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

Abstract

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.

The Leishmania-Macrophage Interactions: Role of E-NTPDases and Purinergic Signaling

“…These results suggested the existence of a possible binding site (like an unknown receptor) for the Lic-E-NTPDase2 in macrophages that could participate in facilitating adhesion and infection [46] . These data are in accordance with previous results also observed in T. cruzi [45] . [80] .…”

“…There is a direct relationship between the ability to hydrolyze nucleotides and the ability to sustain infection [39, 42-44, 47, 74, 78, 115-119] . It has also been shown recently that the E-NTPDase of parasites can act as an adhesion protein during the early stages of infection [45,46,120] . Inhibiting Leishmania molecules that actively modulate the macrophage signaling pathways is essential to prevent the intracellular survival of Leishmania.…”

“…The GP63, a zinc-metalloprotease named glycoprotein 63 (GP63), is another critical virulence factor that has been fully explored [34][35][36][37][38] . However, the E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolases), while less-extensively studied, have been demonstrated and believed to be important infectivity and virulence factors [39][40][41][42][43][44][45][46][47][48] .…”

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase

“…In fact, studies have suggested a role of carbohydrate/lectin interactions on T. rangeli adhesion on insect salivary glands (Basseri et al, 2002;Mello et al, 1999). Interestingly, evidences suggested that T. cruzi NTPDase is possibly involved in host cell adhesion (Mariotini-Moura et al, 2013) and previous studies have showed a plant NTPDase/lectin byfunctional protein that is activated by the presence of carbohydrates (Etzler et al, 1999) . Based on these evidences, researchers have previously investigated the role of carbohydrates on T. rangeli NTPDAse activity.…”

Nucleoside triphosphate diphosphohydrolase1 (TcNTPDase-1) gene expression is increased due to heat shock and in infective forms of Trypanosoma cruzi

“…It is possible that distinct ecto-enzymes could contribute to the parasite adaptation to stress conditions, as nutrients starvation or heat shock. Recently, Mariotini-Moura et al [24] performed heterologous expression, purification and molecular characterization of TcNTPDase-1. By using specific polyclonal antibodies, they confirmed the presence of TcNTPDase-1 not only on the surface of T. cruzi, but also in the kinetoplast, nucleus, intracellular vesicles, flagellum and flagellum insertion region.…”