Recombinant Lactobacillus plantarum Thymidylate synthase (thyA)

What is the biological containment system based on thyA mutation in L. plantarum?

The thyA gene encodes thymidylate synthase, an enzyme essential for DNA synthesis in L. plantarum. When this gene is inactivated through targeted mutation, the resulting thyA- mutants become dependent on external thymidine or thymine for growth and survival. This dependence creates a biological containment system where:

• The strain cannot survive in environments with limited thymidine/thymine concentrations

• Viability is significantly reduced after approximately four hours in thymidine-free media

• Growth and survival are restored when sufficient thymidine (approximately 10 μM) is supplied

This mechanism provides an additional layer of safety beyond physical containment for genetically modified L. plantarum, particularly when used as delivery vehicles in clinical applications. Live-dead population assays and scanning electron microscopy (SEM) analysis can confirm the dependence on external thymidine .

Why is L. plantarum chosen as a host for recombinant protein expression in therapeutic applications?

L. plantarum is frequently selected as a host organism for several scientific reasons:

• GRAS (Generally Recognized As Safe) status for human consumption

• Natural presence in the human intestinal microflora

• Ability to survive passage through the gastrointestinal tract

• Inherent adjuvant properties that enhance immune responses

• Capacity for genetic modification through established techniques

• Potential for mucosal delivery of therapeutics proteins

These characteristics make L. plantarum an excellent candidate for developing recombinant bacteria that can express and deliver therapeutic proteins directly to the intestinal mucosa, offering advantages over other mucosal delivery strategies for vaccines and therapeutic proteins .

What methods are used to construct thyA mutants in L. plantarum?

Construction of thyA mutants in L. plantarum typically employs the following methods:

• Mobile group II intron (Ll.LtrB) for targeted gene disruption

• Computer algorithm prediction of potential intron RNA binding sites in the thyA gene

• PCR modification of wild-type intron Ll.LtrB to create retargeted intron

• Transformation using vectors like pLpACD4C (8.6 kb)

• Selection of insertion locations (e.g., position 188|189s of thyA gene) based on lowest E-value (e.g., -0.134)

• Induction of retargeted intron expression using peptides (e.g., sppIP)

• PCR screening to confirm intron integration in the thyA gene

This process results in ThyA- mutants that require thymidine supplementation for growth, creating a biological containment system. Quantitative real-time PCR confirms successful transformation, with studies showing up to 94-fold increased expression of Ll.LtrB intron and 14-fold increased expression of ltrA gene in recombinant L. plantarum .

How can heterologous proteins be optimally expressed and secreted in recombinant L. plantarum?

Efficient expression and secretion of heterologous proteins in L. plantarum requires careful optimization of several parameters:

Expression Strategies:

• Constitutive promoters (e.g., P_ldhL) for continuous protein expression

• Inducible promoters for controlled expression

• Selection of appropriate signal peptides for protein secretion

Signal Peptide Selection:

• Homologous signal peptides like Lp_0373 and Lp_3050 can be evaluated for secretion efficiency

• Studies show Lp_0373 signal peptide typically achieves higher secretion efficiency than Lp_3050

• Secretion efficiency can reach approximately 25% (as shown with OxdC protein)

Expression Cassette Design:

• For secretory expression: promoter + signal peptide + gene of interest

• For non-secretory (intracellular) expression: promoter + gene of interest without signal peptide

Plasmid Stability Considerations:

• Regular administration of recombinant L. plantarum may be necessary as plasmid segregation analysis reveals 70-90% loss of erythromycin-based plasmids

• Alternative stabilization strategies include constructing mutants lacking essential genes (e.g., alr gene encoding alanine racemase) that can be complemented via the plasmid

These approaches have been successfully demonstrated in studies expressing OxdC, with the OxdC-secreting WCFS1OxdC strain capable of degrading 70% of extracellular oxalate, while the non-secreting NC8OxdC strain degraded 77% of oxalate intracellularly .

What immune responses are elicited by recombinant L. plantarum and how are they measured?

Recombinant L. plantarum elicits diverse immune responses that can be measured through multiple methodologies:

Humoral Immune Responses:

• Serum IgG levels measured by ELISA show significant increases following oral immunization with recombinant L. plantarum expressing heterologous antigens

• Secretory IgA (sIgA) in mucosal surfaces (bile, intestinal lavage, feces) can be quantified by ELISA

• Subclass analysis (e.g., IgG1, IgG2a) provides insights into Th1/Th2 balance of the immune response

Cellular Immune Responses:

• T-cell proliferation assayed by thymidine incorporation following in vitro stimulation with recombinant antigens

• Flow cytometry to quantify CD4+IFN-γ+ and CD8+IFN-γ+ T cells in mesenteric lymph nodes (MLNs) and spleen

• CFSE staining to measure proliferation of CD4+ and CD8+ T cells

• Examination of B cell activation in Peyer's patches, measuring percentages of B220+IgA+ cells

Tissue-Specific Responses:

• Immunofluorescence staining to measure IgA expression in different tissues (lungs, duodenum, jejunum, ileum)

• Changes in gut microbiota composition measured via 16S rRNA sequencing, with analysis of alpha diversity (Chao1 index, Shannon-Wiener index) and beta diversity

Studies have shown that recombinant L. plantarum can significantly increase specific antibody levels compared to control groups, with sustained responses detectable for 10+ weeks after primary immunization .

What strategies can enhance the genetic stability of recombinant L. plantarum for therapeutic applications?

Maintaining genetic stability of recombinant L. plantarum is critical for therapeutic applications. Several strategies can be employed:

Chromosomal Integration Techniques:

• Integration of expression cassettes into the thyA locus through homologous recombination

• Using mobile genetic elements like Ll.LtrB group II intron for site-specific integration

• Creation of double crossover events using targeting sequences flanking the gene of interest

• Recombinase-assisted crossovers to improve integration efficiency

Selection Systems:

• Thymidine auxotrophy (thyA mutants) requiring thymidine supplementation

• Complementation of essential gene deletions through plasmid-based expression

Plasmid Stabilization Methods:

• Construction of balanced-lethal systems where the plasmid complements a chromosomal deletion

• Development of food-grade selection systems without antibiotic resistance markers

• Integration of auxotrophic marker genes (e.g., alr encoding alanine racemase) into the plasmid

Stability Assessment:

• Plasmid segregation analysis under non-selective conditions

• PCR verification of integrated genes following multiple generations

• Protein expression analysis through western blotting over extended cultivation periods

• In vivo stability determination through recovery and analysis of bacteria from animal models

Studies have shown that chromosomal integration provides superior stability compared to plasmid-based expression, with maintenance of heterologous gene expression even in the absence of selective pressure .

How does the efficacy of recombinant L. plantarum compare in different disease models?

Recombinant L. plantarum has shown variable efficacy across different disease models, with performance depending on the specific therapeutic protein expressed and the target condition:

Calcium Oxalate Stone Disease:

• OxdC-expressing L. plantarum reduced urinary oxalate by 53% in vitro

• In rat models, recombinant WCFS1OxdC and NC8OxdC strains significantly reduced urinary parameters:

  Parameter	Group I (Control)	Group II (Lithiatic)	Group IV (WCFS1OxdC)	Group V (NC8OxdC)
  Urine pH (day 28)	7.25 ± 0.11	6.09 ± 0.07	6.90 ± 0.17	6.79 ± 0.13
  Uric acid (mg/24h)	0.17 ± 0.02	0.46 ± 0.02	0.12 ± 0.01	0.18 ± 0.02
  Creatinine (mg/24h)	1.77 ± 0.23	3.69 ± 0.30	2.52 ± 0.14	3.07 ± 0.61

• Microscopic examination showed high CaOx crystal scores (4+) in control groups versus no crystals or low scores (1+) in treatment groups

Allergic Disorders:

• Recombinant L. plantarum expressing T-cell epitopes from Der p 1 (house dust mite allergen) inhibited both IFN-γ and IL-5 production

• Non-specific inhibition of IFN-γ was observed with L. plantarum, but IL-5 inhibition was specific to the recombinant expressing Der p 1 peptide

• No significant effect on antibody responses was observed

Viral Infections:

• L. plantarum expressing influenza virus HA1 with dendritic cell-targeting peptide (DCpep) showed enhanced immune responses

• Recombinant L. plantarum expressing ALV-J gp85 protein significantly reduced viremia in chickens after viral challenge, with protection correlating with increased antibody titers

These varied outcomes suggest disease-specific optimization is necessary for maximum therapeutic efficacy .

What are the current limitations in using thyA-mutant L. plantarum for therapeutic delivery in clinical settings?

Despite promising results in preclinical studies, several limitations must be addressed before thyA-mutant L. plantarum can be widely applied in clinical settings:

Genetic Stability Challenges:

• Long-term stability of chromosomally integrated genes remains a concern

• Potential for horizontal gene transfer to gut microbiota must be minimized

• Reversion of thyA mutation could compromise biological containment

Dosing and Administration Considerations:

• Optimal dosing regimens remain undefined for different applications

• Plasmid segregation analysis reveals 70-90% loss of erythromycin-based plasmids, necessitating frequent administration

• Standardization of viable bacterial count in preparations is challenging

Variable Immune Responses:

• Inter-individual variability in immune responses to recombinant L. plantarum

• Potential for immunological tolerance with repeated administration

• Balancing immunogenicity of the bacterial vector versus the heterologous antigen

Regulatory and Safety Concerns:

• Demonstration of reliable biological containment under various environmental conditions

• Comprehensive safety assessment including potential for unexpected immune reactions

• Requirements for non-antibiotic selection markers acceptable for human use

Manufacturing and Quality Control:

• Development of consistent production methodologies

• Stability during storage and transport

• Validation of expression and functionality of the therapeutic protein

Researchers are addressing these limitations through improved genetic engineering techniques, novel selection systems, and comprehensive preclinical safety studies to advance thyA-mutant L. plantarum toward clinical applications .

Advanced Methodologies Section

What techniques are used to verify successful construction and function of recombinant L. plantarum strains?

Multiple complementary techniques are employed to verify the successful construction and proper function of recombinant L. plantarum strains:

Genetic Verification:

• PCR screening to confirm integration of heterologous genes or disruption of thyA

• Quantitative real-time PCR to measure expression levels of integrated genes

• DNA sequencing to verify the integrity of the insert and absence of mutations

• Plasmid stability assays to determine retention under non-selective conditions

Protein Expression Verification:

• Western blotting to confirm expression of the target protein (e.g., 44 kDa OxdC protein)

• Flow cytometry for surface-displayed proteins (e.g., pgsA-fused antigens)

• ELISA to quantify secreted proteins in culture supernatants

• N-terminal protein sequencing by Edman degradation to confirm correct processing

Functional Assessment:

• Enzyme activity assays (e.g., OxdC activity measured by substrate degradation)

• Growth curves in thymidine-supplemented versus thymidine-free media

• Live-dead population assays to confirm thymidine dependence

• Scanning electron microscopy (SEM) to analyze cellular morphology

In Vivo Functionality:

• Colonization studies to assess persistence in the gastrointestinal tract

• Immune response evaluation through antibody titers and T-cell assays

• Challenge studies with relevant pathogens or disease models

• Environmental survival assessment to confirm biological containment

These comprehensive verification methods ensure both the genetic integrity and functional properties of the recombinant strains before proceeding to more complex applications .

How can one optimize the immune response generated by recombinant L. plantarum vaccines?

Optimizing immune responses generated by recombinant L. plantarum vaccines involves multiple strategies targeting both the bacterial vector and the expressed antigen:

Antigen Design and Expression Optimization:

• Fusion with immunostimulatory molecules (e.g., dendritic cell-targeting peptide DCpep)

• Co-expression with adjuvants like IL-33 or CTA1-DD to enhance specific immune pathways

• Surface display versus secretion of antigens affects the type of immune response generated

• Expression level optimization through promoter selection and codon optimization

Vaccination Protocol Refinement:

• Determination of optimal dose through dose-response studies

• Prime-boost strategies with varying intervals between administrations

• Route of administration (oral, nasal, other mucosal surfaces)

• Co-administration with other adjuvants or delivery systems

Immune Response Monitoring and Analysis:

• Comprehensive measurement of both mucosal and systemic immune responses:

  Immune Parameter	Sample Type	Measurement Method
  IgG (total)	Serum	ELISA
  IgG subclasses (IgG1, IgG2a)	Serum	ELISA
  Secretory IgA	Bile, feces, intestinal lavage	ELISA
  CD4+IFN-γ+ T cells	MLNs, spleen	Flow cytometry
  CD8+IFN-γ+ T cells	MLNs, spleen	Flow cytometry
  T cell proliferation	Splenocytes	Thymidine incorporation
  B220+IgA+ B cells	Peyer's patches	Flow cytometry

• Tissue-specific responses through immunofluorescence staining