Eduardo Alves

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.

The Structurally Related Auxin and Melatonin Tryptophan‐Derivatives and their Roles in Arabidopsis thaliana and in the Human Malaria Parasite Plasmodium falciparum

Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin‐proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co‐participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT‐PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation.

Unlike the synchronous Plasmodium falciparum and P. chabaudi infection, the P. berghei and P. yoelii asynchronous infections are not affected by melatonin

We have previously reported that Plasmodium chabaudi and P. falciparum sense the hormone melatonin and this could be responsible for the synchrony of malaria infection. In P. chabaudi and P. falciparum, melatonin induces calcium release from internal stores, and this response is abolished by U73122, a phospholipase C inhibitor, and luzindole, a melatonin-receptor competitive antagonist. Here we show that, in vitro, melatonin is not able to modulate cell cycle, nor to elicit an elevation in intracellular calcium concentration of the intraerythrocytic forms of P. berghei or P. yoelii, two rodent parasites that show an asynchrononous development in vivo. Interestingly, melatonin and its receptor do not seem to play a role during hepatic infection by P. berghei sporozoites either. These data strengthen the hypothesis that host-derived melatonin does not synchronize malaria infection caused by P. berghei and P. yoelii. Moreover, these data explain why infections by these parasites are asynchronous, contrary to what is observed in P. falciparum and P. chabaudi infections.

The PfNF-YB transcription factor is a downstream target of melatonin and cAMP signalling in the human malaria parasite Plasmodium falciparum.

Abstract:  Plasmodium falciparum causes the most severe form of malaria and is responsible for the majority of deaths worldwide. The mechanism of cell cycle control within intra‐erythrocytic stages has been examined as a potential means of a promising way to identifying how to stop parasite development in red blood cells. Our group determined that melatonin increases parasitemia in P. falciparum and P. chabaudi through a complex signalling cascade. In vertebrates, melatonin controls the expression of transcription factors, leading us to postulate rather that the indoleamine would affect PfNF‐YB expression in human malaria parasites. We show here that PfNF‐YB transcription factor is highly expressed and colocalized in the nucleus in mature parasites during intra‐erythrocytic stages, thus suggesting an important role in cell division. Moreover, we demonstrate for the first time that melatonin and cAMP modulate the PfNF‐YB transcription factor expression in P. falciparum at erythrocytic stages. In addition, PfNF‐YB is found to be more ubiquitinated in the presence of melatonin. Finally, the proteasome inhibitor bortezomib is able to modulate PfNF‐YB expression as well. Taken together, our dada reinforce the role played by melatonin in the cell cycle control of P. falciparum and point this indolamine as a target to develop new antimalarial drugs.

InsP3 Signaling in Apicomplexan Parasites.

Background: Phosphoinositides (PIs) and their derivatives are essential cellular components that form the building blocks for cell membranes and regulate numerous cell functions. Specifically, the ability to generate myo-inositol 1,4,5-trisphosphate (InsP3) via phospholipase C (PLC) dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to InsP3 and diacylglycerol (DAG) initiates intracellular calcium signaling events representing a fundamental signaling mechanism dependent on PIs. InsP3 produced by PI turnover as a second messenger causes intracellular calcium release, especially from endoplasmic reticulum, by binding to the InsP3 receptor (InsP3R). Various PIs and the enzymes, such as phosphatidylinositol synthase and phosphatidylinositol 4-kinase, necessary for their turnover have been characterized in Apicomplexa, a large phylum of mostly commensal organisms that also includes several clinically relevant parasites. However, InsP3Rs have not been identified in genomes of apicomplexans, despite evidence that these parasites produce InsP3 that mediates intracellular Ca2+ signaling. Conclusion: Evidence to supporting IP3-dependent signaling cascades in apicomplexans suggests that they may harbor a primitive or non-canonical InsP3R. Understanding these pathways may be informative about early branching eukaryotes, where such signaling pathways also diverge from animal systems, thus identifying potential novel and essential targets for therapeutic intervention.

Encapsulation of metalloporphyrins improves their capacity to block the viability of the human malaria parasite Plasmodium falciparum

UNLABELLED Several synthetic metallated protoporphyrins (M-PPIX) were tested for their ability to block the cell cycle of the lethal human malaria parasite Plasmodium falciparum. After encapsulating the porphyrin derivatives in micro- and nanocapsules of marine atelocollagen, their effects on cultures of red blood cells infected (RBC) with P. falciparum were verified. RBCs infected with synchronized P. falciparum incubated for 48 h showed a toxic effect over a micromolar range. Strikingly, the IC50 of encapsulated metalloporphyrins reached nanomolar concentrations, where Zn-PPIX showed the best antimalarial effect, with an IC50=330 nM. This value is an 80-fold increase in the antimalarial activity compared to the antimalarial effect of non-encapsulated Zn-PPIX. These findings reveal that the incubation of P. falciparum infected-RBCs with 20 μM Zn-PPIX reduced the size of hemozoin crystal by 34%, whereas a 28% reduction was noticed with chloroquine, confirming the importance of heme detoxification pathway in drug therapy. FROM THE CLINICAL EDITOR In this study, synthetic metalloporphyrins were tested as therapeutics that target Plasmodium falciparum. The IC50 of encapsulated metalloporphyrins was found to be in the nanomolar concentration range, with encapsulated Zn-PPIX showing an 80-fold increase in its antimalarial activity compared to the non-encapsulated form.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely, E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium.

Efeito de extratos e óleos essenciais de plantas na germinação de urediniósporos de Phakopsora pachyrhizi

Alternative methods to control plant diseases and plagues have been studied, in order to cause less damages to the environment and human health. The purpose of this study was to identify plant extracts and essential oils as possible alternative products that affect the urediniospore germination of Phakopsora pachyrhizi, the causal agent of the Asian soybean rust. Plant extracts (61) from the native flora of the Alto Rio Grande region, State of Minas Gerais, Brazil, as well as five commercial essential oils were used with the purpose of testing their effects on the germination of urediniospores of the referred fungus. 6cm Petri dishes were used, with water-agar medium, added with 150 μL of plant extract or 6 μL of essential oil mixed to the culture medium. As control, 150 μL of Tween 20 at 0.7% and 150 μL of distilled water were used. Then, 50 μL of a suspension of urediniospores were added, at a concentration of 2mg5mL-1 and spread with Drigalsky spatula. Then, the plates were put in BOD, at 23(±2oC), for 4 hours. Germination was paralyzed by adding lactoglicerol. The percentage of spore germination was evaluated in a light microscope. Among the vegetal extracts tested, ten of them presented a germination percentage below 15%. The extracts from the Pelargonium sp., Salvia officinalis, Lavandula officinalis, Mentha pulegium and M. arvensis plants presented 3.0%, 3.5%, 3.0%, 2.0% and 4.0% of urediniospore germination, respectively. All the essential oils evaluated were effective in inhibiting germination, especially the Caryophilus aromaticus essential oil that presented 5.0% of germination, with 50.0% of inhibition.

REPLY to Nanomedicine: NMB, 2015; 11:1035

The observations raised by Somsri Wiwanitkit and Viroj Wiwanitkit concerning our recent report about the encapsulation of metalloporphyrins and their effect on human malaria parasite Plasmodium falciparum highlighted the increasing concern in finding new and safe antimalarial therapies and allow an opportunity to better clarify some aspects of our work. It is well known that one of the effects of chloroquine treatment is to impair the hemozoin formation. In our experiments comparing the hemozoin area in living red blood cells (RBC) infected with P. falciparum, we have confirmed that 20 μM of Zn-PPIX had a similar effect as 1 μM chloroquine on hemozoin growth inhibition after 2 hours incubation. Under these conditions, no relevant differences were observed between Zn-PPIX and chloroquine, however both treatments induced a statistically significant (P b 0.05) decrease on hemozoin area when those groups are compared with a control group treated with DMSO only, as shown in figure and methods section (a full comparison between all groups for this experiment is shown on Table 1). In fact, our work confirmed the results previously obtained by Martiney et al listed as reference 8 of the article. The decrease of hemozoin activity by Zn-PPIX reported by