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. 2019 Mar;66(2):e12484.
doi: 10.1111/jpi.12484. Epub 2018 Oct 5.

Melatonin activates FIS1, DYN1, and DYN2 Plasmodium falciparum related-genes for mitochondria fission: Mitoemerald-GFP as a tool to visualize mitochondria structure

Pedro H Scarpelli  1   2 , Giulliana Tessarin-Almeida  2 , Kênia Lopes Viçoso  2 , Wania Rezende Lima  3 , Lucas Borges-Pereira  2 , Kamila Anna Meissner  1 , Carsten Wrenger  1 , Anna Raffaello  4 , Rosario Rizzuto  4 , Tullio Pozzan  4 , Celia R S Garcia  2   2
Affiliations

Melatonin activates FIS1, DYN1, and DYN2 Plasmodium falciparum related-genes for mitochondria fission: Mitoemerald-GFP as a tool to visualize mitochondria structure

Pedro H Scarpelli et al. J Pineal Res. 2019 Mar.

Erratum in

  • Corrigendum.
    [No authors listed] [No authors listed] J Pineal Res. 2019 Sep;67(2):e12595. doi: 10.1111/jpi.12595. Epub 2019 Jul 26. J Pineal Res. 2019. PMID: 31393648 Free PMC article. No abstract available.

Abstract

Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild-type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild-type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild-type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra-erythrocyte cell cycle progression.

Keywords: GFP; Plasmodium falciparum; malaria parasites; melatonin; mitochondria.

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Figures

Figure 1
Figure 1
Protein alignment of FIS1 and DRP1 from Homo sapiens with FIS1 and DRP1 candidates from Plasmodium falciparum. Numbers shows length and positions, and residues that may regulate FIS1 by phosphorylation are evidenced. Regions in red shows TPR domain of FIS1, which are important for protein‐protein interactions. Regions in blue show GTP/co‐factors binding and dimerization domain of DRP1; Green regions represent GTPase activity motif; Yellow region is Forkhead domain for DNA interaction of DYN1
Figure 2
Figure 2
Relative expression of FIS1, DYN1, and DYN2 throughout 3D7, PfPk7 knockout, and PfPk7 complement strains. Parasites were synchronized to isolate ring, trophozoite, and schizont stages using sorbitol, and RNA samples were extracted, purified, and submitted to RT‐qPCR. Seryl‐tRNA Synthetase expression was quantified to normalize mRNA levels. The graphics reveal an increase in FIS1, DYN1, and DYN2 expression in mature forms of 3D7 parasites. In PfPk7 knockouts, DYN2 presented no change in expression throughout stages, while FIS1 had a small change only in trophozoite stage and DYN1 was expressed similarly to 3D7 strain. For PfPk7 complement strain, both FIS1 and DYN2 did not show increase in expression
Figure 3
Figure 3
Relative expression of FIS1, DYN1, and DYN2 in 3D7, PfPk7−, and PfPk7 complement after melatonin treatment. Parasites at trophozoite stage (30 hours after invasion) were treated with 100 nmol L−1 of melatonin for 5 and 17 hours, and RNA samples were extracted, purified, and submitted to RT‐qPCR. Seryl‐tRNA Synthetase expression was quantified to normalize mRNA levels. The graphics reveal an increase in FIS1, DYN1, and DYN2 expression of 3D7 parasites. In PfPk7 knockouts and PfPk7 complement, we could not observe any change in expression of FIS1, DYN1, and DYN2 after treatment (A). Giemsa stained smears of infected red blood cells were made to make sure that the treatment did not interfered in parasites stage or morphology (B)
Figure 4
Figure 4
Mitochondria from Plasmodium falciparum seen by fluorescent microscopy using Mito‐Emerald construction. Images were taken from ring, trophozoites, and schizonts stages of Pf3D7 (A) The parasite nucleus was stained by HOECHST 33342 and mitochondria with MitoTracker Red CMX Ros, to demonstrate co‐localization with Mito‐Emerald. Mitochondria shape changes from small dots in ring stages to an elongated form in trophozoites and multiple mitochondria within segmented schizonts. MitoTracker Red CMX Ros was used for staining Pf PK7 mitochondria (B). In Pf PK7, mitochondria starts as an elongated form in ring, and then, it grows bigger until segmentation in schizogony (B)
Figure 5
Figure 5
Oxygen consumption in Plasmodium falciparum Pf3D7 wild‐type and Pf PK7 strains at ring, trophozoites and schizonts. 108 synchronized parasites of each strain were incubated in O2K (Oroboros Instruments) at 37°C in complete RPMI media, oxygen concentration was monitored for 10‐30 minutes, and oxygen consumption rates were determined. Figures A‐F are typical recordings of O2K instrument, showing a time course of total oxygen concentration (A, C, and E), and its derivative function showing consumption ratio in pmol per second (B, D, and F). Total oxygen consumption is equal to consumption of parasites (3) minus consumption of RPMI media alone (1). Addition of cells to O2K instrument causes a disturbance in oxygen quantification (2). The consumption ratio of parasites is shown in (4), and mean corrected values of 3 independent experiments for each stage are in (G). Pf3D7 showed a consumption that increases along with the cycle, as expected. Pf PK7 has an unusual oxygen consumption: Rings of this strain have a twofold higher consumption when compared to wild‐type strain, and it is not observable a direct correlation between oxygen consumption and cycle development
Figure 6
Figure 6
Oxygen consumption over time and after treatments with Oligomycin and carbonyl cyanide m‐chlorophenylhydrazone (CCCP) in 5 × 108 3D7 wild‐type parasites. Results of Oligomycin 10 μmol L−1 and CCCP 20 μmol L−1 were used for plotting bar graphs and analyzing the results because these were the lowest concentrations with highest consumption. Three independent experiments were performed for each stage. *Statistical significant differences by Student's t test. Results show that mitochondria operates in an uncoupled state in trophozoites and schizont stages, and ATP production is responsible for 50% or less O2 consumption in these stages. Ring stages do not use oxygen for ATP synthesis. Blue line shows total oxygen concentration inside the O2K chamber over time, and red line shows its derivative curve, oxygen consumption rate in pmol/s. Each arrow represents addition of cells (first), oligomycin (second), CCCP (third to sixth). The intervals used to calculate oxygen consumption in each timepoint are shown between dotted lines: basal respiration (A), with 10 μmol L−1 oligomycin (B), with 5, 10, 20, and 40 μmol L−1 CCCP (C, D, E, and F, respectively)
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