Host–Pathogen Cellular Communication: The Role of Dynamin, Clathrin, and Macropinocytosis in the Uptake of Giardia-Derived Extracellular Vesicles

<i>Giardia intestinalis</i>, a protozoan causing giardiasis, disrupts gastrointestinal health through complex host-parasite interactions. This study explores the differential uptake mechanisms of extracellular vesicles (EVs) derived from <i>Giardia</i> (gEVs), host cells (hEVs), and the host-parasite interaction (intEVs) in intestinal Caco-2 cells. Results show that intEVs are internalized more rapidly than gEVs and hEVs, underscoring their pivotal role in pathogenesis. To delineate uptake pathways, various endocytosis inhibitors were applied, and clathrin-mediated endocytosis inhibition using monodansylcadaverine (MDC) significantly reduced intEV and gEV uptake, confirming the role of clathrin-mediated endocytosis (CME). The use of dynasore, a dynamin inhibitor, strongly reduced the internalization of all EV types, demonstrating that uptake is dynamin-dependent. In contrast, methyl-β-cyclodextrin (MβCD), which disrupts lipid rafts and caveolae-mediated pathways, had no effect on EV uptake, indicating that caveolae are not involved in this process. Furthermore, inhibition of Na⁺/H⁺ exchange and phosphoinositide 3-kinase activity, both essential for macropinocytosis, also led to a significant reduction in intEV internalization. These findings strongly support that gEVs are internalized primarily through a dynamin- and clathrin-dependent pathway, independent of caveolae and lipid rafts, but modulated by tyrosine kinase signaling and macropinocytosis. These insights into selective and comprehensive inhibition pathways offer promising therapeutic targets to mitigate giardiasis.