The flagellated protozoan Trypanosoma cruzi is the causative agent of Chagas' disease, which affects about 7 million people worldwide. T. cruzi trypomastigotes infect host cells and differentiate into relatively immotile intracellular amastigotes which lack a flagellum. After replication, and once the cell is full of amastigotes they differentiate into trypomastigotes and escape (egress from) the spent cell, disrupting the plasma membrane accessing the extracellular space and circulation thereby enabling a cycle of reinfection. Over the years, several studies addressed cell invasion by T. cruzi but less is known about its egress. Using confocal microscopy of living cells and later prepared for scanning electron microscopy (correlative), we investigated some aspects of T. cruzi egress. Our results showed that parasite egress is a sudden event; infected cells maintain plasma membrane integrity until parasites are released, though immediately after parasite egress little remains of host cytoplasmic structures. In addition, there are apparent actin cytoskeleton modifications from normal F-actin meshwork in non-infected cells to "circular-shaped" F-actin rings that seem to be formed in cells infected with amastigotes and which become even more evident in cells containing trypomastigotes - indicating that the construction of a cellular infrastructure upon which egress relies may initiate early in the cycle of infection, while parasites are still amastigotes. We also observed that egress is not entirely restricted to trypomastigotes, egress of amastigotes was also observed and might be related to the origin of infective extracellular amastigotes. Finally, in experiments using a protocol to remove membrane components but preserving host cell actin cytoskeleton we observed a disorganized actin cytoskeleton that was seemingly disrupted where it lay adjacent to intracellular parasites. In order to continue my investigations, I believe that spending time in Dr. Tyler´s laboratory at the University of East Anglia would provide access to technology not currently available in Brazil and thereby enable a series of novel experiments. During the stay, I hope to evaluate plasma membrane composition and fluidity during trypomastigote intracellular development, and changes in cytoskeletal and nuclear architecture associated with vacuolar escape, amastigote proliferation and trypomastigogenesis. Finally, I hope to identify the conditions, mechanism and specific signals which trigger amastigote differentiation and trypomastigote egress.
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