RESUMO
Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed.
Assuntos
Anticorpos Antiprotozoários/imunologia , Cromatina/metabolismo , Toxoplasma/genética , Toxoplasmose/parasitologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/isolamento & purificação , Replicação do DNA , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Epitopos/imunologia , Fibroblastos/parasitologia , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/imunologia , Toxoplasma/fisiologiaRESUMO
BACKGROUND INFORMATION: Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat-shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. RESULTS: The localization of Hsp20 was further analysed using high-resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding-associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate phospholipids could be observed. CONCLUSIONS: Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped-arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.