Estudo das vesículas extracelulares de Toxoplasma gondii e a correlação com a infecção humana

Quiarim, Talita Motta

Toxoplasma gondii é causador da toxoplasmose, uma das doenças mais prevalentes no mundo. Estudos recentes mostraram que vesículas extracelulares (EVs) liberadas por parasitas participam no processo de invasão e replicação no hospedeiro, porém o mecanismo de infecção ainda não está completamente elucidado. O objetivo desse trabalho foi identificar e caracterizar EVs produzidas por taquizoítos das cepas RH, ME-49 e VEG de T. gondii e a participação na patogênese da toxoplasmose. Purificação de EVs liberadas das três cepas de T. gondii foi realizada por cromatografia de exclusão de tamanho seguida por ELISA. Concentração e tamanho das vesículas isoladas foram analisados por Nanoparticle Tracking Analysis, o qual mostrou que as três cepas possuem perfis de liberação de EVs similares, com maior produção observada pela cepa RH. Quando analisados diferentes tempos de incubação, observou-se que em 2 horas de incubação ocorreu maior liberação de EVs do que em 24 horas de incubação, para as três cepas. A maior parte das vesículas encontradas possuía tamanho entre 100-200 nm, caracterizadas como microvesículas. Observou-se através de imagens capturadas por Microscopia Eletrônica de Varredura que a cepa RH liberou mais EVs do que as cepas VEG e ME-49. Após a análise de taquizoítos da cepa RH por Microscopia Eletrônica de Transmissão, observou-se que no...(AU)

Toxoplasma gondii is the agent of toxoplasmosis, one of the most prevalent diseases in the world. Recent studies show that extracellular vesicles (EVs) released by parasites are involved in the invasion and replication mechanisms in the host, however they are not completely clear. The aim of this study was to identify and characterize EVs released by tachyzoites from RH, ME-49 and VEG strains of T. gondii and their role in toxoplasmosis pathogenesis. EVs purification was performed by size exclusion chromatography followed by ELISA. Size and concentration of EVs was analysed by Nanoparticle Tracking Analysis, which showed similar EVs release profile from the three strains, however RH strain showed higher production of EVs. When analysed different incubation periods, it was observed higher production of EVs in 2 hours rather than 24 hours of incubation, for the three strains. The majority size of EVs found was of 100-200 nm which is classified by microvesicles. Images captured by Scanning Electron Microscopy showed that tachyzoites from RH strain released more EVs than tachyzoites from ME-49 and VEG strains. Also, images captured by Transmission Electron Microscopy of tachyzoites from RH strain showed that in the beginning of incubation period starts the formation of multivesicular bodies with vesicles inside ready to be released in the lumen. After 24 hours, it was able to observe intense release of EVs from the plasmatic membrane, as well as from posterior pore and apical ring. Furthermore, it was found that T. gondii was able to express the same miRNAs found in infected hosts. miR-155-5p, miR-125b-5p e miR-423-3p were the most expressed in tachyzoites as well as in EVs released from them in the three strains. Experiments with laboratory mice infected with tachyzoites of RH strain mixed with EVs, especially for EVs released from RH strain, showed that EVs can enhance parasitemia and virulence, decreasing mice’s survival. Protein extracted from EVs of the three strains demonstrated similar electrophoretic profiles, but when EVs were incubated with sera from patients with toxoplasmosis, in Immunoblot analysis, EVs from ME-49 and VEG strains reacted poorly, unlike EVs from RH strains which reacted with sera from patients with gestational and cerebral toxoplasmosis. Stimulus of EVs released form the three strains in mice splenocytes in vitro produced similar concentrations of IL-10 and IFN- after 24 and 48 hours, in the three strains. EVs released from RH strain stimulated the production of more TNF- than EVs released from ME-49 and VEG strains. Finally, these results suggest that EVs released from tachyzoites of three T. gondii strains, especially the ones released from RH strain, were able to stablish communication between host cells and parasites, modulating host immune system, although they unbalance the host immune response since they carry virulence factors. (AU)