Célia R.S. Garcia

Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites.

he development of malarial parasites is a complex process involving both intracellular and extracellular phases. Intraerythrocytic maturation proceeds through well-defined stages, termed rings, trophozoites and schizonts. In vivo, transition to a new stage and invasion of new erythrocytes are highly synchronous. The timing of these processes varies between Plasmodium species, but is always a multiple of 24 h. The simultaneous appearance of billions of individual parasites in the bloodstream may represent an efficient evolutionary strategy to escape the defence mechanisms of the host. The synchronicity of these processes is rapidly lost in culture, indicating the possible involvement of a host-derived signal, although the nature of this signal is presently unknown. Here we show that the hormone melatonin modifies the development of malarial parasites in vitro, that in vivo surgical ablation of the pineal gland leads to reduced synchronicity in the maturation process of Plasmodium, an effect that is reversed upon treatment with melatonin, and that in vivo inhibition of melatonin receptors mimics the effect of pinealectomy. We also demonstrate that melatonin, through activation of specific receptors coupled to phospholipase C activation, causes release of Ca from the intracellular stores of Plasmodium grown in vitro. We therefore propose that circadian changes in melatonin concentration in the host represent a key signal that controls synchronous maturation of Plasmodium in vivo. In search of a host-derived signal that undergoes circadian changes in mammals (and other vertebrates), we considered melatonin as a potential candidate. Melatonin is synthesized and released by the pineal gland during darkness, and this hormone is thought to participate in regulation of circadian rhythms in many eukaryotes, including vertebrates, invertebrates, higher plants and dinoflagellates. Not only does melatonin release exhibit a circadian rhythm, but the molecule is also sufficiently hydrophobic to cross T

Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem

Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be ∼40 μM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP3-dependent Ca2+ signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca2+ upon addition of exogenous IP3. In the present study, we investigated whether the IP3 signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca2+ indicator Fluo-4/AM and caged-IP3. Photolytic release of IP3 elicited a transient Ca2+ increase in the cytosol of the intact parasite within the RBC. The intracellular Ca2+ pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca2+ and the antimalarial chloroquine to deplete Ca2+ from acidocalcisomes. These data show that the ER is the major IP3-sensitive Ca2+ store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca2+-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca2+ responses to melatonin and uncaging of IP3 were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP3 and open ER-localized IP3-sensitive Ca2+ channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

PLASMODIUM IN THE POSTGENOMIC ERA : NEW INSIGHTS INTO THE MOLECULAR CELL BIOLOGY OF MALARIA PARASITES

In this review, we bring together some of the approaches toward understanding the cellular and molecular biology of Plasmodium species and their interaction with their host red blood cells. Considerable impetus has come from the development of new methods of molecular genetics and bioinformatics, and it is important to evaluate the wealth of these novel data in the context of basic cell biology. We describe how these approaches are gaining valuable insights into the parasite-host cell interaction, including (1) the multistep process of red blood cell invasion by the merozoite; (2) the mechanisms by which the intracellular parasite feeds on the red blood cell and exports parasite proteins to modify its cytoadherent properties; (3) the modulation of the cell cycle by sensing the environmental tryptophan-related molecules; (4) the mechanism used to survive in a low Ca(2+) concentration inside red blood cells; (5) the activation of signal transduction machinery and the regulation of intracellular calcium; (6) transfection technology; and (7) transcriptional regulation and genome-wide mRNA studies in Plasmodium falciparum.

Products of tryptophan catabolism induce Ca2+ release and modulate the cell cycle of Plasmodium falciparum malaria parasites.

Abstract:  Intraerythrocytic malaria parasites develop in a highly synchronous manner. We have previously shown that the host hormone melatonin regulates the circadian rhythm of the rodent malaria parasite, Plasmodium chabaudi, through a Ca2+‐based mechanism. Here we show that melatonin and other molecules derived from tryptophan, i.e. N‐acetylserotonin, serotonin and tryptamine, also modulate the cell cycle of human malaria parasite P. falciparum by inducing an increase in cytosolic free Ca2+. This occurs independently of the extracellular Ca2+ concentration, indicating that these molecules induce Ca2+ mobilization from intracellular stores in the trophozoite. This in turn leads to an increase in the proportion of schizonts. The effects of the indolamines in increasing cytosolic free Ca2+ and modulating the parasite cell cycle are both abrogated by an antagonist of the melatonin receptor, luzindole, and by the phospholipase inhibitor, U73122.

Acidic calcium pools in intraerythrocytic malaria parasites

Calcium uptake by permeabilized P. chabaudi malaria parasites was measured at the trophozoite stage to assess calcium accumulation by the parasite organelles. As determined with 45Ca2+, the total calcium in the parasite was found to be 11 pmoles/10(7) cells. When the K+/H+ uncoupling agent, nigericin was present, this level fell to 6.5 pmoles/10(7) cells. A similar regulatory mechanism operates in P. falciparum, since addition of nigericin to intact parasites in calcium free-medium resulted in a transient elevation of free calcium in the parasite cytosol, as judged by fluorescent imaging of single cells loaded with the calcium indicator fluo-3,AM. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and monensin, inhibitors of H+ ATPases and K+/H+ ionophore respectively, induced calcium elevation in fluo-3, AM-labeled intact P. chabaudi parasites. We conclude that malaria parasites utilize acidic intracellular compartments to regulate their cytosolic free calcium concentration.

Human malarial parasite, Plasmodium falciparum, displays capacitative calcium entry: 2-aminoethyl diphenylborinate blocks the signal transduction pathway of melatonin action on the P. falciparum cell cycle.

Abstract:  The malarial parasite senses the environment to modulate its own cycle. Knowledge of the mechanisms for regulation signaling processes at the invasion, maturation, as well as division of Plasmodium falciparum before reinvasion would represent a major breakthrough and, therefore, might open new avenues for therapy. We have previously reported that melatonin modulates the circadian rhythm of malarial parasites through the activation of phospholipase C (PLC), production of InsP3, and induction of calcium release from intracellular stores. To further investigate the molecular mechanism of melatonin’s action, we have used the InsP3 modulator 2‐aminoethyl diphenylborinate (2‐APB) given in a culture of P. falciparum parasites. Here we show that the melatonin acts on Plasmodium cell cycle through InsP3 signaling as 2‐APB blocks melatonin’s effect on calcium release. The function of the InsP3 signaling can be regarded as an important event for parasite invasion and maturation process, since addition of the PLC inhibitor, U73122 into Plasmodium‐infected red blood cells impairs parasite invasion in vitro. By using 8BrcAMP, we also report here that Plasmodia displays a ‘capacitative calcium entry’ mechanism for amplification of calcium signals throughout the cytoplasm.

Tertian and Quartan Fevers: Temporal Regulation in Malarial Infection

The periodicity in the development of Plasmodium parasites in infected animals, including man, has been known for almost 100 years. In turn, this periodicity is a consequence of the synchronous maturation of the parasite during its intracellular development. The cyclic fever that characterizes malarial infections is the outward manifestation of the parasite development. Until recently, little was known about the mechanisms by which parasite synchronicity is established and maintained. This review surveys the recent literature bearing on two main questions. (1) What are the mechanisms involved in the process of parasite synchronicity? (2) Do the circadian rhythms of the host interfere with the parasite cycle?

Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 microM melatonin and 0.1 microM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.