We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7− parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7− parasites.
Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono-and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. ' 2011 International Society for Advancement of Cytometry Key termsPlasmodium falciparum; melatonin; cell cycle; flow cytometry MALARIA is caused by the Apicomplexan parasite Plasmodium falciparum. Its asexual replicative cycle inside red blood cell (RBC) is responsible for pathogenesis and induces several structural and biochemical changes within the host cell (1,2). One striking feature regarding P. falciparum infection is associated with 48-h fever peaks intervals, as a result of from synchronous release of merozoites into the blood stream (3).Melatonin, a hormone produced by the pineal gland of vertebrates, transmit the darkness signal based on circadian and seasonal time measurements (4). The presence of melatonin, however, is not restricted to vertebrates but is also found in phylogenetically divergent species including bacteria, plants and protozoa (4). We have previously shown that this hormone is able to synchronize Plasmodium falciparum and P. chabaudi cell infections (5-7). The synchronicity was lost in vitro when parasites had been incubated with the melatonin antagonist luzindole; the same effect was obtained in vivo in pinealectomized mice and after the injection of luzindole (5). In P. falciparum, melatonin induces a complex signaling pathway with the participation of IP3 generation (8) and subsequent transients in cytosolic calcium concentration, cAMP production and protein kinase A (PKA) (9,10) and protease activation (11).Flow cytometry (FCM) is a powerful method for evaluation of human erythrocyte infection rates as well as for discrimination of Plasmodium falciparum developmental stages (12-16). Several methods of detection of malaria parasite by flow cytometry have been developed taking advantage of the absence of DNA in erythrocytes. Different dyes such as acridine orange (17,18), hydroethidine (19,20), SYTO 16 (21) and thiazole orange (22) were already employed for the determination of parasitemia
Melatonin and its indoles derivatives are central in the synchronization of malaria parasites. In this research, we discovered that melatonin is unable to increase the parasitemia in the human malaria Plasmodium falciparum that lacks the kinase PfeIK1. The PfeIK1 knockout strain is a valuable tool in the screening of indol‐related compound that blocks the melatonin effect in wild‐type (WT) parasite development. The assays were performed by using flow cytometry with simultaneous labeling for mitochondria viability with MitoTracker Deep Red and nucleus staining with SYBR Green. We found that Melatotosil leads to an increase in parasitemia in P. falciparum and blocks melatonin effect in the WT parasite. Using microscopy imaging system, we found that Melatotosil at 500 nM is able to induce cytosolic calcium rise in transgenic PfGCaMP3 parasites. On the contrary, the compound Triptiofen blocks P. falciparum cell cycle with IC50 9.76 µM ± 0.6, inhibits melatonin action, and does not lead to a cytosolic calcium rise in PfGCaMP3 parasites. We also found that the synthetic indol‐related compounds arrested parasite cycle for PfeIK1 knockout and (WT) P. falciparum (3D7) in 72 hours culture assays with the IC50 values slighting lower for the WT strain. We concluded that the kinase PfeIK1 is central for melatonin downstream signaling pathways involved in parasite cell cycle progression. More importantly, the indol‐related compounds block its cycle as an upstream essential mechanism for parasite survival. Our data clearly show that this class of compounds emerge as an alternative for the problem of resistance with the classical antimalarials.
Agradeço à Profª. Drª. Célia Regina da Silva Garcia pela oportunidade oferecida e acolhimento em seu laboratório, pelas discussões e credibilidade ao me oferecer um projeto tão interessante e desafiador. Aos colegas e amigos de laboratório por todo ensinamento, paciência e momentos de descontração ao longo do mestrado:
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