Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo

SIMPLE SUMMARY: Extracellular vesicles are vesicles produced by cells and organisms, capable of developing different functions. In this sense, these vesicles have been involved in communication, in the production of damage during an infection and in the modulation of different cell pathways. These vesicles have also been proposed as key players for the diagnosis and treatment of several diseases. In this study, we report for the first time the production of extracellular vesicles by Naegleria fowleri, the free-living amoeba that causes primary amoebic meningoencephalitis, a severe infection of the central nervous system that is fatal in more than 95% of cases. The vesicles were isolated and size, surface charge and protein content were analyzed. The report of the production of extracellular vesicles by this amoeba is relevant for different reasons: i. these vesicles could be implicated during the processes of invasion and damage to the brain; ii. they could influence the environment to trigger specific responses by cells and, iii. specific vesicles secreted by the amoeba could be employed for diagnostic purposes. Several studies to reveal the role of extracellular vesicles of this pathogen are being performed in our laboratory. ABSTRACT: Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eukaryotic cells, involved in intercellular communication, immunomodulation and pathogenesis. In this study, we performed a characterization of the EVs produced by trophozoites of a clinical isolate of the free-living amoeba Naegleria fowleri (N. fowleri). Size distribution, zeta potential, protein profile and protease activity were analyzed. Under our incubation conditions, EVs of different sizes were observed, with a predominant population ranging from 206 to 227 nm. SDS-PAGE revealed protein bands of 25 to 260 KDa. The presence of antigenic proteins was confirmed by Western blot, which evidenced strongest recognition by rat polyclonal antibodies raised against N. fowleri in the region close to 80 KDa and included peptidases, as revealed by zymography. Proteins in selected immunorecognized bands were further identified using nano-ESI-MS/MS. A preliminary proteomic profile of the EVs identified at least 184 proteins as part of the vesicles’ cargo. Protease activity assays, in combination with the use of inhibitors, revealed the predominance of serine proteases. The present characterization uncovers the complexity of EVs produced by N. fowleri, suggesting their potential relevance in the release of virulence factors involved in pathogenicity. Owing to their cargo’s diversity, further research on EVs could reveal new therapeutic targets or biomarkers for developing rapid and accurate diagnostic tools for lethal infections such as the one caused by this amoeba.