Extracellular vesicles of Trypanosoma cruzi tissue-culture cell-derived trypomastigotes: Induction of physiological changes in non-parasitized culture cells

Background Trypanosoma cruzi is the obligate intracellular parasite that causes Chagas disease. The pathogenesis of this disease is a multifactorial complex process that involves a large number of molecules and particles, including the extracellular vesicles. The presence of EVs of T. cruzi was first described in 1979 and, since then, research regarding these particles has been increasing. Some of the functions described for these EVs include the increase in heart parasitism and the immunomodulation and evasion of the host immune response. Also, EVs may be involved in parasite adhesion to host cells and host cell invasion. Methodology/Principal findings EVs (exosomes) of the Pan4 strain of T. cruzi were isolated by differential centrifugation, and measured and quantified by TEM, NTA and DLS. The effect of EVs in increasing the parasitization of Vero cells was evaluated and the ED50 was calculated. Changes in cell permeability induced by EVs were evaluated in Vero and HL-1 cardiomyocyte cells using cell viability techniques such as trypan blue and MTT assays, and by confocal microscopy. The intracellular mobilization of Ca2+ and the disruption of the actin cytoskeleton induced by EVs over Vero cells were followed-up in time using confocal microscopy. To evaluate the effect of EVs over the cell cycle, cell cycle analyses using flow cytometry and Western blotting of the phosphorylated and non-phosphorylated protein of Retinoblastoma were performed. Conclusion/Significance The incubation of cells with EVs of trypomastigotes of the Pan4 strain of T. cruzi induce a number of changes in the host cells that include a change in cell permeability and higher intracellular levels of Ca2+ that can alter the dynamics of the actin cytoskeleton and arrest the cell cycle at G0/G1 prior to the DNA synthesis necessary to complete mitosis. These changes aid the invasion of host cells and augment the percentage of cell parasitization. Author summary Extracellular vesicles (EVs) are a diverse group of nanoparticles involved in intercellular communication under physiological and pathological conditions. Trypanosoma cruzi, the protozoan that causes Chagas disease, releases EVs that facilitate parasite invasion of the host cell, immunomodulate the host response, and help the parasite to evade this response. However, little is known about how the host cell is altered. In this work, we confirm that EVs of tissue-culture cell-derived trypomastigotes of the Pan4 strain increase cell parasitism. We also demonstrate that EVs affect cell permeability in Vero cells and cardiomyocytes and raise intracellular Ca2+ levels, altering the actin filaments and arresting the cell cycle at the G0/G1 phases. This work seeks to elucidate the way in which EVs influence certain aspects of the cell physiology that favour the establishment of this parasite inside the host cell.