Comprehensive molecular profiling and functional retargeting of Helicobacter pylori outer membrane vesicles

Osteosarcoma is a primary malignant bone tumor for which innovative therapeutic strategies are currently under investigation. Bacterial outer membrane vesicles (OMVs) have emerged as promising nanocarriers owing to their ability to mediate intercellular transfer of proteins, lipids, and nucleic acids. Among Gram-negative bacteria, Helicobacter pylori represents an attractive model because of its compact genome, extensive regulation by small RNAs, and constitutive production of OMVs during gastric colonization. This thesis provides a comprehensive molecular and biophysical characterization of OMVs derived from the H. pylori G27 strain. Vesicle morphology and size distribution were assessed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and nano-flow cytometry (nanoFCM), confirming the production of heterogeneous nanosized vesicles. Molecular analyses demonstrated that OMVs contain DNA, diverse RNA species, and proteins. Transcriptomic profiling revealed selective enrichment of small non-coding RNAs and specific mRNA subsets, supporting the existence of regulated RNA sorting mechanisms. Proteomic analysis identified enrichment of outer membrane-associated proteins and virulence-related factors, indicating that OMV composition is dynamically regulated rather than resulting from random membrane shedding. To investigate the feasibility of targeted OMV delivery, a single-chain variable fragment (scFv) was genetically fused to the outer membrane adhesin BabA. Comparative proteomic analysis revealed a marked reduction of BabA in engineered OMVs, suggesting that the fusion protein impaired its incorporation into the outer membrane. Consequently, modified OMVs failed to show detectable uptake by U2OS cells. In contrast, fluorescently labelled OMVs were rapidly internalized by THP-1 monocytes and primary peripheral blood mononuclear cells, although fluorescence markedly decreased after prolonged incubation, suggesting rapid intracellular processing or vesicle degradation. Overall, this work advances the understanding of H. pylori OMV composition, RNA cargo selection, and the molecular constraints affecting OMV engineering, providing insights that may support the future development of OMV-based therapeutic platforms.