Ubiquitin proteasome system and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum

Abstract

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.

Figure 1

Melatonin does not affect the cell cycle of pfpk7⁻ parasites. Parasite culture was treated with melatonin (MLT) (or vehicle) for 24 h. Proportions of the different parasite forms (ring, trophozoite and schizont) were determined by analyzing 2,000 cells in triplicates. The data show the average of three independent experiments. Addition of melatonin in Plasmodium falciparum 3D7 causes a modulation in the distribution of developmental stages in the intraerythrocytic cycle (A and C). However, this modulation is lost in pfpk7⁻ parasites (clone B9) (B). Upon complementation of the KO parasite with an extra copy of the pfpk7 gene, the effect of melatonin is restored (D). Statistical analysis by ANOVA and Newman-Keuls Multiple Comparison Test. * p<0.05.

Figure 2

Knockout of pfnek-2 and 3 does not affect melatonin signaling. Parasite culture was treated with melatonin (MLT) (or vehicle) for 24 h. Proportions of the different parasite forms (ring, trophozoite and schizont) were determined by analyzing 2,000 cells in triplicates. The data show the average of three independent experiments. Addition of melatonin in Plasmodium falciparum 3D7 causes a modulation in the distribution of developmental stages in the intraerythrocytic cycle (A and C). The modulation persist in pfnek-2⁻ (B) and pfnek-3⁻ parasites (D). Statistical analysis by ANOVA and Newman-Keuls Multiple Comparison Test. * p<0.05.

Figure 3

Calcium rise in response to melatonin in pfpk7⁻ strain. Fluorimetric calcium measure in melatonin treated wild-type 3D7, pfpk7⁻ parasites (clone B9), and in the pfpk7-complemented clone B9 (see Materials and Methods). The data show the average of three independent experiments.

Figure 4

Plasmodium UPS genes are upregulated upon melatonin treatment. Differential transcription of UPS genes from parasites subjected to melatonin treatment compared to control was analyzed by real time PCR (A) and in the presence of luzindole, both using 18S ribosomal RNA for normalization. At least three independent experiments were performed (See Supplementary table 2). See experimental procedures for statistical treatment. * p value < 0.05, ** p value < 0.01. The criterion for altered gene expression was twofold (up or downregulation). The ID of the genes are available in supplemental material.

Figure 5

Melatonin-induced up-regulation of UPS gene transcript levels is mediated by PfPK7. The effect of 0.1 μM melatonin treatment on UPS gene transcript levels observed in wild-type 3D7 parasites (see Fig. 1) was assessed by real time PCR in pfpk7⁻ parasites (clone B9 (A) and the pfpk7-complemented clone B9 (B) The effect of melatonin treatment is abolished in the knockout clone and partially restored in the complemented parasites (PfPK7 – complement) (B) * p<0.05 and ** p<0.01. The data show the average of at least three independent experiments.