Purification and characterization of extracellular vesicles from tachyzoites of Toxoplasma gondii

• Main Author: Oliveira Silva, Valéria
• Publication Date: 2020
• Other Authors: Lucia Pereira-Chioccola, Vera
• Format: Article
• Language: por
• Source: BEPA. Boletim epidemiológico paulista (Online)
• Download full: https://periodicos.saude.sp.gov.br/BEPA182/article/view/34274
• topic: Toxoplasma. Toxoplasmose. Cromatografia. Vesículas extracelulares. Microscopia eletrônica e Nanopartículas; Toxoplasma. Toxoplasmosis. Chromatography. Extracellular Vesicles. Electron Microscopy and Nanoparticles

Toxoplasma gondii, a protozoan that causes toxoplasmosis, is transmitted from animals to humansthrough ingestion of infected meat or oocysts released by felines into the environment. In humans, theinfection is usually asymptomatic, but in cases where primary infection occurs during pregnancy itcan lead to neonatal malformations. In patients such as AIDS, reactivation of latent infection occurs,with episodes of parasite proliferation, causing symptomatic disease, such as cerebral or disseminatedtoxoplasmosis. In the last decades, small structures secreted by prokaryotic and eukaryotic cells calledextracellular vesicles (EVs) were studied. These small structures can be isolated by ultracentrifugation,chromatography and examined by electron microscopy. EVs participate in the communication betweencells, transfer of proteins, lipids and nucleic acids. In addition, them can carry disease biomarkers,bioreactive macromolecules contributing to the pathogenesis of diseases. In view of the above, thepresent study aimed to establish a protocol to isolate and characterize extracellular vesicles producedand excreted by tachyzoites of T. gondii RH strain. To achieve this goal, tachyzoites from VERO cellcultures were isolated from the culture supernatants and centrifuged in five sets of washes. Next, theywere incubated in culture medium for 24 hours for secretion of the EVs. The size and concentration of theisolated EVs by particle scan analysis (NTA) were investigated on the NanoSight (NTA) equipment. Inparallel, the morphology and the release of the EVs were also investigated by transmission and scanningelectron microscopy. Then, EVs were purified by gel-exclusion chromatography in 1 ml aliquots (24-32 fractions) and immuno-selected by ELISA using a pool of reagent sera for toxoplasmosis. Next, thepresence of miRNA in the EVs was investigated; and finally, the protein profile of T. gondii EVs wasevaluated and verify by serological tests if these particles could be recognized by the host immunesystem. The results showed that the protocol for recovery of T. gondii EVs was established from 1 to1010 tachyzoites obtained from cultured cells. Analyzes performed by NTA allowed us to determinethat about 1 x 106 tachyzoites secreted 4 to 8 x 108 EVs / mL within 24 hours of incubation in culturemedium. Additionally, these vesicles presented morphology and size of 165-175 nm of diameter,corresponding microvesicles size. These results were also confirmed by the evaluation of imagesprovided by transmission and scanning electron microscopy. The miRNA purifications from the EVsand subsequent microfluidic electrophoretic run confirmed the presence of smallRNA and miRNA inthe vesicles. SDS-PAGE analyzes show that the proteins carried by the EVs showed an electrophoreticprofile with a spectrum of 15 to 70 kDa. Sera from chronically infected mice (with 2 different strains ofT. gondii) and humans recognized distinct electrophoretic patterns in immunoblotting.