measure specific antibodies (IgG, IgG1, IgG2a) by ELISA

  • Analyze mucosal IgA in fecal samples

  • Perform immunofluorescence staining for IgA in intestinal segments

• Cytokine Profiling:

  • Analyze serum cytokines by multiplex assay

  • Focus on IL-4, IL-10, TNF-α, IFN-γ levels

  • Compare with control groups (PBS, empty vector)

What are the potential applications of lp_2149 in vaccine development and antigen delivery systems?

Recombinant L. plantarum expressing lp_2149 could have significant applications in vaccine development:

• Mucosal Vaccination:

  • L. plantarum can effectively deliver antigens to mucosal surfaces

  • The bacterium's inherent adjuvanticity enhances immune responses

  • Oral administration induces both systemic and mucosal immunity

• Adjuvant Development:

  • If lp_2149 demonstrates immunomodulatory properties, it could be co-expressed with antigens

  • The protein might enhance dendritic cell activation and T cell responses

  • Combination with known adjuvants like dendritic cell-targeting peptide (DCpep) could potentiate effects

• Multivalent Vaccine Platforms:

  • Co-expression of lp_2149 with viral or bacterial antigens

  • Creating fusion proteins with antigens of interest

  • Development of prime-boost strategies combining different delivery systems

• Stability Enhancement:

  • L. plantarum-based vaccines show remarkable stability under various conditions

  • Proteins remain stable at 50°C, pH 1.5, and high salt concentrations

  • This makes them suitable for regions with limited cold chain infrastructure

• Targeted Immune Responses:

  • By understanding the specific immunomodulatory properties of lp_2149

  • Tailoring the vaccine to induce Th1, Th2, or balanced responses

  • Incorporating specific targeting molecules (like DCpep) to enhance delivery to immune cells

How do I resolve contradictory cytokine production results when testing recombinant L. plantarum strains?

Contradictory cytokine production results can arise from multiple factors. A systematic approach to resolution includes:

• Experimental Design Revaluation:

  • Standardize bacterial preparation (growth phase, medium, washing steps)

  • Unify stimulation conditions (cell:bacteria ratio, time points)

  • Use appropriate controls (wild-type strain, empty vector strain)

• Technical Considerations:

  • Sample Preparation: Ensure consistent cell lysis and protein extraction

  • Assay Selection: Different detection methods (ELISA vs. flow cytometry vs. qPCR) may yield different results

  • Reagent Quality: Check antibody specificity and cross-reactivity

• Biological Variation Analysis:

  • Cell Source Variation: Primary cells from different donors show variability

  • Strain-Specific Effects: Compare multiple L. plantarum isolates

  • TLR Polymorphisms: Consider genetic variations in test systems

• Methodological Approach:

  • Test cytokine production using multiple methods:

    • ELISA for protein secretion

    • qRT-PCR for mRNA expression

    • Flow cytometry for intracellular cytokine staining

  • Analyze cytokine production kinetics at multiple time points

• Statistical Considerations:

  • Use appropriate statistical tests (ANOVA followed by post-hoc tests)

  • Account for multiple comparisons

  • Consider biological vs. statistical significance

What methodological modifications are needed when transitioning from in vitro to in vivo studies with recombinant L. plantarum?

Transitioning from in vitro to in vivo studies requires several methodological adaptations:

• Strain Preparation and Administration:

  • Dosage Determination: Typically 10^8-10^10 CFU per animal

  • Administration Route: Oral gavage ensures precise dosing

  • Administration Schedule: Multiple doses (e.g., days 0, 14, and 28) for prime-boost protocols

  • Vehicle Selection: Sodium bicarbonate buffer helps neutralize stomach acid

• Bacterial Survival and Colonization:

  • Gastric Transit: Consider acid tolerance of the strain

  • Colonization Assessment: Fecal sampling and selective plating

  • Persistence Evaluation: Time-course sampling post-administration

• Immune Response Evaluation:

  • Tissue Collection: Harvest relevant tissues (spleen, mesenteric lymph nodes, Peyer's patches)

  • Cell Isolation: Optimize protocols for each tissue type

  • Flow Cytometry Panels: Design comprehensive panels for:

    • T cell subsets (CD4+IFN-γ+, CD8+IFN-γ+)

    • B cell activation (B220+IgA+)

    • Dendritic cell maturation (CD11c+MHC-II+CD80+CD86+)

• Antibody and Cytokine Analysis:

  • Sampling Schedule: Collect serum at weeks 0, 2, 4, and 10 post-immunization

  • ELISA Adaptations: Develop specific assays for IgG, IgG1, IgG2a, and IgA

  • Mucosal Sampling: Collect fecal pellets for SIgA analysis

  • Tissue Cytokine Analysis: Use immunohistochemistry or tissue homogenates

• Ethical and Practical Considerations:

  • Sample Size Calculation: Power analysis based on expected effect size

  • Minimizing Animal Numbers: Consider longitudinal sampling where possible

  • Refinement Techniques: Minimize stress during handling and sampling

  • Endpoint Selection: Define humane endpoints based on ethical guidelines

When analyzing in vivo data, use statistical approaches that account for individual variation and repeated measures. Consider including additional control groups (untreated, empty vector, non-recombinant L. plantarum) to disambiguate strain-specific from protein-specific effects.