Recombinant Lactobacillus plantarum 50S ribosomal protein L32 (rpmF)

What is Lactobacillus plantarum and why is it suitable for recombinant protein expression?

Lactobacillus plantarum is a lactic acid bacterium recognized as Generally Recognized As Safe (GRAS) with noninvasive properties, good adhesion capabilities, and inherent immunogenicity. These characteristics make it an ideal candidate for recombinant protein expression, particularly for vaccine development. L. plantarum functions as a probiotic with immunomodulatory properties, allowing it to serve as an effective vector for carrying antigens. The use of L. plantarum as a recombinant vector for developing novel vaccines has gained significant attention in recent years due to these beneficial properties .

What is the 50S ribosomal protein L32 (rpmF) and what is its function in bacteria?

The 50S ribosomal protein L32 (rpmF) is a component of the large subunit of bacterial ribosomes. While specific research on L32 in L. plantarum is limited, studies on this protein in other bacterial species provide valuable insights. In Glaesserella parasuis, L32 has been shown to be important for bacterial growth, stress resistance, and virulence. Although L32 is non-essential for cell proliferation, its deletion results in altered growth patterns and increased susceptibility to environmental stressors such as osmotic pressure, oxidation, and heat shock . Additionally, L32 plays a role in antibiotic resistance, particularly against aminoglycosides, as deletion mutants demonstrate increased sensitivity to antibiotics like spectinomycin and apramycin .

How does recombinant protein expression in L. plantarum affect its probiotic properties?

When L. plantarum is engineered to express recombinant proteins, its probiotic properties can be maintained or even enhanced depending on the expressed protein. Research shows that recombinant L. plantarum strains retain their ability to colonize the intestinal mucosa and interact with host immune cells. For example, recombinant L. plantarum expressing heterologous proteins can effectively stimulate both mucosal and systemic immune responses, as demonstrated by increased levels of specific antibodies (IgG in serum and sIgA in intestinal samples) following oral administration . Additionally, these strains enhance the proliferation of spleen lymphocytes and stimulate the differentiation of T cells, particularly CD3+CD4+ and CD3+CD8+ T cells, further supporting their immunomodulatory function .

What are the optimal expression systems for producing recombinant L. plantarum expressing 50S ribosomal protein L32?

The optimal expression systems for producing recombinant L. plantarum expressing 50S ribosomal protein L32 involve carefully selected plasmid vectors and surface display elements. Based on current methodologies, the pSIP expression system has proven effective for heterologous protein expression in L. plantarum. This system typically incorporates:

• An inducible promoter system (such as sakacin P-based induction system)

• Surface display elements like poly-γ-glutamic acid synthetase A (pgsA') or fibronectin-binding protein A (FnBPA)

• Appropriate restriction sites for gene insertion

The expression vector construction typically begins with gene synthesis of the target protein (in this case, L32) with appropriate restriction sites. For example, in similar recombinant L. plantarum studies, researchers have used HindIII and XbaI restriction sites for gene insertion into vectors like pSIP409 . Following vector construction, the recombinant plasmid is introduced into L. plantarum strain NC8 through electroporation using standardized protocols (such as program 4-1-9 for lactic acid bacteria). After electroporation, transformed bacteria are selected on appropriate antibiotic-containing media, and positive colonies are verified through PCR, Western blot, and immunofluorescence techniques .

What immunological effects are observed when recombinant L. plantarum expressing L32 is administered orally?

Based on studies of recombinant L. plantarum expressing other proteins, we can predict potential immunological effects of L. plantarum expressing L32. Oral administration of recombinant L. plantarum typically elicits:

• Enhanced humoral immunity, as evidenced by increased levels of specific IgG in serum and secretory IgA (sIgA) in intestinal samples

• Stimulated cellular immunity, including increased proliferation of spleen lymphocytes when exposed to the recombinant protein

• Elevated proportions of CD3+CD4+ and CD3+CD8+ T cells in spleen lymphocytes, indicating enhanced T cell differentiation

• Modulated cytokine production, typically including increased levels of immunoregulatory cytokines like IL-4 and IL-10, and potentially decreased levels of pro-inflammatory cytokines like IL-17, IL-22, and TNF-α

These immunological effects highlight the potential of recombinant L. plantarum expressing L32 as an oral vaccine or immunotherapeutic agent, particularly if L32 is found to have immunogenic properties in the context of specific diseases or conditions.

What are the key steps and considerations for constructing recombinant L. plantarum expressing 50S ribosomal protein L32?

Constructing recombinant L. plantarum expressing 50S ribosomal protein L32 requires a systematic approach with several critical steps:

• Gene Synthesis and Vector Preparation:

  • Synthesize the L32 gene with appropriate restriction sites (e.g., HindIII and XbaI)

  • Select a suitable expression vector (e.g., pSIP409-pgsA' or pSIP409-FnBPA-pgsA')

  • Digest both the vector and target gene with appropriate restriction enzymes

  • Ligate the target gene into the prepared vector

• Transformation and Selection:

  • Prepare competent L. plantarum NC8 cells

  • Introduce the recombinant plasmid via electroporation (using standard parameters for lactic acid bacteria)

  • Allow for recovery in MRS culture medium at 37°C

  • Select transformants on antibiotic-containing media

  • Verify positive colonies through PCR and sequencing

• Expression Verification:

  • Induce protein expression using appropriate inducers (e.g., sakacin P-inducing peptide)

  • Verify surface expression through:

    • Immunofluorescence experiments

    • Western blot analysis (expected protein size for L32 is approximately 6-7 kDa)

    • Flow cytometry to quantify expression levels

Key considerations include selecting the appropriate surface-anchoring domain (pgsA' or FnBPA-pgsA'), optimizing codon usage for L. plantarum, and ensuring proper protein folding and presentation on the bacterial surface.

How can researchers effectively measure the expression and surface display of L32 on recombinant L. plantarum?

Effective measurement of L32 expression and surface display on recombinant L. plantarum requires multiple complementary techniques:

• Immunofluorescence Microscopy:

  • Fix bacterial cells with 4% paraformaldehyde

  • Block with appropriate blocking buffer (e.g., 2% BSA in PBS)

  • Incubate with primary antibodies against L32

  • Detect with fluorescently-labeled secondary antibodies

  • Visualize using fluorescence microscopy to confirm surface localization

• Western Blot Analysis:

  • Extract total or surface proteins from recombinant L. plantarum

  • Separate proteins by SDS-PAGE

  • Transfer to PVDF membrane

  • Probe with anti-L32 antibodies

  • Detect with appropriate secondary antibodies and visualization system

• Flow Cytometry:

  • Incubate bacterial cells with anti-L32 antibodies

  • Label with fluorescent secondary antibodies

  • Analyze using flow cytometry to quantify the percentage of bacteria expressing L32 and the relative expression levels

  • Compare with appropriate controls (non-recombinant L. plantarum)

• ELISA:

  • Coat plates with whole bacteria or extracted surface proteins

  • Detect L32 using specific antibodies

  • Quantify expression levels compared to standards

These combined approaches provide comprehensive information about both the presence and quantity of L32 on the bacterial surface, as well as confirmation of the correct protein size and localization.

What animal models are most appropriate for studying the effects of recombinant L. plantarum expressing L32?

The selection of appropriate animal models for studying recombinant L. plantarum expressing L32 depends on the specific research questions:

• Mouse Models:

  • Most commonly used due to well-characterized immune system and cost-effectiveness

  • Suitable for immunogenicity studies, including analysis of antibody production and T cell responses

  • Effective for preliminary safety and efficacy assessments

  • Can be used to evaluate intestinal colonization by recombinant L. plantarum

• Specialized Disease Models:

  • If investigating the protective effects against specific conditions, specialized models may be required

  • For example, dextran sodium sulfate (DSS)-induced colitis models can be used to study inflammatory bowel disease protection

  • Challenge models with specific pathogens can evaluate protective efficacy

• Study Design Considerations:

  • Route of administration: Oral gavage is typically used for L. plantarum to mimic natural route

  • Dosage and frequency: Often 10^8-10^10 CFU/mouse, administered daily or on alternating days

  • Duration: Short-term studies (7-14 days) for acute responses, longer studies (4-8 weeks) for sustained effects

  • Sample collection: Serum for IgG, intestinal washes for sIgA, spleen and mesenteric lymph nodes for cellular responses

The immunological readouts should include both humoral (IgG, IgA) and cellular (T cell subsets, cytokine profiles) parameters to comprehensively assess the immune response to recombinant L. plantarum expressing L32.

How might recombinant L. plantarum expressing L32 be applied in vaccine development?

Recombinant L. plantarum expressing L32 presents several potential applications in vaccine development:

Future research should focus on evaluating the immunogenicity of L32 in various disease contexts and optimizing expression systems for maximum immune stimulation.

What role might L32 expression play in the stress resistance and antibiotic sensitivity of L. plantarum?

Based on findings from other bacterial species, L32 expression likely plays significant roles in stress resistance and antibiotic sensitivity of L. plantarum:

• Stress Resistance Mechanisms:

  • L32 may contribute to structural integrity of ribosomes under stress conditions

  • Its presence could be critical for translation efficiency during environmental challenges

  • Based on studies in G. parasuis, L32 may influence resistance to:

    • Osmotic stress

    • Oxidative stress

    • Heat shock

    • Other environmental stressors commonly encountered by L. plantarum in food fermentation or gut colonization

• Antibiotic Sensitivity Profiles:

  • L32 might mediate resistance to specific antibiotics, particularly aminoglycosides

  • This could influence the selection of appropriate marker antibiotics for recombinant strain development

  • Modification of L32 expression might alter the antibiotic sensitivity profile of probiotic L. plantarum strains

• Interaction with Membrane Structure:

  • In G. parasuis, L32 deletion resulted in increased production of outer membrane vesicles with irregular morphology

  • Similar effects in L. plantarum could alter surface properties and interaction with host cells

  • Changes in membrane composition might affect adhesion properties and colonization capabilities

Understanding these roles could provide insights for optimizing L. plantarum as a probiotic or vaccine vector, particularly in applications where stress resistance or antibiotic compatibility is important.

How might the immune response to recombinant L. plantarum expressing L32 differ between animal models and human applications?

Several factors influence the translation of immune responses from animal models to human applications:

• Species-Specific Differences:

  • Mice and humans have distinct gut microbiota compositions, potentially affecting L. plantarum colonization

  • The distribution and phenotype of immune cells in gut-associated lymphoid tissue differ between species

  • Receptor-ligand interactions may have species-specific affinities affecting immune recognition

• Dosing and Administration Considerations:

  • Scaling doses from small animal models to humans requires careful consideration

  • The transit time and pH conditions of the gastrointestinal tract differ significantly

  • Formulation requirements may change to ensure bacterial viability in human applications

• Immune Response Variations:

  • Baseline immune parameters may differ between laboratory animals and humans

  • Human populations show greater genetic diversity in immune response genes

  • Pre-existing immunity to L. plantarum or cross-reactive antigens may influence responses in humans

• Clinical Translation Requirements:

  • Moving from animal models to human applications requires additional safety testing

  • Standardization of bacterial preparations becomes more stringent for human use

  • Production processes must meet good manufacturing practice (GMP) requirements

Researchers should consider these factors when designing preclinical studies and interpreting results for potential human applications of recombinant L. plantarum expressing L32.