Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri

As global temperatures rise to unprecedented levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri . This opportunistic parasite causes a rare, but > 97% fatal, neurological infection called Primary Amoebic Meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans. To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and secretion mechanism of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases. Herein we show that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Fusion assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed > 2,000 proteins within the N. fowleri -secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intracellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.