RESUMO
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.
Assuntos
Antígenos CD/fisiologia , Apirase/fisiologia , Adesão Celular/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Trypanosoma cruzi/enzimologia , Animais , Antígenos CD/química , Apirase/química , Western Blotting , Imuno-HistoquímicaRESUMO
In this study, the morphology of spermatozoa of Bethylidae Dissomphalus connubialis (Pristocerinae) was analyzed using light and transmission electron microscopy. Spermatozoa of this species are thin, measure approximately 130 µm in length and comprise a head region and flagellum region. The head is formed by the acrosome and nucleus. The acrosome consists of the acrosome vesicle and the perforatorium, the posterior portion of which is inserted into a cavity at the anterior extremity of the nucleus. The nucleus is compact, electron-dense and measures 15 µm in length. The flagellum possesses two mitochondrial derivatives, two accessory bodies and one axoneme with a 9+9+2 microtubular pattern. The nucleus is connected to the flagellum by the centriole adjunct. Mitochondrial derivatives are compact, apparently without paracrystalline material and with rare mitochondrial cristae. They are asymmetric in length, such that the larger mitochondrial derivative begins parallel to the posterior region of the nucleus and the smaller mitochondrial derivative begins just below the centriole adjunct. The basic structure of spermatozoa of D. connubialis is similar to that of other Aculeata studied. However, this species shows characteristics not seen in other Hymenoptera, such as the wide electron-lucid region that separates the acrosomal vesicle from the perforatorium and the depth of the cavity in the anterior extremity of the nucleus, into which the base of the perforatorium is inserted. There are also characteristics that distinguish this species from Bethylidae Prorops nasuta, including the fact that one of the mitochondrial derivatives lies to parallel to the nucleus over a long distance, the small quantity of cristae, the absence of paracrystalline material in these organelles, and the fact that the accessory microtubules are the first to terminate in the final portion of the flagellum.