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Most protocols used to study the dynamics of calcium (Ca 2+ ) 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 Ca 2+ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca 2+ 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 Ca 2+ fluctuations. These spontaneous cytosolic Ca 2+ 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 Ca 2+ oscillations during the asexual stage in P. falciparum, independent from external stimuli., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The nucleotides were discovered in the early 19th century and a few years later, the role of such molecules in energy metabolism and cell survival was postulated. In 1972, a pioneer work by Burnstock and colleagues suggested that ATP could also work as a neurotransmitter, which was known as the "purinergic hypothesis". The idea of ATP working as a signaling molecule faced initial resistance until the discovery of the receptors for ATP and other nucleotides, called purinergic receptors. Among the purinergic receptors, the P2Y family is of great importance because it comprises of G proteincoupled receptors (GPCRs). GPCRs are widespread among different organisms. These receptors work in the cells' ability to sense the external environment, which involves: to sense a dangerous situation or detect a pheromone through smell; the taste of food that should not be eaten; response to hormones that alter metabolism according to the body's need; or even transform light into an electrical stimulus to generate vision. Advances in understanding the mechanism of action of GPCRs shed light on increasingly promising treatments for diseases that have hitherto remained incurable, or the possibility of abolishing side effects from therapies widely used today., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

•Discuss molecular components for the coordination of circadian rhythm of malaria parasites inside the vertebrate host.•Synthetic indole compounds show antimalarial activity in vitro against P.falciparum 3D7.• Plasmodium falciparum synchronizes in cell culture upon melatonin treatment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Published by Elsevier B.V.)

Cytosolic Ca 2+ regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage Plasmodium falciparum , yet our understanding of Ca 2+ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically encoded Ca 2+ indicator GCaMP3, we have investigated the dynamics of Ca 2+ release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca 2+ from intracellular stores in P. falciparum parasites; (ii) Thg is able to induce Ca 2+ release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca 2+ influx from extracellular media, through a mechanism resembling store-operated Ca 2+ entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca 2+ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m -chlorophenyl hydrazone or the release of acidic Ca 2+ stores with nigericin. These data suggest the presence of two Ca 2+ pools in P. falciparum with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase pump inhibitors, and only the release of the Thg-sensitive Ca 2+ store induces Ca 2+ influx. Activation of the store-operated Ca 2+ entry-like Ca 2+ influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca 2+ levels for their activation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Borges-Pereira et al.)

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., (© 2018 The Authors. Journal of Pineal Research Published by John Wiley & Sons Ltd.)

Studying Ca 2+ dynamics in protozoan parasites is not an easy task. Loading of parasites with commonly used Ca 2+ fluorescent dyes (such as Fuo4-AM) remains as the major protocol to measure the Ca 2+ oscillations inside the cell. In this chapter, we describe an alternative method to study Ca 2+ signaling in Plasmodium falciparum parasite. This method employs the construction of transgenic parasites (through standard molecular biology techniques), selection of the transfected population, and use of those parasites in spectrofluorometric Ca 2+ assays.

Inositol 1,4,5 trisphosphate (IP 3 ) signaling plays a crucial role in a wide range of eukaryotic processes. In Plasmodium falciparum, IP 3 elicits Ca 2+ release from intracellular Ca 2+ stores, even though no IP 3 receptor homolog has been identified to date. The human host hormone melatonin plays a key role in entraining the P. falciparum life cycle in the intraerythrocytic stages, apparently through an IP 3 -dependent Ca 2+ signal. The melatonin-induced cytosolic Ca 2+ ([Ca 2+ ] cyt ) increase and malaria cell cycle can be blocked by the IP 3 receptor blocker 2-aminoethyl diphenylborinate (2-APB). However, 2-APB also inhibits store-operated Ca 2+ entry (SOCE). Therefore, we have used two novel 2-APB derivatives, DPB162-AE and DPB163-AE, which are 100-fold more potent than 2-APB in blocking SOCE in mammalian cells, and appear to act by interfering with clustering of STIM proteins. In the present work we report that DPB162-AE and DPB163-AE block the [Ca 2+ ] cyt rise in response to melatonin in P. falciparum, but only at high concentrations. These compounds also block SOCE in the parasite at similarly high concentrations suggesting that P. falciparum SOCE is not activated in the same way as in mammalian cells. We further find that DPB162-AE and DPB163-AE affect the development of the intraerythrocytic parasites and invasion of new red blood cells. Our efforts to episomally express proteins that compete with native IP 3 receptor like IP 3 -sponge and an IP 3 sensor such as IRIS proved to be lethal to P. falciparum during intraerythrocytic cycle. The present findings point to an important role of IP 3 -induced Ca 2+ release in intraerythrocytic stage of P. falciparum., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plasmodium falciparum is the causative agent of the most dangerous form of malaria in humans. It has been reported that the P. falciparum genome encodes for a single ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), an enzyme that hydrolyzes extracellular tri- and di-phosphate nucleotides. The E-NTPDases are known for participating in invasion and as a virulence factor in many pathogenic protozoa. Despite its presence in the parasite genome, currently, no information exists about the activity of this predicted protein. Here, we show for the first time that P. falciparum E-NTPDase is relevant for parasite lifecycle as inhibition of this enzyme impairs the development of P. falciparum within red blood cells (RBCs). ATPase activity could be detected in rings, trophozoites, and schizonts, as well as qRT-PCR, confirming that E-NTPDase is expressed throughout the intraerythrocytic cycle. In addition, transfection of a construct which expresses approximately the first 500 bp of an E-NTPDase-GFP chimera shows that E-NTPDase co-localizes with the endoplasmic reticulum (ER) in the early stages and with the digestive vacuole (DV) in the late stages of P. falciparum intraerythrocytic cycle.

The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca 2+ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca 2+ ] cyt increase in isolated parasites and this Ca 2+ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite's internal Ca 2+ pools. This response persists even in the absence of free extracellular Ca 2+ and cannot be elicited by addition of Na + , Mg 2+ or Ca 2+ . However, when the PfSR25 gene was deleted, no effect on [Ca 2+ ] cyt was observed in response to changing KCl concentration in the knocked out (PfSR25 - ) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25 - parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25 - parasites, wt parasites showed a better survival in albumax-deprived condition.

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection., Methodology/principal Findings: We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis., Conclusions/significance: In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.

Calcium (Ca(2+)) signaling pathways are vital for all eukaryotic cells. It is well established that changes in Ca(2+) concentration can modulate several physiological processes such as muscle contraction, neurotransmitter secretion and metabolic regulation (Giacomello et al. (2007) [1], Rizzuto and Pozzan (2003) [2]). In the complex life cycle of Plasmodium falciparum, the causative agent of human malaria, Ca(2+) is involved in the processes of protein secretion, motility, cell invasion, cell progression and parasite egress from red blood cells (RBCs) (Koyama et al. (2009) [3]). The generation of P. falciparum expressing genetically encoded calcium indicators (GECIs) represents an innovation in the study of calcium signaling. This development will provide new insight on calcium homeostasis and signaling in P. falciparum. In addition, these novel transgenic parasites, PfGCaMP3, is a useful tool for screening and identifying new classes of compounds with anti-malarial activity. This represents a possibility of interfering with signaling pathways controlling parasite growth and development. Our new method differs from previous loading protocols (Garcia et al. (1996) [4]; Beraldo et al. (2007) [5]) since:•It provides a novel method for imaging calcium fluctuations in the cytosol of P. falciparum, without signal interference from the host cell and invasive loading protocols.•This technique could also be expanded for imaging calcium in different subcellular compartments.•It will be helpful in the development of novel antimalarials capable of disrupting calcium homeostasis during the intraerythrocytic cycle of P. falciparum.

Canine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leishmaniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI=92.60-100.0%) and a high specificity of 100% (95% CI=86.77-100.0%), with a high degree of confidence (k=1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)