Recombinant Lactobacillus plantarum Cell cycle protein GpsB (gpsB)

Advanced Research Questions

• What strategies can enhance stability of GpsB-expressing recombinant L. plantarum under various environmental conditions?

Enhancing stability of recombinant L. plantarum expressing GpsB requires optimization at multiple levels:

Genetic stability:

• Integration into the chromosome rather than plasmid-based expression

• Use of selection markers that maintain pressure for retention

• Avoiding toxic expression levels through careful promoter selection

Protein stability:

• Fusion partners that enhance folding (e.g., thioredoxin)

• Directed evolution to select for stable GpsB variants

• Codon harmonization to match translational rhythm of L. plantarum

Environmental resistance strategies:
Research indicates recombinant proteins expressed in L. plantarum can remain stable under challenging conditions including high temperatures (50°C), acidic pH (as low as 1.5), and high salt concentrations . These properties make L. plantarum an excellent chassis for GpsB expression in applications requiring environmental robustness.

Methodological approach: Perform stability testing using accelerated storage conditions and stress challenges. Monitor protein expression and function over time using immunodetection and activity assays. Sequence confirmation should be performed after multiple passages to verify genetic stability.

• How can GpsB be engineered in L. plantarum to enhance its potential as a mucosal vaccine delivery platform?

Engineering GpsB in L. plantarum for vaccine applications requires strategic modifications:

GpsB fusion protein design:

• Selection of appropriate immunogenic epitopes to fuse with GpsB

• Optimization of linker sequences to maintain both GpsB function and epitope presentation

• Addition of dendritic cell-targeting peptides (DCpep) to enhance immune responses

Expression optimization:

• Balancing expression level to avoid metabolic burden

• Subcellular targeting (surface vs. secreted) depending on desired immune response

• Co-expression with immune-stimulating molecules

When dendritic cell-targeting peptides are incorporated into recombinant L. plantarum constructs, significantly enhanced immune responses are observed. Flow cytometry data shows that such constructs can increase CD4+IFN-γ+ T cells in mouse mesenteric lymph nodes (P<0.0001 compared to controls) and activate B220+IgA+ B cells in Peyer's patches (P<0.0001) .

Methodological workflow: Gene fusion construction followed by transformation into L. plantarum, expression verification, stability assessment in simulated gastrointestinal conditions, and finally, immune response evaluation in animal models through measurement of specific antibodies and T-cell responses.

• What experimental approaches can elucidate the GpsB interactome in L. plantarum?

Understanding the GpsB interaction network requires multifaceted approaches:

Based on findings in other bacteria, GpsB likely interacts with peptidoglycan synthases, division proteins, and shape determinants in L. plantarum. In B. subtilis, researchers discovered new GpsB-interacting partners by using a sequence motif identified from structural studies . This approach can be adapted for L. plantarum by identifying conserved motifs in putative interaction partners.

Another powerful approach is comparative interactomics—comparing GpsB interactions across different bacteria (B. subtilis, L. monocytogenes, S. pneumoniae) to identify conserved partners that are likely to also interact with GpsB in L. plantarum.

• How does modification of GpsB expression affect cell morphology and division in L. plantarum?

GpsB modification can significantly impact bacterial morphology and division, as demonstrated in multiple bacteria:

Overexpression effects:

• Potential disruption of normal protein complex formation

• Sequestration of interaction partners from their native locations

• Altered division site selection

Depletion/deletion effects:

• Based on studies in related bacteria, we would expect elongated cells with division defects

• In L. monocytogenes, gpsB deletion causes growth defects at 37°C and lethality at 42°C

• Altered peptidoglycan structure and increased susceptibility to cell wall-targeting antibiotics

Visualization methodology: Phase contrast microscopy for basic morphology assessment, followed by fluorescence microscopy of membrane and septum markers. For detailed analysis, transmission electron microscopy can reveal fine alterations in cell envelope structure. Time-lapse microscopy allows tracking of division dynamics in GpsB-modified strains.

Quantitative analysis should include cell length distribution, width measurements, and division frequency. Cell wall composition can be analyzed through HPLC of peptidoglycan fragments to detect structural changes resulting from altered GpsB function.

• What are the immunological implications of using recombinant L. plantarum expressing modified GpsB for vaccine delivery?

Recombinant L. plantarum strains can induce robust immune responses, making them promising vaccine vectors. When considering GpsB-modified strains, several immunological factors must be evaluated:

T-cell responses:

• Recombinant L. plantarum can significantly increase CD4+IFN-γ+ T cells in mesenteric lymph nodes and spleen

• Enhanced T-cell proliferation is observed after oral administration

• Both CD4+ and CD8+ T-cell populations can be activated, with particularly strong responses when dendritic cell-targeting peptides are included

B-cell activation:

• Significant increases in B220+IgA+ B cells in Peyer's patches following oral administration (P<0.0001 compared to controls)

• Sustained antibody production can be achieved, with highest responses typically peaking at week 4 post-immunization

The immunological profile can be optimized by:

• Selection of appropriate antigen fusion partners with GpsB

• Incorporation of adjuvant molecules or targeting peptides

• Optimization of dosing regimen for sustained immune responses

Methodological assessment requires comprehensive immune profiling, including flow cytometry analysis of T and B cell populations, cytokine measurements, antibody titer determination, and functional assays such as hemagglutination inhibition for influenza antigen constructs .