Nocaute no gene da proteína P21 de Trypanossoma Cruzi modula a invasão, multiplicação intracelular e eclosão do parasita de cepa pouco virulenta

Abstract

Trypanosoma cruzi protozoan is the etiologic agent of Chagas disease. The process of intracellular invasion and multiplication involves different molecules from the parasite and the host cell to perpetuate its life cycle within the mammalian host. P21 is a protein secreted by T. cruzi and is involved in parasite host cell invasion and intracellular multiplication. Previous studies demonstrate that treatment of the host cell with the recombinant form of P21 (rP21) during cell invasion increases parasite internalization. Furthermore, when the treatment is performed at different times of intracellular multiplication of the parasite, we observed a negative regulation of the multiplication rate. Corroborating these data, TcP21-/- parasites of the Y strain showed a higher multiplication rate compared to controls. To understand whether the same occurs with another, less virulent strain, we generated knockout parasites of the G lineage for P21 expression using the CRISPR/Cas9 system and evaluated the ability to invade the host cell, multiply intracellularly and hatch into the supernatant. Our results showed that TcP21 -/- parasites had a lower ability to invade the host cell compared to controls (WT and Cas9). Furthermore, we observed that the TcP21 -/- parasites multiplied more during the first 24- and 48-hours post-infection and released a greater number of trypomastigote and amastigote forms into the host cell supernatant during a hatch kinetics assay. Taken together, our results corroborate the data obtained previously, in which P21 plays a relevant role in the invasion of host cells by the parasite and in intracellular multiplication. Furthermore, we observed for the first time that the lack of P21 expression increases the parasite egress from the host cell.