Authors

Discussion

Trypanosoma cruzi possesses a complex life cycle, with different infective and invasive forms in different hosts. Among these infective forms, extracellular amastigotes (EAs), present in mammalian hosts, have been shown to be able to invade host cells without the need to differentiate back into trypomastigotes. Previous studies showed that these same parasite forms release vesicles and other molecules, in the presence of the host cell, which may be associated with the process of invasion and modulation of infectivity. These events were observed in previous results, in which vesicles and other released molecules from the G strain (highly infective) positively modulated the Y strain (low infective) host cell invasion in vitro and vice-versa. Therefore, this study aims to identify molecules released by extracellular vesicles and investigate their importance for the invasion of different strains of T. cruzi with different levels of infectivity in host cells. First, extracellular vesicles and proteins free of vesicles released from the G and Y strain were obtained by ultracentrifugation in three fractions, named V2, V16 and vesicle free (VF). The fractions were assessed relative to the vesicles sizes by NanoSight NS300 (Malvern Panalytical), which showed that V2 fractions (obtained after 2 hours of centrifugation) had an average size of 192 nm while V16 (obtained after 16 hours of centrifugation) had an average size of 90 nm in both strains. Following this analysis, the same fractions were assessed relative to their protein content by mass spectrometry. Preliminary gene onthology analysis showed that the V2, V16 and VF of G strain had a higher percentage of proteins related to T. cruzi virulence (18%, 12% and 56%, respectively), when compared to the Y strain (7%, 26% and 11%, respectively). The virulence proteins found in both strains comprise trans-sialidases (TS), mucins and mucin-associated surface proteins (MASPS) and gp63 (only present on Y strain). Notably, TS showed to be the most present in G strain with 78% of the total of virulence proteins, while in the Y strain it was identified 41% of TS, which was the same amount of MASPs for that strain. Furthermore, in the next steps of this study, the most frequent protein among the total of TS, MASP and mucins, will be chosen to be deleted through the CRISPR/CAS9 method. The parasite will then be evaluated for their capacity to invade host cells and modulate the invasion of other strains. MicroRNA searches are also being performed to identify other molecules that might also be involved in modulating the host cell during the infection process. The results from these analyzes will contribute for the identification of the main components released by the parasite during the invasion process and will lead to a better understanding of the invasion mechanism of T. cruzi extracellular amastigotes.