Recombinant Lactobacillus plantarum Peptide chain release factor 2 (prfB)

What is peptide chain release factor 2 (prfB) and what is its function in bacterial translation?

Peptide chain release factor 2 (prfB or RF2) is a soluble protein that plays a critical role in translation termination. In bacteria, RF2 specifically recognizes the UGA and UAA stop codons and catalyzes the hydrolysis of the peptidyl-tRNA bond, releasing the completed polypeptide chain from the ribosome. This process is essential for proper protein synthesis and cellular function .

Unlike release factor 1 (RF1), which recognizes UAG and UAA stop codons, RF2 has specificity for UGA and UAA codons. Both factors are essential for bacterial viability and demonstrate significant sequence homology, reflecting their evolutionary relationship and similar functions .

How is recombinant L. plantarum typically constructed for protein expression?

Construction of recombinant L. plantarum typically involves several key steps:

• Vector selection: Common vectors for L. plantarum include pWCF series, pMG36e, and pIB184 vectors containing constitutive promoters (like P23) or inducible promoters

• Gene optimization: Codon optimization based on L. plantarum's codon usage bias significantly improves expression efficiency

• Cloning strategies: Gibson assembly or restriction enzyme-based cloning methods are used to insert the target gene into the selected vector

• Transformation: Electroporation is the primary method for introducing recombinant plasmids into L. plantarum

• Selection: Transformants are selected using appropriate antibiotics (chloramphenicol, erythromycin, or rifampin)

• Expression verification: Protein expression is verified using Western blot, flow cytometry, or immunofluorescence analysis

What techniques are used to verify successful expression of recombinant proteins in L. plantarum?

Multiple complementary techniques are used to confirm successful expression:

Stability of recombinant proteins expressed in L. plantarum varies depending on the protein and environmental conditions. Research has demonstrated that:

• Temperature stability: Some recombinant proteins (like SARS-CoV-2 spike protein) maintain stability at temperatures up to 50°C for 20 minutes

• pH stability: Many recombinant proteins remain stable at acidic pH (as low as pH 1.5) for 30 minutes, which is crucial for oral delivery applications

• Bile salt resistance: Recombinant proteins can withstand bile salt concentrations up to 0.5%, with some showing increased stability at 0.2% bile salt

• Long-term expression: Stability of expression can be maintained for multiple weeks, as demonstrated in studies measuring antibody responses over 10-week periods post-immunization

The exceptional stability of recombinant proteins in L. plantarum makes it particularly suitable for oral vaccine development, as proteins need to withstand the harsh gastrointestinal environment .

What are the challenges in expressing prfB in L. plantarum and how can they be overcome?

Expressing functional prfB in L. plantarum presents several challenges:

• Autogenous regulation: Like in E. coli, prfB may undergo autogenous regulation where high levels of functional RF2 can inhibit its own synthesis through frameshift suppression mechanisms . This natural regulation can limit overexpression attempts.

• Codon optimization requirements: The different codon usage preferences between the source organism and L. plantarum can significantly impact expression efficiency. Studies show that codon optimization according to L. plantarum's bias can dramatically improve protein expression levels .

• Protein toxicity concerns: Overexpression of translation-related proteins like RF2 may disrupt normal translation termination patterns and potentially be toxic to the host.

• Surface display challenges: If attempting surface display of prfB, fusion partners and appropriate anchoring domains must be carefully selected to ensure proper folding and functionality.

These challenges can be addressed through:

• Inducible expression systems: Using tightly regulated, inducible promoters like the SppIP-inducible system, which allows controlled expression

• Codon optimization: Implementing host-specific codon optimization to enhance translation efficiency

• Fusion strategies: Creating fusion proteins with well-characterized surface display anchors like the pgsA gene product

• Signal sequence selection: Optimizing signal peptides for efficient secretion or surface display

How does codon optimization affect the expression efficiency of heterologous genes in recombinant L. plantarum?

Codon optimization significantly impacts expression efficiency in L. plantarum:

Table 2: Effects of codon optimization on protein expression in L. plantarum

Research on SARS-CoV-2 spike protein expression in L. plantarum demonstrated that codon optimization according to L. plantarum's usage bias significantly enhanced expression efficiency, with flow cytometry showing a 37.5% positive rate compared to baseline levels .

The optimization process typically involves:

What regulatory elements can enhance the expression of recombinant proteins in L. plantarum?

Several regulatory elements can optimize recombinant protein expression in L. plantarum:

• Promoters:

  • Constitutive promoters: P23 from lactic acid bacteria provides strong, continuous expression

  • Inducible promoters: SppIP-inducible system allows controlled expression with 50 ng/mL SppIP induction

  • Phage-derived promoters: Recent research identified phage promoters with expression levels nearly 9-fold higher than previously reported strongest promoters in L. plantarum

• Signal sequences:

  • Native signal peptides from L. plantarum secreted proteins

  • Heterologous signal sequences optimized for L. plantarum

• Ribosome binding sites (RBS):

  • Optimization of RBS strength and spacing from start codon

• Terminators:

  • Strong transcription terminators prevent read-through and increase mRNA stability

• Repressor systems:

  • Phage-derived repressor systems can provide tight regulation with nearly 500-fold repression capability

The selection of appropriate regulatory elements should be guided by the specific requirements of the expression system and the characteristics of the target protein.

How can surface display techniques be applied to express recombinant proteins on the surface of L. plantarum?

Surface display in L. plantarum can be achieved through several approaches:

• pgsA-based display: The poly-γ-glutamic synthetase complex component (pgsA) has been successfully used to display proteins like ALV-J gp85 on L. plantarum surfaces .

• Fibronectin binding protein A (FnBPA)-based display: FnBPA from Staphylococcus aureus significantly improves adhesion and invasion capabilities of L. plantarum, enhancing its interaction with host cells approximately two-fold compared to control strains .

• Dendritic cell-targeting peptide (DCpep) fusion: DCpep fusion enhances targeting to dendritic cells in Peyer's patches, improving immune responses to displayed antigens like influenza virus HA1 .

Recombinant L. plantarum expressing heterologous antigens induces comprehensive immune responses:

• Cellular immunity:

  • Enhanced activation of dendritic cells (DCs) in Peyer's patches with increased expression of CD80, CD86, and MHC-II

  • Increased numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in spleen and mesenteric lymph nodes

  • Enhanced T cell proliferation capability in response to specific antigens

  • Increased IL-4+ and IL-17A+ T helper cells in splenocytes

• Humoral immunity:

  • Elevated levels of antigen-specific IgG, IgG1, and IgG2a in serum

  • High production of specific IgA antibodies in feces, bile, and intestinal lavage samples

• Mucosal immunity:

  • Increased B220+IgA+ cells in Peyer's patches

  • Enhanced levels of IgA in lungs and different intestinal segments

  • Protection against mucosal infection by pathogens

For example, recombinant L. plantarum expressing influenza HA1 with DCpep significantly enhanced DC activation markers (CD80, CD86, MHC-II) and increased IFN-γ production by CD4+ and CD8+ T cells compared to control groups (P < 0.01) .

How can in vivo expression technology (IVET) be used to study gene expression in L. plantarum?

Resolvase-based in vivo expression technology (R-IVET) has been successfully applied to study L. plantarum gene expression in the gastrointestinal tract:

• Methodology:

  • Generation of genomic library fused to promoterless genes (e.g., resolvase)

  • Integration of constructs into L. plantarum chromosome

  • Administration to mice followed by recovery from fecal samples

  • Identification of promoters activated in vivo

• Applications:

  • Identification of 72 L. plantarum genes induced during passage through the GI tract

  • Discovery of genes involved in sugar metabolism, amino acid acquisition, and stress responses

  • Characterization of niche-specific gene expression

• Key findings:

  • Nine genes encoding sugar-related functions (including ribose, cellobiose, sucrose transporters) are upregulated in vivo

  • Nine genes involved in amino acid and vitamin acquisition are induced, indicating limited availability in the GI tract

  • Four stress-related genes are activated, reflecting the harsh conditions L. plantarum encounters in vivo

This approach could be applied to study in vivo expression of recombinant prfB to understand its regulation and function under physiological conditions.

What is the role of prfB in autogenous regulation and how might this affect recombinant expression?

The prfB gene encoding RF2 demonstrates a unique autogenous regulation mechanism that has implications for recombinant expression:

• Frameshift-dependent regulation:

  • An in-frame UGA stop codon in the prfB coding region requires a +1 frameshift for complete translation

  • When RF2 levels are low, the frameshift occurs at a high rate (~50%), allowing expression

  • When RF2 levels are high, efficient termination at the UGA codon prevents full-length protein synthesis

• Implications for recombinant expression:

  • Deletion of the internal stop codon leads to overproduction of RF2 fusion proteins

  • Modifications to the frameshift site can disrupt regulation and potentially increase expression

  • Codon optimization must consider maintaining or modifying this regulatory mechanism

• Potential solutions:

  • Synthetic constructs without the internal stop codon for constitutive expression

  • Alternate promoters and regulatory elements to bypass autogenous control

  • Fusion strategies that preserve protein function while altering regulatory sequences

Understanding this regulatory mechanism is crucial when designing recombinant L. plantarum expressing prfB, as modifications may dramatically affect expression levels and potentially cell viability.

How does vancomycin sensitivity in L. plantarum relate to peptidoglycan precursor modifications?

L. plantarum demonstrates intrinsic vancomycin resistance due to its unique peptidoglycan precursor composition, which has implications for genetic modification approaches:

• Molecular basis of resistance:

  • L. plantarum produces peptidoglycan precursors ending in D-lactate (D-Lac) instead of D-alanine (D-Ala)

  • This modification significantly reduces vancomycin binding affinity

  • The process involves D-Ala-D-Lac ligase (Ddl Lp) and D-Lac dehydrogenase

• Experimental modifications:

  • Knockout of ldhL and ldhD genes (encoding L-Lac and D-Lac dehydrogenases) dramatically increases vancomycin sensitivity

  • Expression of heterologous D-Ala-D-Ala ligase (Ddl Lc) from L. lactis renders L. plantarum sensitive to vancomycin

  • Exogenous D-Lac can restore resistance in dehydrogenase mutants

• Applications in recombinant systems:

  • Vancomycin sensitivity can serve as a selectable marker for genetic modifications

  • The peptidoglycan pathway offers targets for expression regulation

  • Modified peptidoglycan alters immunomodulatory properties, with decreased D-Ala in teichoic acids significantly reducing pro-inflammatory cytokine induction

These findings suggest that modifications to peptidoglycan biosynthesis can have profound effects on both antibiotic resistance and immunological properties of recombinant L. plantarum.

What novel delivery systems can enhance the efficacy of recombinant L. plantarum vaccines?

Several innovative delivery approaches have been developed to enhance recombinant L. plantarum vaccine efficacy:

• Dendritic cell-targeting peptide (DCpep) fusion:

  • DCpep fusion significantly enhances DC activation in Peyer's patches

  • Increases CD4+/CD8+ T cell responses and IgA production in multiple tissues

  • Improves hemagglutination inhibition potency against influenza

• Invasive L. plantarum with FnBPA:

  • Surface display of FnBPA from S. aureus creates invasive L. plantarum

  • Improves adhesion and invasion by approximately 2-fold

  • Stimulates DCs and increases IL-6, IL-4, and IL-17A production

  • Enhances B220+ B cell production in mesenteric lymph nodes and Peyer's patches

• Food-grade oral vaccine formulations:

  • Recombinant L. plantarum remains stable at low pH (1.5) and elevated temperatures (50°C)

  • Withstands bile salt concentrations up to 0.5%

  • Can be delivered through food matrices for enhanced stability and palatability

• Bactofection approaches:

  • Recombinant L. plantarum can deliver DNA to host cells (bactofection)

  • Has potential applications in autoimmune diseases like rheumatoid arthritis by delivering FOXP3 to restore regulatory T-cell functionality

These delivery strategies can be adapted to optimize recombinant L. plantarum expressing prfB for specific applications in vaccination or therapeutic protein delivery.

How can quorum sensing mechanisms in L. plantarum be exploited for recombinant protein expression?

L. plantarum possesses sophisticated quorum sensing (QS) systems that can be harnessed for recombinant protein expression:

• Dual-trigger quorum sensing system:

  • L. plantarum has a unique QS system triggered by both auto-inducing peptide (PlnA1) and acetate

  • PlnA1 activates QS during logarithmic growth phase via binding to histidine kinase PlnB1

  • Acetate independently activates QS during stationary phase by binding to a different region of PlnB1

• Molecular interactions:

  • PlnA1 binds to hydrophobic region Phe-Ala-Ser-Gln-Phe of extracytoplasmic loop 2 of PlnB1

  • Acetate binds to positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1

  • Side chain of Phe143 determines specificity and affinity for acetate

• Applications for recombinant expression:

  • Development of inducible expression systems based on PlnA1 peptide or acetate concentration

  • Creation of growth phase-specific expression systems (log phase via PlnA1, stationary phase via acetate)

  • Fine-tuning expression by modifying the histidine kinase binding regions

These QS mechanisms offer promising approaches for creating sophisticated, environmentally responsive expression systems for recombinant proteins, including prfB, in L. plantarum.

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

Optimization of expression conditions is crucial for maximizing recombinant protein yield in L. plantarum:

A comprehensive experimental design for evaluating immunogenicity should include:

• Animal models:

  • Mice are commonly used (BALB/c, C57BL/6)

  • Specific models for target diseases (e.g., SCID mice with human RA synovium for rheumatoid arthritis studies)

• Immunization schedule:

  • Primary immunization followed by 2-3 booster doses at 2-week intervals

  • Oral administration of 10^9-10^10 CFU per dose

• Sample collection timeline:

  • Serum: Pre-immunization, 2 weeks post-primary, 2 weeks post-booster, and extended timepoints (up to 10 weeks)

  • Fecal samples: Similar timeline as serum for mucosal IgA analysis

  • Tissue samples: Collection at experimental endpoint from:

    • Peyer's patches

    • Mesenteric lymph nodes

    • Spleen

    • Intestinal segments

    • Other relevant tissues (lungs, etc.)

• Immune response analysis:

  • Humoral immunity:

    • ELISA for antigen-specific IgG, IgG subtypes, and IgA in serum and mucosal secretions

    • Functional assays (e.g., hemagglutination inhibition for influenza antigens)

  • Cellular immunity:

    • Flow cytometry for:

      • DC activation markers (CD80, CD86, MHC-II)

      • T cell populations (CD4+IFN-γ+, CD8+IFN-γ+, Th subtypes)

      • B cell populations (B220+IgA+)

    • T cell proliferation assays with specific antigen stimulation

    • Cytokine measurement (IFN-γ, IL-4, IL-6, IL-17A) by ELISA and qRT-PCR

  • Mucosal immunity:

    • Immunofluorescence staining for IgA in tissue sections (lungs, intestinal segments)

    • Analysis of lymphoid tissues associated with mucosa

• Challenge studies (where applicable):

  • Pathogen challenge to assess protection

  • Monitoring of clinical parameters, pathogen load, and survival

This comprehensive approach provides a holistic assessment of the immune responses induced by recombinant L. plantarum vaccines.

What are common challenges in recombinant L. plantarum research and how can they be addressed?

Researchers working with recombinant L. plantarum commonly encounter several challenges:

Rigorous quality control is essential for ensuring reproducible results with recombinant L. plantarum:

• Genetic stability assessment:

  • PCR verification of the recombinant construct after multiple passages

  • Sequencing to confirm absence of mutations

  • Restriction enzyme analysis of extracted plasmids

• Expression consistency:

  • Western blot analysis to verify consistent protein expression levels

  • Flow cytometry to quantify the percentage of expressing cells (for surface-displayed proteins)

  • ELISA to measure secreted protein levels

• Functional verification:

  • Activity assays specific to the expressed protein

  • Binding assays for adhesins or receptor-targeting proteins

  • Immunological assays for antigenic proteins

• Viability and growth characteristics:

  • Growth curve analysis to ensure consistent growth patterns

  • CFU counts to verify viable cell numbers

  • Acid and bile resistance testing for oral application candidates

• Purity assessment:

  • Gram staining and microscopy

  • Species-specific PCR

  • Selective plating to ensure absence of contamination

• Storage stability:

  • Protein expression stability after freeze-thaw cycles

  • Long-term storage evaluation at different temperatures

  • Accelerated stability testing under stress conditions

Implementation of these quality control measures ensures the reliability and reproducibility of results obtained with recombinant L. plantarum strains.