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

Acknowledgments: Transmission electron microscopy was performed at the “Centro de Instrumentación Científica” of the “Universidad de Granada”. Dynamic light scattering was performed at the “Laboratorio de Polímeros” of the School of Chemistry, “Universidad Nacional” and atomic force microscopy imaging was performed at the Physics Department of the same university. Proteomic analyses were performed at the “Instituto Clodomiro Picado”, of the “Universidad de Costa Rica”. Special thanks to Oscar Rojas Carrillo for allowing the access to the equipment and for his methodological support during the dynamic light scattering analyses and Desire Arrieta Murillo and Jocelyn Cortés Espínola for the technical support during the atomic force microscopy analyses. G.S.-A. wants to thank “Plan de Mejoramiento Institucional UNA BM-04”, Consejo Nacional de Rectores (FEES-CONARE) and the “Vicerrectoría de Investigación” of Universidad Nacional de Costa Rica for funding for equipment.

Data Availability Statement: Mass spectrometry raw files of the data presented in this work are available from the authors upon reasonable request.

Funding: This research was funded by “Vicerrectoría de Investigación” of the “Universidad de Costa Rica”, by supporting the research projects C-1061: “Caracterización de antígenos de excreción/ secreción y antígenos somáticos en amebas de vida libre mediante empleo de anticuerpos policlonales producidos en roedores” and C-2600: “Secreción de vesículas extracelulares por Naegleria fowleri y evaluación de su potencial rol inmunomodulador en un modelo in vitro”.

Institutional Review Board Statement: The use of animals was performed following the institutional guidelines (Spanish government regulations (Real Decreto RD1201/05)) and the guidelines of the European Union (European Directive 2010/63/EU). These experiments were approved by the Ethical Committee of the University of Granada (235-CEEA-OH-2018) and by the authorities of the Regional Government of Andalucía (JJAA) (number 12/11/2017/162).

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 byWestern 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.

Vicerrectoría de Investigación” of the Universidad de Costa Rica by supporting the research projects C-1061: “Caracterización de antígenos de excreción/ secreción y antígenos somáticos en amebas de vida libre mediante empleo de anticuerpos policlonales producidos en roedores” and C-2600: “Secreción de vesículas extracelulares por Naegleria fowleri y evaluación de su potencial rol inmunomodulador en un modelo in vitro”