Recombinant Lactobacillus plantarum Probable inorganic polyphosphate/ATP-NAD kinase (ppnK)

What is Lactobacillus plantarum and how has its classification changed in recent years?

Lactobacillus plantarum is a gram-positive probiotic bacterium naturally found in the human gut and mouth, as well as in fermented foods. In April 2020, taxonomic reclassification moved this species from the Lactobacillus genus to the Lactiplantibacillus genus, making its current scientific name Lactiplantibacillus plantarum. Many scientific publications and product labels still use the previous nomenclature .

The bacterium serves as an important probiotic that helps break down food, absorb nutrients, and compete against pathogenic organisms. L. plantarum has been extensively studied as a recombinant expression system due to its GRAS (Generally Recognized As Safe) status and ability to survive gastrointestinal transit .

What is inorganic polyphosphate/ATP-NAD kinase (ppnK) and what is its function in bacterial metabolism?

Inorganic polyphosphate/ATP-NAD kinase (ppnK) is an enzyme that catalyzes the phosphorylation of NAD to NADP using either ATP or inorganic polyphosphate as a phosphoryl donor. The enzyme plays a critical role in regulating the NAD/NADP ratio, which affects numerous metabolic pathways including redox reactions and energy production.

The dual-substrate specificity of ppnK is particularly significant, as demonstrated in studies of similar enzymes from other bacterial species such as Micrococcus flavus. Analysis of the primary structure of ppnK has revealed candidate amino acid residues, primarily charged ones, that may be related to inorganic polyphosphate utilization .

What are the structural characteristics of ppnK enzymes in bacteria?

Structural analysis of inorganic polyphosphate/ATP-NAD kinase from Micrococcus flavus has revealed several important features that may be conserved in L. plantarum ppnK:

• The enzyme typically contains a core catalytic domain responsible for phosphoryl transfer

• Some ppnK variants feature a protruding C-terminal polypeptide that may be dispensable for kinase activities

• The entire primary structure shows homology with the ATP synthase β chain, suggesting evolutionary relationships between these energy-related enzymes

• The alignment of ppnK sequences across bacterial species reveals conserved charged amino acid residues likely involved in substrate binding and catalysis

What are effective strategies for constructing recombinant L. plantarum expressing heterologous proteins like ppnK?

Based on established protocols for recombinant L. plantarum construction, researchers can follow these methodological steps:

• Gene selection and optimization: The ppnK gene should be codon-optimized for expression in L. plantarum

• Vector selection: Vectors such as pWCF have been successfully used for heterologous protein expression in L. plantarum

• Cloning strategy: The target gene can be amplified by PCR using primers containing appropriate restriction sites (e.g., XbaI and HindIII)

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

• Selection and verification: Transformants should be selected using appropriate antibiotics and verified through restriction enzyme digestion, PCR, and immunoblotting

Research has demonstrated successful construction of recombinant L. plantarum expressing viral antigens using similar approaches, which can be adapted for ppnK expression .

How can researchers verify successful expression of ppnK in recombinant L. plantarum?

Verification of ppnK expression requires multiple complementary techniques:

• Protein detection:

  • Western blotting using specific antibodies against ppnK or epitope tags

  • Mass spectrometry to confirm protein identity

  • Enzyme activity assays measuring NAD kinase function

• Functional verification:

  • Measuring changes in NAD/NADP ratios in the recombinant strain

  • Assessing polyphosphate utilization capacity

  • Evaluating changes in stress resistance profiles

In previous studies with recombinant L. plantarum, researchers have successfully employed immunoblotting to detect expressed proteins using specific antibodies. The recombinant protein can be detected following cell sonication or freeze-thaw cycles to release intracellular proteins .

How does recombinant L. plantarum activate immune responses in mucosal tissues?

Research has demonstrated that recombinant L. plantarum can effectively stimulate immune responses in mucosal tissues through several mechanisms:

• Activation of dendritic cells (DCs) in Peyer's patches, as evidenced by increased expression of activation markers CD80, CD86, and MHC-II on the surface of DCs

• Induction of CD4+IFN-γ+ and CD8+IFN-γ+ T cells in the spleen and mesenteric lymph nodes, indicating activation of cellular immunity

• Promotion of B220+IgA+ cells in Peyer's patches, contributing to mucosal antibody production

• Stimulation of IgA production in the lungs and different intestinal segments (duodenum, jejunum, and ileum)

These immune-activating properties suggest that recombinant L. plantarum expressing ppnK could potentially serve as an effective delivery system for therapeutic proteins or vaccine antigens, with the metabolic advantages conferred by ppnK potentially enhancing these immunological effects.

What techniques can be used to evaluate immune responses induced by recombinant L. plantarum?

Based on established immunological research methods, several techniques can be employed to evaluate immune responses:

• Flow cytometry analysis:

  • Quantification of activated dendritic cells (CD11c+CD80+, CD11c+CD86+, CD11c+MHC-II+)

  • Enumeration of T cell subsets (CD4+IFN-γ+, CD8+IFN-γ+)

  • Assessment of B cell populations (B220+IgA+)

• ELISA assays:

  • Measurement of specific antibodies (IgG, IgG1, IgG2a, IgA) in serum and mucosal secretions

  • Quantification of cytokine production

• Immunofluorescence staining:

  • Visualization of IgA-producing cells in tissue sections

  • Assessment of lymphoid tissue architecture and cellular distribution

• Functional assays:

  • T cell proliferation assays

  • Hemagglutination inhibition (HI) assays to evaluate functional antibody responses

How might overexpression of ppnK affect the metabolic capabilities of L. plantarum?

Overexpression of ppnK in L. plantarum may induce several metabolic changes:

• Altered NAD/NADP ratio: Increased ppnK activity would likely enhance NADP production, potentially shifting metabolism toward anabolic pathways that require NADPH as a cofactor.

• Enhanced polyphosphate utilization: Improved ability to use polyphosphate as a phosphoryl donor could provide metabolic advantages under conditions where ATP is limited.

• Redox balance modulation: Changes in NAD/NADP ratio would affect cellular redox state, potentially influencing fermentation pathways and end-product profiles.

• Stress response enhancement: Higher NADPH availability may improve antioxidant capacity through systems like glutathione reductase and thioredoxin reductase, potentially conferring resistance to oxidative stress.

Experimental approaches to investigate these effects should include comparative metabolomics, growth studies under various stress conditions, and analysis of fermentation end-products.

What kinetic parameters should be assessed when studying ppnK enzyme activity with different substrates?

Comprehensive kinetic characterization of ppnK should include:

• Substrate affinity: Determination of Km values for:

  • NAD+ as the phosphoryl acceptor

  • ATP as the phosphoryl donor

  • Polyphosphate as an alternative phosphoryl donor

• Catalytic efficiency: Calculation of kcat and kcat/Km for each substrate combination to understand substrate preference.

• Influence of polyphosphate chain length: Assessment of how polyphosphate chain length affects enzyme kinetics, as longer chains may have different binding properties.

• Reaction mechanism: Investigation of ordered versus random binding mechanisms and potential allosteric effects.

• pH and temperature optima: Determination of optimal environmental conditions for enzyme activity.

Based on studies of similar enzymes, the table below illustrates hypothetical kinetic parameters that might be expected:

What common challenges might researchers encounter when expressing ppnK in L. plantarum?

Several technical challenges may arise when expressing ppnK in L. plantarum:

• Expression level optimization:

  • Low expression due to poor codon usage or inefficient promoters

  • Potential toxicity from overexpression

  • Protein instability or degradation

• Enzyme activity verification:

  • Distinguishing recombinant ppnK activity from native NAD kinase activity

  • Ensuring proper folding and post-translational modifications

  • Developing specific assays for polyphosphate-dependent activity

• Genetic stability:

  • Plasmid loss during prolonged cultivation

  • Mutational inactivation of the expressed gene

  • Metabolic burden affecting growth and survival

• Batch-to-batch variability:

  • Inconsistent expression levels between experiments

  • Variable enzyme activity under different growth conditions

  • Reproducibility challenges in complex assays

What strategies can researchers employ to optimize expression and stability of recombinant ppnK?

To address the challenges in ppnK expression, researchers can implement several optimization strategies:

• Genetic optimization:

  • Codon optimization for L. plantarum

  • Use of strong, constitutive promoters like PldH or inducible systems when appropriate

  • Inclusion of stabilizing sequences or fusion partners

  • Chromosomal integration for long-term stability

• Expression conditions optimization:

  • Screening different growth media compositions

  • Testing various induction parameters (timing, concentration)

  • Optimizing growth temperature and pH

  • Harvest timing optimization to capture peak enzyme activity

• Protein engineering approaches:

  • Addition of affinity tags for easier purification and detection

  • Fusion to stability-enhancing protein domains

  • Site-directed mutagenesis to improve stability without compromising activity

  • Directed evolution to select variants with improved expression

How might systems biology approaches enhance understanding of ppnK function in L. plantarum?

Systems biology offers comprehensive frameworks to understand the global effects of ppnK overexpression:

• Multi-omics integration:

  • Transcriptomics to identify gene expression changes resulting from altered NAD/NADP ratios

  • Proteomics to detect shifts in enzyme abundance across metabolic pathways

  • Metabolomics to map changes in metabolite flux and pool sizes

  • Fluxomics to quantify changes in metabolic pathway activities

• Mathematical modeling:

  • Genome-scale metabolic models incorporating ppnK activity

  • Kinetic models of NAD/NADP metabolism

  • Regulatory network reconstruction

  • Predictive models for optimizing expression and activity

• Comparative studies:

  • Analysis of ppnK function across different Lactobacillus species

  • Investigation of ppnK variants from diverse bacterial sources

  • Evolutionary analysis of ppnK adaptation in different ecological niches

What potential applications exist for recombinant L. plantarum expressing ppnK in vaccine development?

Based on successful applications of L. plantarum in vaccine delivery, recombinant strains expressing ppnK might offer several advantages:

• Enhanced survival and colonization:

  • If ppnK overexpression improves stress resistance, the strain may show better survival in gastrointestinal conditions

  • Improved metabolic capabilities might enhance persistence in mucosal tissues

• Immunological advantages:

  • Similar to findings with other recombinant L. plantarum, ppnK-expressing strains might effectively activate dendritic cells in Peyer's patches

  • Potential for inducing balanced Th1/Th2 responses, as evidenced by IgG1 and IgG2a antibody production

  • Ability to stimulate mucosal IgA production across multiple sites (intestine, lungs)

• Delivery capabilities:

  • Co-expression of ppnK with vaccine antigens might create metabolically optimized vaccine vectors

  • Potential for improved antigen production due to enhanced NADPH availability

  • Possible synergistic effects between ppnK-mediated metabolic changes and immunostimulatory properties

The successful expression of influenza virus antigen HA1 in L. plantarum and the resulting immune activation provides a methodological framework that could be adapted for future vaccine development using ppnK-expressing strains .