The morphological analysis of autophagy in primary skeletal muscle cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.

Trypanosoma cruzi nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase-1) biochemical characterization, immunolocalization and possible role in host cell adhesion.

Previous work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite-host cell adhesion. These data open new frontiers to future studies on this specific parasite-host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.