Changes in K+ Concentration as a Signaling Mechanism in the Apicomplexa Parasites Plasmodium and Toxoplasma.

During their life cycle, apicomplexan parasites pass through different microenvironments and encounter a range of ion concentrations. The discovery that the GPCR-like SR25 in Plasmodium falciparum is activated by a shift in potassium concentration indicates that the parasite can take advantage of its development by sensing different ionic concentrations in the external milieu. This pathway involves the activation of phospholipase C and an increase in cytosolic calcium. In the present report, we summarize the information available in the literature regarding the role of potassium ions during parasite development. A deeper understanding of the mechanisms that allow the parasite to cope with ionic potassium changes contributes to our knowledge about the cell cycle of Plasmodium spp.

THE GENETICALLY ENCODED CALCIUM INDICATOR GCAMP3 REVEALS SPONTANEOUS CALCIUM OSCILLATIONS AT ASEXUAL STAGES OF THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM.

Most protocols used to study the dynamics of calcium (Ca2+) in the malaria parasite are based on dyes, which are invasive and do not allow discrimination between the signal from the host cell and the parasite. To avoid this pitfall, we have generated a parasite line expressing the genetically encoded calcium sensor GCaMP3. The PfGCaMP3 parasite line is an innovative tool for studying spontaneous intracellular Ca2+ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca2+ oscillations in the ring, trophozoite, and schizont stages in Plasmodium falciparum. Using the Fourier transform to fluorescence intensity data extracted from different experiments, we observe cytosolic Ca2+ fluctuations. These spontaneous cytosolic Ca2+ oscillations occur in the three intraerythrocytic stages of the parasite, with most oscillations occurring in the ring and trophozoite stages. A control parasite line expressing only a GFP control did not reveal such fluctuations, demonstrating the specificity of the observations. Our results clearly show dynamic, spontaneous Ca2+ oscillations during the asexual stage in P. falciparum, independent from external stimuli.

Molecular basis of synchronous replication of malaria parasites in the blood stage.

The search for host factors that leads to malaria parasite synchronization has been the focus of several laboratories. The host hormone melatonin synchronizes Plasmodium falciparum in culture by increasing the number of mature parasite stages through a PLC-IP3 activation. Melatonin signaling is linked to crosstalk between Ca2+-cAMP that results in PKA activation. Two other kinases, PfPK7 and PfeIK1, and the nuclear protein PfMORC that lacks melatonin sensitivity in the inducible knock-down parasites are also identified as part of the hormone-signal transduction pathways. Melatonin also modulates P. falciparum mitochondrial fission genes FIS1, DYN1, and DYN2 in a stage-specific manner. How these multiple molecular mechanisms are orchestrated to lead to parasite synchronization is a fascinating and opened biological question.

The Knockout for G Protein-Coupled Receptor-Like PfSR25 Increases the Susceptibility of Malaria Parasites to the Antimalarials Lumefantrine and Piperaquine but Not to Medicine for Malaria Venture Compounds.

Previously we have reported that the G protein-coupled receptor (GPCR)-like PfSR25 in Plasmodium falciparum is a potassium (K+) sensor linked to intracellular calcium signaling and that knockout parasites (PfSR25-) are more susceptible to oxidative stress and antimalarial compounds. Here, we explore the potential role of PfSR25 in susceptibility to the antimalarial compounds atovaquone, chloroquine, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, primaquine, and pyrimethamine and the Medicine for Malaria Venture (MMV) compounds previously described to act on egress/invasion (MMV006429, MMV396715, MMV019127, MMV665874, MMV665878, MMV665785, and MMV66583) through comparative assays with PfSR25- and 3D7 parasite strains, using flow cytometry assays. The IC50 and IC90 results show that lumefantrine and piperaquine have greater activity on the PfSR25- parasite strain when compared to 3D7. For MMV compounds, we found no differences between the strains except for the compound MMV665831, which we used to investigate the store-operated calcium entry (SOCE) mechanism. The results suggest that PfSR25 may be involved in the mechanism of action of the antimalarials lumefantrine and piperaquine. Our data clearly show that MMV665831 does not affect calcium entry in parasites after we depleted their internal calcium pools with thapsigargin. The results demonstrated here shed light on new possibilities on the antimalarial mechanism, bringing evidence of the involvement of the GPCR-like PfSR25.

Plasmodium falciparum Knockout for the GPCR-Like PfSR25 Receptor Displays Greater Susceptibility to 1,2,3-Triazole Compounds That Block Malaria Parasite Development.

The search for new compounds with antimalarial activity is urgent, as resistance to ones in the classical drug, has already been described in more than one continent. Compounds derived from 1,2,3-triazoles are effective against parasites and bacteria. Here, we evaluated the potential antimalarial activity against the human malaria parasite Plasmodium falciparum in a culture of fifty-four triazole compounds derived from 1H-and 2H-1,2,3-triazole. We identified thirty-one compounds with potential antimalarial activity at concentrations in the micromolar order (µM) and IC50 values ranging from 2.80 µM (9) to 29.27 µM (21). Then, we selected some of these compounds to perform the same tests on the PfSR25- strain (knockout for P. falciparum G-protein coupled receptor-like, SR25). Our experiences with the PfSR25- strain showed that both compounds with higher antimalarial activity for the 3D7 strain and those with less activity resulted in lower IC50 values for the knockout strain. The cytotoxicity of the compounds was evaluated in human renal embryonic cells (HEK 293), using MTT assays. This demonstrated that the compounds with the highest activity (9, 13, 19, 22, 24, 29), showed no toxicity at the tested concentrations.