Abstract

Salmonella enterica serovar Typhi (S. Typhi) produces outer membrane vesicles (OMVs) that remain comparatively underexplored as potential biotechnological tools. Here, we investigated how hypervesiculating S. Typhi mutants (?tolR and ?degS) can be engineered to load and deliver the fluorescent reporter protein mCherry, targeting human epithelial cells and the murine immune system. Deletions in tolR and degS led to distinct OMV phenotypes characterized by higher vesicle production and altered cargo composition, underscoring the impact of disrupted membrane integrity and envelope stress on OMV biogenesis. By fusing mCherry with the S. Typhi OmpA signal peptide (SPompA), we achieved robust and functionally intact intravesicular packaging in all strains. Flow cytometry and confocal microscopy revealed that the ?tolR mutant exhibited particularly high cargo loading in the OMV fraction and pronounced mCherry delivery to epithelial cells, highlighting the potential of hypervesiculation to enhance OMV-based protein transport. However, immunization studies in mice showed that wild-type OMVs, despite carrying less mCherry than their hypervesiculating counterparts, induced the strongest anti-mCherry IgG responses. These findings indicate that, at least under these conditions, antigen loading alone is not sufficient to fully determine immunogenicity. Instead, the intrinsic composition or adjuvant-like properties of OMVs play a pivotal role in driving robust immune activation. Our results establish S. Typhi OMVs, especially when genetically modified with a Sec-dependent targeting signal (SPompA), as versatile platforms for heterologous protein delivery. Although hypervesiculation facilitates increased protein encapsulation and delivery to epithelial cells, native OMVs appear to better preserve and/or present antigens for effective immunogenic responses in vivo. These insights set the stage for further optimization of S. Typhi OMVs in vaccine development and protein therapeutics, where balancing cargo loading with immunostimulatory features may be key to achieving maximal efficacy. © 2025 by the authors.