Hypervesiculation Meets Sec-Targeting: Enhancing Heterologous Protein Loading in Salmonella Typhi Outer Membrane...

Area of Science:

• Microbiology and Immunology
• Bacterial Outer Membrane Vesicles
• Biotechnology

Background:

• Salmonella enterica serovar Typhi (S. Typhi) produces outer membrane vesicles (OMVs) with underexplored biotechnological potential.
• OMVs are promising platforms for drug delivery and vaccine development.
• Understanding OMV biogenesis and cargo loading is crucial for optimizing their applications.

Purpose of the Study:

• To engineer S. Typhi mutants for enhanced OMV production and cargo loading of the fluorescent protein mCherry.
• To investigate the impact of hypervesiculation on OMV cargo composition and delivery to host cells.
• To evaluate the immunogenicity of engineered OMVs compared to wild-type OMVs in a murine model.

Main Methods:

• Genetic engineering of S. Typhi to create hypervesiculating mutants (ΔtolR and ΔdegS).
• Fusion of mCherry with an OmpA signal peptide for intravesicular packaging.
• Flow cytometry and confocal microscopy for cargo analysis and cellular delivery assessment.
• Immunization of mice with wild-type and engineered OMVs to assess immune responses.

Main Results:

• ΔtolR and ΔdegS mutants exhibited increased OMV production and altered cargo.
• Fusion with SP-mCherry enabled robust intravesicular packaging in all strains.
• ΔtolR mutants showed high mCherry loading and delivery to epithelial cells.
• Wild-type OMVs induced stronger anti-mCherry IgG responses than hypervesiculating mutants, despite lower cargo loading.

Conclusions:

• S. Typhi OMVs are versatile platforms for heterologous protein delivery, especially when engineered with targeting signals.
• Hypervesiculation enhances protein encapsulation and cellular delivery but may not equate to superior immunogenicity.
• Intrinsic OMV composition or adjuvant properties play a critical role in immune activation, suggesting a balance between cargo and immunostimulatory features is key for vaccine development.