Deciphering Extracellular Vesicle Uptake Mechanisms in Giardia-host Interactions

INTRODUCTION: G. intestinalis is a flagellated protozoan responsible for giardiasis, one of the most common parasitic diseases in humans, primarily affecting the gastrointestinal tract. This parasitic infection is prevalent worldwide, with a notable incidence in underdeveloped regions. Giardiasis is recognized as a neglected disease by the World Health Organization due to its significant global burden of morbidity and the lack of adequate attention in terms of research and therapy development. The pathogenesis of the disease is multifactorial, and the mechanisms are not fully elucidated. However, the parasite's ability to adhere to and colonize the intestinal epithelium leads to gastrointestinal symptoms. Additionally, it has been recently revealed that this process is facilitated by the release of extracellular vesicles (EVs). These EVs, consisting of microvesicles and exosomes surrounded by a lipid bilayer, play a crucial role in intercellular communication by transporting essential biomolecules. OBJECTIVE: Understand the mechanism of EV internalization and communication during interaction for the development of novel therapeutic strategies targeting parasite-host interactions. MATERIALS AND METHODS: We defined three types of EVs during parasite-host cell interaction, Giardia-originated EVs (gEV), host cell-released EVs (hEV), and parasite-host cell interaction EVs (intEV). Uptake assays were performed with three types of purified EVs labeled with fluorochrome DiI or PKH26 in Caco-2 cells and G. intestinalis. We performed a dose-dependent and kinetic titration of vesicles in contact with host cells. The results were analyzed by flow cytometry and Nanotracking analysis. The pathways involved in the internalization of these EVs were analyzed using a different inhibitors comparing the internalization of vesicles in the host cell treated and not treated with the inhibitor. RESULTS AND CONCLUSION: Our results demonstrated that Caco-2 cells are internalized EVs in an energy- and dose-dependent manner, with different patterns for each EV type. The inhibitor Dynasore blocked the internalization of EVs by host cells. This study confirms that EVs are taken up by both host cells and the parasite, validating these findings using different methods. Furthermore, it suggests that endocytosis may be the route by which host cells capture EVs. SUPPORT: Capes.