Recombinant Lactobacillus plantarum Probable transaldolase (tal)

What is transaldolase (tal) and what is its role in Lactobacillus plantarum metabolism?

Transaldolase (Tal, lp_3539 in L. plantarum ATCC BAA-793/WCFS1) is a key enzyme in the non-oxidative phase of the pentose phosphate pathway (PPP). It catalyzes the reversible transfer of a three-carbon dihydroxyacetone unit from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, yielding erythrose 4-phosphate and fructose 6-phosphate . In L. plantarum metabolism, transaldolase serves as a rate-limiting enzyme in the non-oxidative part of the PPP, connecting this pathway with glycolysis .

Research shows that transaldolase regulation significantly affects NADPH levels, which are crucial for managing oxidative stress. Decreased TAL activity leads to increased NADPH levels, inhibiting the production of reactive oxygen species (ROS) and reducing cell damage under oxidative stress conditions . This regulatory role makes transaldolase an important component in L. plantarum's adaptive response to environmental stresses.

What are the established protocols for cloning and expressing the L. plantarum tal gene?

For cloning and expressing L. plantarum tal, researchers should consider the following evidence-based protocol:

• Vector selection:

  • pNZ8048 or pNZ8110 vectors have proven successful for gene expression in Lactobacillus species

  • Both high and low copy number origins of replication can be employed depending on desired expression levels

• Cloning strategy:

  • Amplify the tal gene using high-fidelity polymerase and primers containing appropriate restriction sites

  • For example, one study used primers with NcoI and XbaI restriction sites for cloning a related protein

  • Include epitope tags (e.g., His6-tag) for detection and purification purposes

• Expression optimization:

  • The optimal spacer between the Shine-Dalgarno sequence and the start codon in L. plantarum consists of 8 nucleotides

  • Elongation or shortening of this sequence gradually down-regulates gene expression

  • Strong promoters such as transcription elongation factor promoters from L. plantarum CD033 and L. buchneri CD034 can enhance expression

• Verification of expression:

  • Western blotting with anti-tag antibodies or specific antibodies against tal

  • Enzyme activity assays to confirm functional expression

Research has demonstrated that combining strong promoters with high copy number plasmids can increase expression levels approximately twofold compared to low copy number vectors .

How can researchers create surface-displayed transaldolase in recombinant L. plantarum?

Surface display of transaldolase in L. plantarum requires careful design of expression constructs and verification strategies:

• Fusion construct design:

  • Select an expression vector compatible with L. plantarum (e.g., pSIP409-pgsA')

  • Create a fusion between tal and a cell-wall anchoring domain like pgsA' (derived from Bacillus subtilis)

  • Include appropriate signal peptides to direct the protein to the cell surface

• Transformation and expression:

  • Transform the recombinant plasmid into L. plantarum using electroporation

  • Verify plasmid stability - studies show that properly constructed plasmids can be inherited stably in L. plantarum

• Surface display verification:

  • Immunofluorescence assay (IFA) to visualize surface-displayed proteins

  • Western blot analysis of cell wall fractions

  • Immunogold electron microscopy for definitive localization evidence

Similar approaches have been successfully used for surface display of other proteins in L. plantarum, including SARS-CoV-2 epitopes and Trichinella spiralis inorganic pyrophosphatase (TsPPase) . These studies confirm that protein functionality can be maintained while achieving efficient surface display.

What purification methods are most effective for isolating recombinant L. plantarum transaldolase?

Based on published methodologies, the following purification strategy is recommended for recombinant L. plantarum transaldolase:

• Affinity chromatography approach:

  • Design the expression construct with a His6-tag as described in studies of related proteins

  • After cell lysis, apply the clarified lysate to nickel or cobalt affinity resin

  • Elute purified protein using an imidazole gradient (20-250 mM)

• Sequential purification strategy:

  • Initial capture: Affinity chromatography using engineered tags

  • Intermediate purification: Ion exchange chromatography based on theoretical pI

  • Polishing step: Size exclusion chromatography (e.g., Superdex 75)

• Quality assessment:

  • SDS-PAGE analysis to confirm purity (>85% purity is achievable)

  • Western blotting to verify protein identity

  • Activity assays to ensure functional integrity

For surface-displayed tal, specialized extraction methods may be required:

• Enzymatic treatment with cell wall hydrolases

• Mild detergent extraction of cell wall proteins

• Careful optimization of extraction conditions to maintain protein structure and function

The choice between E. coli, yeast, baculovirus, or mammalian expression systems should be based on the specific research objectives, as each system offers different advantages for protein folding and post-translational modifications .

What evidence supports the role of transaldolase in adhesion to intestinal mucin?

While direct evidence for L. plantarum transaldolase binding to mucin is limited, compelling data from related bacteria, particularly Bifidobacterium bifidum, suggests this function:

• Identification as a mucin-binding protein in B. bifidum:

  • B. bifidum transaldolase was isolated almost exclusively from the mucin layer, indicating high binding affinity

  • Mass spectrometry analysis identified this protein as transaldolase (Tal, BBPR_1029)

  • Surface localization was confirmed by immunogold electron microscopy

• Experimental validation using recombinant expression:

  • L. lactis strains expressing B. bifidum Tal displayed mucin binding levels more than three times higher than control strains

  • Western blot analysis confirmed the protein's presence in subcellular locations

• Functional implications:

  • Researchers concluded: "These findings suggest that Tal, when exposed on the cell surface, could act as an important colonization factor favoring the establishment of B. bifidum in the intestinal tract"

  • Surface-exposed transaldolase represents an example of bacterial proteins with "moonlighting" functions beyond their primary metabolic roles

These findings suggest that L. plantarum transaldolase might similarly function in mucin adhesion when expressed on the cell surface, though specific studies with L. plantarum tal would be needed for definitive confirmation.

How does transaldolase expression in L. plantarum respond to oxidative stress conditions?

L. plantarum transaldolase expression is significantly modulated under oxidative stress conditions as part of a coordinated metabolic response:

• Downregulation under hydrogen peroxide challenge:

  • Proteomics analysis revealed that transaldolase (TAL) is downregulated when L. plantarum is exposed to H₂O₂

  • This downregulation appears to be part of an adaptive response to oxidative stress

• Metabolic consequences and advantages:

  • TAL acts as a rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway (PPP)

  • Decreased TAL activity leads to increased NADPH levels

  • Higher NADPH concentration inhibits ROS production and reduces cell damage

• Integration with broader stress response:

  • The response involves coordinated changes across multiple metabolic pathways:

    • Upregulation of glycolytic enzymes like PGK to increase ATP production

    • Downregulation of pyruvate oxidase to reduce endogenous H₂O₂ production

    • Modifications to TCA cycle components to maintain cellular homeostasis

• Growth implications:

  • L. plantarum growth slows significantly with increasing H₂O₂ concentration

  • This growth reduction correlates with metabolic adaptations, including TAL downregulation

The precise regulation mechanisms controlling tal expression under oxidative stress remain to be fully elucidated, but the current evidence suggests that TAL downregulation plays an important protective role in L. plantarum's antioxidant defense system.

Can L. plantarum transaldolase be used in mucosal vaccine delivery?

While transaldolase itself hasn't been specifically studied as a vaccine delivery component, research on L. plantarum as a vaccine vector provides insights into how transaldolase might be incorporated:

• L. plantarum as an effective mucosal vaccine vector:

  • Multiple studies demonstrate successful use of L. plantarum for surface display of antigens

  • Examples include SARS-CoV-2 epitopes, ASFV p14.5 protein, and T. spiralis inorganic pyrophosphatase

• Immune responses generated by L. plantarum-based vaccines:

  • Humoral immunity: Increased specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA)

  • Cellular immunity: Enhanced CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in spleen and mesenteric lymph nodes

  • Cytokine production: Elevated levels of IFN-γ and IL-4 from immune tissues

• Potential advantages of transaldolase-based systems:

  • If transaldolase functions as a mucin-binding protein in L. plantarum (as in B. bifidum), it could enhance adherence to intestinal mucosa

  • This improved adherence might increase antigen delivery efficiency

  • A dual-function system (adhesin + antigen carrier) could potentially enhance immune responses

• Experimental support from related research:

  • Oral vaccination with recombinant L. plantarum expressing TsPPase induced protective immunity against T. spiralis infection:

    • 67.18% reduction of intestinal infective larvae

    • 54.78% reduction of adult worms

    • 51.91% reduction of muscle larvae

A transaldolase-based vaccine delivery system would need to be carefully designed to ensure both mucin-binding capability and effective antigen presentation are preserved in the fusion construct.

What are the "moonlighting" functions of transaldolase in L. plantarum beyond its metabolic role?

Transaldolase exemplifies bacterial proteins that perform multiple unrelated functions, known as "moonlighting" proteins. While specific moonlighting functions of L. plantarum transaldolase need further investigation, research from related bacteria suggests several potential additional roles:

• Surface adhesion properties:

  • In B. bifidum, transaldolase functions as a mucin-binding protein despite lacking a signal peptide

  • This adhesion capability likely contributes to colonization of the intestinal tract

  • The mechanism enabling surface localization remains to be fully elucidated

• Comparison with other moonlighting metabolic enzymes:

  • Research shows that "many enzymes of carbon catabolism, either from prokaryotic or eukaryotic cells, are often cell wall associated and are able to perform 'moonlighting' functions"

  • Examples include surface-localized GAPDH acting as a mucin binding protein in several bacteria

  • α-Enolase functions as a plasminogen receptor in some bacteria, facilitating tissue invasion via extracellular matrix degradation

• Potential host interaction roles:

  • Surface-exposed metabolic enzymes can participate in host-microbe interactions

  • This may include interaction with host immune components or extracellular matrix proteins

Understanding these moonlighting functions is significant because they may contribute to probiotic properties and host colonization capabilities of L. plantarum, potentially explaining some of its beneficial effects observed in vivo.

How does the regulation of tal gene expression differ in vivo versus in vitro conditions?

The regulation of gene expression in L. plantarum undergoes substantial changes when comparing laboratory versus gastrointestinal tract conditions, which likely affects tal expression:

• In vivo gene expression patterns:

  • Resolvase-based in vivo expression technology (R-IVET) identified 72 L. plantarum genes whose expression was induced during GI tract passage compared to laboratory media

  • These included genes involved in sugar metabolism, nutrient acquisition, and stress responses

• Nutrient availability as a key regulatory factor:

  • Nine genes encoding sugar-related functions were specifically induced in vivo

  • Limited availability of amino acids, nucleotides, cofactors, and vitamins in the GI tract induced genes involved in their acquisition and synthesis

  • As transaldolase plays a role in carbohydrate metabolism, its regulation might be similarly affected by these environmental differences

• Stress response regulation:

  • The harsh conditions of the GI tract induced stress-related genes

  • Given that transaldolase expression is affected by oxidative stress in vitro , its regulation may be part of the in vivo stress response

• Methodological approaches for studying in vivo expression:

  • Research has employed perfusion of intestinal segments with L. plantarum followed by RNA analysis using DNA microarrays

  • Recovery of bacteria from subjects with ileostomas after consumption of L. plantarum provides valuable in vivo samples

  • Quantitative RT-PCR can assess expression of specific genes like tal in these recovered bacteria

While tal-specific regulation differences between in vivo and in vitro conditions require further investigation, the general pattern suggests that metabolic genes like tal likely show distinct expression patterns in response to the unique challenges of the GI environment.

What is the impact of modifying tal expression levels on L. plantarum's probiotic properties?

Modifying transaldolase expression in L. plantarum could potentially affect several aspects of its probiotic functionality, though direct evidence is limited:

• Oxidative stress resistance:

  • Research demonstrates that TAL downregulation helps L. plantarum cope with oxidative stress by increasing NADPH levels

  • Modifying tal expression could therefore alter the bacterium's ability to survive oxidative stress in the gut

  • This stress resistance is critical for probiotic efficacy and persistence

• Adhesion capabilities:

  • If L. plantarum tal functions similarly to B. bifidum transaldolase as a mucin-binding protein , altering its expression might affect:

    • Adherence to intestinal mucosa

    • Colonization persistence

    • Competitive exclusion of pathogens

• Metabolic adaptability:

  • Transaldolase's role in the pentose phosphate pathway affects carbon flux and NADPH production

  • Modified expression could impact L. plantarum's ability to utilize different carbon sources in the variable gut environment

  • This metabolic flexibility influences survival in the competitive gut ecosystem

• Immune interaction potential:

  • Surface-exposed proteins in probiotics can interact with host immune cells

  • If tal is expressed on the L. plantarum surface, expression modifications might alter immune-modulating properties

  • Studies with other surface-displayed proteins in L. plantarum show they can induce both humoral and cellular immune responses

A systematic investigation involving tal expression variants and comprehensive phenotypic characterization would be needed to fully understand these potential impacts on probiotic functionality.

What are common challenges in achieving stable expression of recombinant transaldolase in L. plantarum?

Researchers working with recombinant L. plantarum face several technical challenges that require careful optimization:

• Plasmid stability issues:

  • Expression vectors can be lost during continuous cultivation without selection pressure

  • Studies evaluate whether recombinant plasmids are "inherited stably in bacteria"

  • Solutions include:

    • Using integrative vectors rather than replicative plasmids

    • Employing food-grade selection markers for continuous selection

    • Optimizing culture conditions to minimize plasmid loss

• Expression level optimization:

  • Research demonstrates that expression levels are affected by:

    • Promoter strength (various constitutive promoters can yield different expression levels)

    • Plasmid copy number (high copy number origins increased expression twofold)

    • Spacing between Shine-Dalgarno sequence and start codon (optimal: 8 nucleotides)

• Surface display challenges:

  • For surface-displayed transaldolase:

    • Ensuring efficient translocation across the cell membrane

    • Proper anchoring to the cell wall while maintaining protein folding

    • Verification requires specialized techniques like immunofluorescence or immunogold electron microscopy

• Metabolic considerations:

  • Overexpression of transaldolase may disrupt metabolic balance

  • As TAL influences the pentose phosphate pathway, its overexpression could affect:

    • Carbon flux distribution

    • NADPH production and oxidative stress response

    • Cell growth and viability

Systematic optimization of these parameters, combined with appropriate stability testing and functional validation, is essential for successful recombinant transaldolase expression in L. plantarum.

How can researchers optimize codon usage for improved tal expression in recombinant systems?

Codon optimization for enhanced tal expression in L. plantarum requires a methodical approach:

• Analysis of L. plantarum codon preferences:

  • Examine codon usage tables for L. plantarum to identify preferred codons for each amino acid

  • Focus particularly on highly expressed genes for optimal codon selection

  • Calculate the Codon Adaptation Index (CAI) for the native tal sequence to assess optimization potential

• Key optimization parameters:

  • Replace rare codons with preferred synonymous codons

  • Pay special attention to the N-terminal region, which significantly impacts translation initiation

  • Maintain appropriate GC content consistent with L. plantarum genome

  • Eliminate potential RNA secondary structures that could impede translation

• L. plantarum-specific considerations:

  • Research has established that "the optimal spacer between the Shine-Dalgarno sequence and the start codon in L. plantarum consists of 8 nucleotides"

  • "Elongation as well as shortening this sequence gradually down-regulates gene expression"

  • Ensure this optimal spacing is preserved in any codon-optimized construct

• Expression validation:

  • Compare expression levels between native and codon-optimized tal sequences

  • Use quantitative methods such as Western blotting or enzyme activity assays

  • Consider testing multiple optimization strategies to determine the most effective approach

When combined with appropriate vector design and expression conditions, codon optimization can significantly enhance recombinant protein yields in L. plantarum expression systems.

What controls should be included when studying the immunomodulatory effects of L. plantarum transaldolase?

Robust experimental design for immunomodulatory studies of L. plantarum transaldolase requires comprehensive controls:

• Bacterial strain controls:

  • Empty vector control: L. plantarum harboring the same plasmid backbone without the tal gene

  • Wild-type L. plantarum without genetic modification

  • L. plantarum expressing an irrelevant protein of similar size

  Control Type	Purpose	Implementation
  Empty vector	Control for vector effects	L. plantarum with plasmid minus tal
  Wild-type	Baseline comparison	Unmodified L. plantarum strain
  Irrelevant protein	Control for protein expression	Express similar-sized non-immunomodulatory protein

• Protein-specific controls:

  • Purified recombinant tal protein (to distinguish effects of the protein from the bacterial carrier)

  • Denatured tal protein (to assess importance of protein conformation)

  • Mutated tal protein lacking enzymatic activity

• Experimental design controls:

  • Medium control: MRS broth used for bacterial growth

  • Time-course sampling to determine response kinetics

  • Dose-dependent responses with varying bacterial or protein concentrations

• Immune assessment parameters:

  • Comprehensive cytokine panel:

    • Pro-inflammatory: TNF-α, IL-6

    • Anti-inflammatory: IL-10

    • T cell-related: IFN-γ, IL-4

  • Both humoral and cellular immunity markers:

    • Antibody responses: IgG (including IgG1, IgG2a), secretory IgA

    • T cell populations: CD3⁺CD4⁺ and CD3⁺CD8⁺ cells

Studies with recombinant L. plantarum have shown that oral administration can induce significant changes in these immune parameters, with different adjuvants affecting the degree of T cell differentiation and cytokine production patterns .