Recombinant Lactobacillus plantarum GTPase Der (der)

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

Lactobacillus plantarum is a versatile probiotic bacterium found in the intestinal microflora of both humans and rodents that has gained significant attention as an expression and delivery system for recombinant proteins. Its significance stems from its GRAS (Generally Recognized As Safe) status, ability to survive gastrointestinal transit, and capacity to interact with mucosal surfaces. As a mucosal delivery vehicle, L. plantarum can effectively present antigens to the immune system, making it particularly valuable for vaccine development and immunomodulatory applications .

For recombinant protein expression, L. plantarum offers several advantages including the ability to express heterologous proteins either intracellularly or display them on its surface, as demonstrated in studies using surface-display motifs like pgsA . This versatility makes it an excellent platform for developing novel biotechnological applications in both research and potential therapeutic contexts.

How does recombinant L. plantarum differ from wild-type strains in research applications?

Recombinant L. plantarum differs from wild-type strains primarily through its engineered capacity to express specific target proteins that confer new functionalities. While wild-type strains possess inherent probiotic properties, recombinant strains are designed to deliver specific antigens or therapeutic proteins to target sites.

Studies have demonstrated that recombinant L. plantarum can significantly alter immune responses compared to natural L. plantarum. For example, recombinant strains expressing viral proteins like gp85 have been shown to elicit stronger specific antibody responses, with significantly higher IgG and IgA titers compared to control groups receiving wild-type L. plantarum . Additionally, recombinant L. plantarum has been demonstrated to modulate gut microbial composition differently than wild-type strains, enhancing species diversity of gut bacteria as measured by the Shannon-Wiener index .

These engineered differences allow researchers to develop targeted interventions for specific conditions while retaining the beneficial core characteristics of the original probiotic strain.

What is the role of GTPases in L. plantarum cellular function?

GTPases in L. plantarum, such as the putative ribosome biogenesis GTPase RsgA 2 (rsgA2), play critical roles in fundamental cellular processes . While the search results don't provide comprehensive details specifically about "GTPase Der (der)," we can infer from related bacterial research that these enzymes likely function in:

• Ribosome assembly and maturation: Bacterial GTPases are often involved in ribosome biogenesis, helping ensure proper assembly of ribosomal subunits.

• Protein synthesis regulation: Many GTPases function as molecular switches controlling translation initiation, elongation, or termination.

• Stress response mechanisms: GTPases may participate in bacterial adaptations to environmental stressors.

These enzymes typically hydrolyze GTP to GDP while undergoing conformational changes that regulate their interactions with other cellular components. Understanding these fundamental processes is essential for developing strategies to manipulate L. plantarum for research and potential therapeutic applications.

What are the optimal vector systems for expressing heterologous proteins in L. plantarum?

The selection of an appropriate vector system is critical for successful heterologous protein expression in L. plantarum. Based on the research literature, several effective systems have been identified:

• pMG36e-based vectors: This shuttle vector has been successfully used to express viral proteins in L. plantarum. For example, in a study expressing gp85 protein of ALV-J, researchers constructed a pMG36e:pgsA:gp85 shuttle vector that effectively displayed the fusion protein on the bacterial surface .

• pLP503-based vectors: This system has been used for expressing allergen peptides, such as Der p 1 peptide 111-139 of house dust mite. The vector contains the l-(+)-lactate dehydrogenase gene (ldh) promoter of L. casei, allowing constitutive expression of the target protein .

When designing expression systems, researchers should consider:

• Promoter selection: Constitutive promoters like the ldh promoter provide stable expression, while inducible promoters offer more controlled expression.

• Signal peptides: For surface display or secretion, appropriate signal sequences must be included.

• Codon optimization: Adapting the coding sequence to L. plantarum's codon usage can significantly improve expression levels.

• Selection markers: Appropriate antibiotic resistance genes (e.g., erythromycin resistance) should be included to enable selection of transformants.

Verification of successful protein expression should include both SDS-PAGE and Western blotting using protein-specific antibodies, as demonstrated in studies where recombinant proteins were confirmed through these methods .

How can researchers optimize mucosal immune responses induced by recombinant L. plantarum?

Optimizing mucosal immune responses induced by recombinant L. plantarum requires careful consideration of several key factors:

• Immunization protocol design:

  • Multiple booster immunizations significantly enhance immune responses. Studies have shown that three booster immunizations with recombinant L. plantarum resulted in peak antibody levels on the 35th day post-immunization .

  • Route of administration is critical—oral administration effectively targets gut-associated lymphoid tissue (GALT), while intranasal delivery targets nasal-associated lymphoid tissue (NALT).

• Adjuvant selection:

  • L. plantarum itself functions as an excellent mucosal adjuvant, enhancing both local and systemic immune responses .

  • Co-expression of immunomodulatory molecules like cytokines can further enhance responses. For example, expression of IL-33 alongside target antigens has been shown to enhance immune responses .

• Antigen design considerations:

  • Surface display versus secretion: Surface-displayed antigens often induce stronger antibody responses due to improved interaction with immune cells.

  • Fusion proteins: Creating fusions with immunogenic carrier proteins or immunostimulatory molecules can enhance response magnitude.

• Assessment of immune responses should be comprehensive:

  • Measure both systemic (serum IgG, IgG1) and mucosal (secretory IgA in bile, intestinal lavage, and feces) antibody responses .

  • Evaluate cellular responses, including CD4+ T cell and IgA+ B cell populations in relevant tissues .

The immune response data from recombinant L. plantarum studies demonstrate the capability to induce robust mucosal protection, making it a promising platform for vaccine development against mucosal pathogens.

What methodological approaches are most effective for studying the impact of recombinant L. plantarum on gut microbiota?

Investigating the impact of recombinant L. plantarum on gut microbiota requires sophisticated methodological approaches to capture the complex ecological changes. Based on research findings, the following methodologies are particularly effective:

Research has shown that recombinant L. plantarum can significantly alter gut microbial structure, enhancing species diversity and modulating functional capabilities related to metabolism and immune regulation . These methodological approaches provide robust tools for characterizing these complex interactions.

How does recombinant L. plantarum modulate tight junction integrity in respiratory epithelium?

Recombinant L. plantarum has demonstrated significant effects on tight junction integrity in respiratory epithelium, particularly through the upregulation of key tight junction proteins. Research findings with Lactiplantibacillus plantarum IS-10506 in a mouse model of allergic airway inflammation provide important insights:

• Mechanism of action:

  • L. plantarum significantly enhances the expression of critical tight junction proteins, including claudin-18, occludin, and zonula occludens-1 (ZO-1) (p<0.05) .

  • These proteins are essential structural components of tight junctions that maintain epithelial barrier integrity in the respiratory tract.

  • The enhanced expression of these proteins contributes to fortifying the airway epithelial barrier, which constitutes the initial structural defense against inhaled allergens.

• Experimental evidence:

  • Immunohistochemistry analysis of bronchial epithelial tissue from treated mice revealed increased immunoreactive scores (IRS) for tight junction proteins compared to control groups .

  • The upregulation of tight junction proteins was observed in the context of an allergic airway inflammation model using Dermatophagoides pteryonissinus (Der p) challenge, indicating the probiotic's protective effect even under inflammatory conditions.

• Research implications:

  • These findings suggest that recombinant L. plantarum could potentially be developed for therapeutic applications in respiratory conditions characterized by compromised epithelial barrier function.

  • The ability to enhance tight junction integrity may represent a novel approach to treating or preventing allergic airway diseases by addressing a fundamental aspect of disease pathophysiology—barrier dysfunction.

This research demonstrates an important non-immunological mechanism through which L. plantarum may confer health benefits, highlighting its potential applications beyond traditional vaccine or immune-stimulatory approaches.

What antibody responses can be expected following immunization with recombinant L. plantarum expressing heterologous antigens?

Immunization with recombinant L. plantarum expressing heterologous antigens induces a robust antibody response profile that includes both mucosal and systemic components. Based on experimental data, the following patterns can be expected:

• Systemic antibody responses:

  • Significant increase in serum IgG levels, with peak titers typically observed after multiple immunizations .

  • Differential IgG subclass responses, with studies showing elevation of both IgG1 (associated with Th2 responses) and IgG2a (associated with Th1 responses), though often with IgG1 predominance .

  • IgG responses appear to be antigen-specific and significantly higher than those observed with control (non-recombinant) L. plantarum administration.

• Mucosal antibody responses:

  • Strong secretory IgA (sIgA) production in mucosal secretions, including:

    • Elevated sIgA in bile samples

    • Increased sIgA in duodenal-mucosal fluid

    • Enhanced sIgA in fecal samples

  • The sIgA response typically reaches its highest level after multiple immunizations, with one study showing peak levels on the 35th day following a third booster immunization .

• Response kinetics:

  • Initial antibody responses are detectable within 1-2 weeks after first immunization

  • Boosters significantly enhance both magnitude and duration of antibody responses

  • Sustained elevation of antibodies for several weeks following the final immunization

Representative antibody data from studies using L. plantarum expressing Der p1 peptide 111-139:

*ND = Not Detected

These antibody response patterns indicate that recombinant L. plantarum serves as an effective delivery system for inducing both mucosal and systemic immunity, with particular strength in generating mucosal antibody responses that are critical for protection against pathogens at mucosal surfaces.

How do recombinant L. plantarum strains influence T-cell responses and cellular immunity?

Recombinant L. plantarum strains exert significant and multifaceted effects on T-cell responses and cellular immunity, displaying both immunostimulatory and immunomodulatory properties depending on the experimental context:

• CD4+ T-cell modulation:

  • Research demonstrates that recombinant L. plantarum administration leads to enrichment of CD4+ T cells in gut-associated lymphoid tissues, suggesting enhanced cellular immune activation .

  • These strains can modulate the Th1/Th2 balance, with various studies showing capabilities to either enhance or suppress specific T-cell subsets depending on the expressed antigen and experimental model.

• Allergen-specific T-cell response modulation:

  • In house dust mite allergen models, recombinant L. plantarum expressing Der p 1 peptide 111-139 has been shown to inhibit allergen-specific T-cell responses, potentially through interference with antigen presentation or induction of regulatory mechanisms .

  • This inhibitory effect on allergen-specific T-cells represents a potential therapeutic avenue for allergic diseases.

• Regulatory T-cell induction:

  • Some recombinant L. plantarum strains may induce regulatory T-cells that help maintain immune homeostasis and prevent excessive inflammatory responses.

  • This effect appears to be strain-specific and dependent on the specific proteins being expressed.

• Cellular immunity coordination:

  • Beyond direct T-cell effects, recombinant L. plantarum influences cellular immunity by enhancing IgA+ B cell populations in gut-associated lymphoid tissues, creating a coordinated immune response .

  • The increased presence of IgA+ B cells works in concert with T-cell modulation to strengthen mucosal immunity.

• Cytokine response patterns:

  • Recombinant L. plantarum can influence cytokine production profiles, with some strains enhancing proinflammatory cytokines while others promote anti-inflammatory responses.

  • These cytokine patterns further shape the quality and magnitude of T-cell responses.

These findings highlight the sophisticated immunomodulatory capabilities of recombinant L. plantarum strains and suggest their potential applications in various contexts, from vaccine development to therapeutic interventions for immunological disorders.

What are the critical steps for successful transformation of L. plantarum with recombinant expression vectors?

Successful transformation of L. plantarum with recombinant expression vectors requires meticulous attention to several critical steps:

• Vector design and construction:

  • Selection of appropriate vectors compatible with L. plantarum, such as pMG36e or pLP503-based vectors .

  • Incorporation of suitable promoters that function efficiently in L. plantarum (e.g., the ldh promoter from L. casei for constitutive expression) .

  • Inclusion of appropriate selection markers, with erythromycin resistance (5 μg/ml) being commonly used .

  • Proper design of fusion proteins, ensuring correct reading frame and inclusion of necessary elements like surface-display motifs (e.g., pgsA) if surface expression is desired .

• Preparation of competent L. plantarum cells:

  • Growth of L. plantarum cultures to optimal density in MRS medium.

  • Washing steps to remove components that may interfere with transformation.

  • Carefully controlled preparation conditions to enhance competence without reducing viability.

• Transformation procedure:

  • Electroporation is the preferred method for transforming L. plantarum, as indicated in multiple studies .

  • Critical parameters include:

    • Voltage settings appropriate for L. plantarum

    • Proper cell density and DNA concentration ratios

    • Pre-chilling of cells and cuvettes

    • Immediate recovery in appropriate media following electric pulse

• Selection and verification of transformants:

  • Plating on selective media containing appropriate antibiotics (e.g., erythromycin at 5 μg/ml) .

  • Screening of colonies using PCR to confirm the presence of the insert.

  • Verification of recombinant protein expression through:

    • SDS-PAGE analysis

    • Western blotting using specific antibodies against the target protein

    • Flow cytometry for surface-displayed proteins

• Optimization of expression conditions:

  • Adjusting growth conditions (temperature, media composition) to maximize recombinant protein yield.

  • Determining optimal induction timing if using inducible promoters.

  • Monitoring protein stability and potential degradation.

Adherence to these critical steps is essential for achieving successful transformation and expression of heterologous proteins in L. plantarum, which forms the foundation for subsequent experimental applications.

How can researchers effectively analyze the impact of recombinant L. plantarum on gut microbial diversity?

Effective analysis of recombinant L. plantarum's impact on gut microbial diversity requires a comprehensive methodological approach combining multiple analytical techniques:

• Experimental design considerations:

  • Include appropriate control groups: untreated controls, groups receiving wild-type L. plantarum, and groups receiving the recombinant strain to isolate specific effects of the recombinant constructs .

  • Establish clear sampling timepoints to capture both acute and long-term effects on microbial communities.

  • Standardize sample collection procedures to minimize technical variability.

• Sample processing and sequencing:

  • Extract high-quality DNA using protocols optimized for fecal or intestinal samples.

  • Target the V3-V4 hypervariable regions of the 16S rRNA gene for bacterial community profiling.

  • Ensure adequate sequencing depth (typically >10,000 reads per sample) to capture less abundant community members.

  • Include appropriate negative controls for sequencing and extraction.

• Diversity analysis methods:

  • Alpha diversity metrics:

    • Shannon-Wiener index for assessing species diversity, which has been shown to increase significantly with recombinant L. plantarum treatment .

    • Simpson's index for evaluating community evenness.

    • Observed OTUs (Operational Taxonomic Units) for measuring species richness.

  • Beta diversity analyses:

    • Principal Coordinates Analysis (PCoA) to visualize differences in microbial community structure between treatment groups.

    • PERMANOVA testing to statistically assess differences in community composition.

• Taxonomic and functional analysis:

  • Identify specific bacterial taxa that change in abundance following recombinant L. plantarum administration.

  • Perform functional prediction analysis to assess potential metabolic pathway alterations.

  • Correlate taxonomic changes with immune parameters (e.g., IgA levels, T-cell populations) to establish mechanistic connections.

• Validation approaches:

  • qPCR targeting specific bacterial groups to validate sequencing results.

  • In vitro co-culture experiments to confirm direct interactions between recombinant L. plantarum and identified key bacterial species.

Research has demonstrated that recombinant L. plantarum can significantly alter the gut microbiota composition, enhancing bacterial diversity and modifying the functional capacity of the microbiome, particularly in pathways related to metabolism and immune regulation . These analytical approaches provide robust tools for characterizing these complex microbial community changes.

What are the best practices for evaluating the stability and functionality of recombinant proteins expressed in L. plantarum?

Evaluating the stability and functionality of recombinant proteins expressed in L. plantarum requires rigorous analytical approaches that assess both expression levels and biological activity:

• Expression stability assessment:

  • Time-course analysis of protein expression using Western blotting to monitor potential degradation or expression decline over multiple bacterial generations .

  • Evaluation of plasmid retention in the absence of selective pressure through growth on non-selective media followed by replica plating onto selective media.

  • Quantitative PCR to measure plasmid copy number and assess potential plasmid loss during extended cultivation.

• Protein localization confirmation:

  • Surface display verification through flow cytometry using protein-specific antibodies, as demonstrated in studies with recombinant L. plantarum expressing gp85 .

  • Fractionation studies separating membrane, cytoplasmic, and secreted fractions to confirm proper protein localization.

  • Immunofluorescence microscopy to visualize protein distribution on bacterial cells.

• Structural integrity analysis:

  • SDS-PAGE combined with Western blotting to confirm correct molecular weight and absence of truncated products .

  • Mass spectrometry to verify protein sequence and identify any post-translational modifications.

  • Circular dichroism spectroscopy to assess secondary structure elements for proteins where folding is critical for function.

• Functional activity assays:

  • Immunological function: ELISA-based assays to confirm antigen recognition by specific antibodies.

  • Enzymatic activity: Specific activity assays for enzyme-based recombinant proteins.

  • Binding assays: For proteins intended to interact with specific receptors or ligands.

• In vivo functionality assessment:

  • Animal studies measuring specific immune responses (antibody production, T-cell responses) to confirm biological activity of expressed antigens .

  • Verification of protective effects in challenge models, such as the ALV-J challenge model used to assess recombinant L. plantarum expressing gp85 .

  • Evaluation of physiological effects, such as changes in gut microbiota composition or tight junction protein expression .

Research has shown that properly expressed and stable recombinant proteins in L. plantarum can elicit significant biological responses, including enhanced antibody production, modulation of T-cell responses, and alteration of gut microbial communities. These comprehensive evaluation methods ensure that the expressed proteins maintain their intended functionality, which is critical for research applications and potential therapeutic development.

What are the most promising applications of recombinant L. plantarum in respiratory disease research?

Recombinant L. plantarum shows significant promise in respiratory disease research, with several emerging applications supported by current evidence:

• Epithelial barrier enhancement therapy:

  • Recent research demonstrates that L. plantarum IS-10506 significantly enhances tight junction protein expression (claudin-18, occludin, and ZO-1) in bronchial epithelium .

  • This barrier-strengthening effect could be leveraged to develop novel therapies for conditions characterized by compromised epithelial integrity, such as asthma, chronic obstructive pulmonary disease (COPD), and respiratory viral infections.

  • Future research could focus on engineering recombinant strains that express additional factors that specifically target and enhance respiratory epithelial barrier function.

• Mucosal vaccine delivery systems:

  • L. plantarum's ability to survive transit through the gastrointestinal tract and elicit both mucosal and systemic immune responses makes it an excellent candidate for developing mucosal vaccines against respiratory pathogens .

  • Recombinant strains could be engineered to express antigens from respiratory viruses or bacteria, potentially offering a non-invasive alternative to injectable vaccines.

  • The dual stimulation of IgA production at mucosal surfaces and systemic IgG responses could provide comprehensive protection against respiratory infections.

• Immunomodulatory applications:

  • The demonstrated ability of recombinant L. plantarum to modulate allergen-specific T-cell responses suggests potential applications in allergic respiratory conditions .

  • Engineering strains to express immunomodulatory molecules or allergen epitopes could lead to novel approaches for allergic rhinitis, allergic asthma, or other hypersensitivity disorders affecting the respiratory tract.

• Microbiome-respiratory axis research:

  • Growing evidence of gut-lung axis interconnections suggests that modulation of gut microbiota through recombinant L. plantarum could influence respiratory health .

  • Future research could explore how gut microbiome changes induced by recombinant L. plantarum translate to alterations in respiratory immune responses and susceptibility to pulmonary diseases.

These promising directions represent significant opportunities for advancing respiratory disease research using recombinant L. plantarum as both an investigative tool and potential therapeutic agent.

How might advances in genetic engineering techniques enhance the utility of L. plantarum as a research tool?

Advances in genetic engineering techniques are poised to substantially enhance the utility of L. plantarum as a research tool through several innovative approaches:

• CRISPR-Cas9 application in L. plantarum:

  • Implementation of CRISPR-Cas9 technology would enable precise genomic modifications, allowing researchers to:

    • Create clean deletion mutants without antibiotic resistance markers

    • Perform targeted gene insertions at specific genomic loci

    • Develop inducible gene expression systems with tighter regulation

  • This precision would overcome current limitations of traditional transformation methods that rely heavily on plasmid-based expression systems .

• Development of advanced expression control systems:

  • Creation of tunable promoter systems that allow fine control over expression levels of recombinant proteins.

  • Design of environmentally responsive promoters that activate only under specific conditions (e.g., in response to inflammation markers or at particular anatomical sites).

  • Implementation of orthogonal expression systems that enable simultaneous, independent control of multiple recombinant proteins.

• Surface display technology enhancements:

  • Current research has utilized surface display motifs such as pgsA , but further refinements could include:

    • Development of novel anchoring domains with improved stability and display efficiency

    • Creation of systems allowing controlled orientation of displayed proteins

    • Engineering of multivalent display platforms for presenting complex antigens or antigen combinations

• Stable chromosome integration strategies:

  • Moving beyond plasmid-based expression to develop tools for stable chromosomal integration of recombinant genes.

  • This approach would eliminate concerns about plasmid loss during in vivo applications and reduce the need for antibiotic selection.

  • Site-specific integration systems could target neutral genomic locations to minimize unintended effects on bacterial physiology.

• Biosensor development:

  • Engineering L. plantarum to function as biological sensors that respond to specific environmental stimuli by producing measurable outputs.

  • This would enable real-time monitoring of conditions in complex biological environments like the gastrointestinal or respiratory tracts.

  • Combining sensing and response capabilities could create "smart" probiotic systems that deliver therapeutic molecules only when and where needed.

These advances would significantly expand researchers' ability to utilize L. plantarum as a sophisticated research tool for investigating complex biological processes and developing novel therapeutic approaches across multiple disease areas.

What challenges remain in optimizing recombinant L. plantarum for immunological research applications?

Despite significant progress, several important challenges remain in optimizing recombinant L. plantarum for immunological research applications:

• Expression system limitations:

  • Current expression systems often face stability issues during long-term in vivo applications, with potential plasmid loss in the absence of selective pressure .

  • Protein expression levels may be inconsistent or decline over time, complicating dose-response studies and reducing reproducibility.

  • Development of stable chromosomal integration systems or improved plasmid stability mechanisms is needed to overcome these limitations.

• Immunological variability challenges:

  • Host-specific variations in immune responses to L. plantarum complicate translation between animal models and human applications.

  • Pre-existing immunity to lactobacilli in some subjects may alter responses to recombinant strains.

  • Individual differences in microbiome composition can influence colonization efficiency and subsequent immune modulation .

• Delivery optimization issues:

  • Optimal dosing regimens (frequency, quantity, duration) remain poorly defined for many applications.

  • Route-specific delivery challenges exist, with different formulation requirements for oral, intranasal, or other mucosal routes.

  • Survival of bacteria through gastrointestinal transit varies between strains and conditions, affecting reproducibility.

• Standardization and regulatory considerations:

  • Lack of standardized protocols for measuring colonization efficiency, protein expression levels, and immune responses complicates cross-study comparisons.

  • Regulatory uncertainties regarding genetically modified organisms in research and potential therapeutic applications present practical challenges.

  • Batch-to-batch variability in bacterial preparations can introduce experimental noise.

• Mechanistic understanding gaps:

  • The precise mechanisms by which recombinant L. plantarum modulates specific aspects of immunity remain incompletely understood.

  • The interplay between direct effects of expressed proteins and the inherent immunomodulatory properties of the bacterial vector requires further elucidation.

  • Understanding how gut microbiota changes translate to systemic or mucosal immune effects in distant sites (e.g., respiratory tract) remains challenging .

Addressing these challenges will require multidisciplinary approaches combining advances in genetic engineering, immunology, microbiology, and computational modeling to fully realize the potential of recombinant L. plantarum in immunological research applications.