Guidelines for the purification and characterization of extracellular vesicles of parasites

Abstract

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells.

Keywords: EVs methodology; extracellular vesicles; helminth; host‐parasite interaction; infection; protocols; protozoan parasites.

FIGURE 1

EVs from distinct pathogenic parasites (different forms and stages of the life cycle). Parasite EVs are composed of a lipid bilayer and a several molecules derived from the plasma membrane and/or cytosol. EVs can participate in the parasite‐host relationship since they on carry proteins, lipids, and nucleic acids distributed inside, outside or merged in the lipid bilayer (a). EVs from pathogenic parasites functions. Parasite EVs participate in the parasite‐parasite and parasite‐host relationship. EVs have different roles depending on the species or the evolutive form. They can induce an immune response (activating or suppressing the immune cells), facilitate the parasite attachment to the host cells, participate in parasite‐parasite communication, and contribute to the parasite‐parasite communications, among other functions (b). (Evans‐Osses et al., 2017)1 and 2; (Ma'ayeh et al., 2017)−3; (Sharma et al., 2020)−4; (Costa et al., 2021)−5; (Goncalves et al., 2018)−6; (Olmos‐Ortiz et al., 2017)−7; (Twu et al., 2013)−8; (Silva et al., 2018)−9; (Atayde et al., ; Silverman et al., 2010b)−10; (Madeira et al., ; Torrecilhas et al., ; Trocoli Torrecilhas et al., 2009)−11 and 12; (Mantel & Marti, 2014)−13; (Toda et al., 2020)−14; (Sisquella et al., 2017)–15; (Regev‐Rudzki et al., 2013)–16; (Marcilla et al., 2014)−17.

FIGURE 2

Flowchart to obtain, characterize and perform functional studies with parasite extracellular vesicles. The different methods suggested in each step are highlighted to the left of the directional arrows.