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Malaria is a parasitic disease caused by protozoan parasites from the genus

Background The intra-erythrocytic development of the malaria parasite Plasmodium falciparum depends on the uptake of a number of essential nutrients from the host cell and blood plasma. It is widely recognized that the parasite imports low molecular weight solutes from the plasma and the consumption of these nutrients by P. falciparum has been extensively analysed. However, although it was already shown that the parasite also imports functional proteins from the vertebrate host, the internalization route through the different infected erythrocyte membranes has not yet been elucidated. In order to further understand the uptake mechanism, the study examined the trafficking of human plasminogen from the extracellular medium into P. falciparum-infected red blood cells. Methods Plasmodium falciparum clone 3D7 was cultured in standard HEPES-buffered RPMI 1640 medium supplemented with 0.5% AlbuMAX. Exogenous human plasminogen was added to the P. falciparum culture and the uptake of this protein by the parasites was analysed by electron microscopy and Western blotting. Immunoprecipitation and mass spectrometry were performed to investigate possible protein interactions that may assist plasminogen import into infected erythrocytes. The effect of pharmacological inhibitors of different cellular physiological processes in plasminogen uptake was also tested. Results It was observed that plasminogen was selectively internalized by P. falciparum-infected erythrocytes, with localization in plasma membrane erythrocyte and parasite’s cytosol. The protein was not detected in parasitic food vacuole and haemoglobin-containing vesicles. Furthermore, in erythrocyte cytoplasm, plasminogen was associated with the parasite-derived membranous structures tubovesicular network (TVN) and Maurer’s clefts. Several proteins were identified in immunoprecipitation assay and may be involved in the delivery of plasminogen across the P. falciparum multiple compartments. Conclusion The findings here reported reveal new features regarding the acquisition of plasma proteins of the host by P. falciparum-infected erythrocytes, a mechanism that involves the exomembrane system, which is distinct from the haemoglobin uptake, clarifying a route that may be potentially targeted for inhibition studies.

Malaria is among the tropical diseases that cause the most deaths in Africa. Around 500,000 malaria deaths are reported yearly among African children under the age of five. Chloroquine (CQ) is a low-cost antimalarial used worldwide for the treatment of Plasmodium vivax malaria. Due to resistance mechanisms, CQ is no longer effective against most malaria cases caused by P. falciparum. The World Health Organization recommends artemisinin combination therapies for P. falciparum malaria, but resistance is emerging in Southeast Asia and some parts of Africa. Therefore, new medicines for treating malaria are urgently needed. Previously, our group identified the 4-aminoquinoline DAQ, a CQ analog containing an acetylenic bond in its side chain, which overcomes CQ resistance in K1 P. falciparum strains. In this work, the antiplasmodial profile, drug-like properties, and pharmacokinetics of DAQ were further investigated. DAQ showed no cross-resistance against standard CQ-resistant strains (e.g., Dd2, IPC 4912, RF12) nor against P. falciparum and P. vivax isolates from patients in the Brazilian Amazon. Using drug pressure assays, DAQ showed a low propensity to generate resistance. DAQ showed considerable solubility but low metabolic stability. The main metabolite was identified as a mono N-deethylated derivative (DAQM), which also showed significant inhibitory activity against CQ-resistant P. falciparum strains. Our findings indicated that the presence of a triple bond in CQ-analogues may represent a low-cost opportunity to overcome known mechanisms of resistance in the malaria parasite.

Cell-to-cell interactions mediated by intercellular junctions (IJs) are crucial for beta-cell functioning and proper insulin secretion, however, their role in type-2 diabetes is still unclear. This work aimed to evaluate the cellular distribution and expression of proteins associated with adherens (AJs) and gap junctions (GJs) in pancreatic islets of C57BL6 mice fed a high-fat (HF) diet. The administration of HF diet for 30 days induced an increase in body weight, post-prandial glycemia, insulinemia, glucose intolerance, and moderate insulin resistance associated with mild perturbations in insulin secretion. The intercellular content of the AJ-associated proteins (namely, E-, N-cadherins, and α-, β-catenins) was significantly higher in islet cells of HF-fed mice. Inversely, the gap junctional content of Cx36 was significantly decreased, as revealed by immunofluorescence, which was paralleled by a reduction in the frequency of calcium oscillations in islets of prediabetic mice. In conclusion, the endocrine pancreas displays significant changes in the content of several junctional proteins at the cell-cell contact region following short-term HF diet administration, indicating that IJs may be involved in the adaptive response of beta cells seen during this state.

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in P. falciparum.

Malaria is an infectious disease caused by protozoan parasites of the Plasmodium genus through the bite of female Anopheles mosquitoes, affecting 228 million people and causing 415 thousand deaths in 2018. Artemisinin-based combination therapies (ACTs) are the most recommended treatment for malaria; however, the emergence of multidrug resistance has unfortunately limited their effects and challenged the field. In this context, the ocean and its rich biodiversity have emerged as a very promising resource of bioactive compounds and secondary metabolites from different marine organisms. This systematic review of the literature focuses on the advances achieved in the search for new antimalarials from marine sponges, which are ancient organisms that developed defense mechanisms in a hostile environment. The principal inclusion criterion for analysis was articles with compounds with IC50 below 10 µM or 10 µg/mL against P. falciparum culture. The secondary metabolites identified include alkaloids, terpenoids, polyketides endoperoxides and glycosphingolipids. The structural features of active compounds selected in this review may be an interesting scaffold to inspire synthetic development of new antimalarials for selectively targeting parasite cell metabolism.

Background: Interaction of nuclear-distribution element-like 1 with disrupted-in-schizophrenia 1 protein is crucial for neurite outgrowth/neuronal migration, and this interaction competitively inhibits nuclear-distribution element-like 1 peptidase activity. Nuclear-distribution element-like 1 activity is reduced in antipsychotic-naïve first-episode psychosis and in medicated chronic schizophrenia, with even lower activity in treatment-resistant schizophrenia. Aims: The purpose of this study was to investigate in a rat model overexpressing human non-mutant disrupted-in-schizophrenia 1, with consequent dysfunctional disrupted-in-schizophrenia 1 signaling, the relation of nuclear-distribution element-like 1 activity with neurodevelopment and dopamine-related phenotypes. Methods: We measured cell distribution in striatum and cortex by histology and microtomography, and quantified the basal and amphetamine-stimulated locomotion and nuclear-distribution element-like 1 activity (in blood and brain) of transgenic disrupted-in-schizophrenia 1 rat vs wild-type littermate controls. Results: 3D assessment of neuronal cell body number and spatial organization of mercury-impregnated neurons showed defective neuronal positioning, characteristic of impaired cell migration, in striatum/nucleus accumbens, and prefrontal cortex of transgenic disrupted-in-schizophrenia 1 compared to wild-type brains. Basal nuclear-distribution element-like 1 activity was lower in the blood and also in several brain regions of transgenic disrupted-in-schizophrenia 1 compared to wild-type. Locomotion and nuclear-distribution element-like 1 activity were both significantly increased by amphetamine in transgenic disrupted-in-schizophrenia 1, but not in wild-type. Conclusions: Our findings in the transgenic disrupted-in-schizophrenia 1 rat allow us to state that decreased nuclear-distribution element-like 1 activity reflects both a trait (neurodevelopmental phenotype) and a state (amphetamine-induced dopamine release). We thus define here a role for decreased nuclear-distribution element-like 1 peptidase activity both for the developing brain (the neurodevelopmental phenotype) and for the adult (interaction with dopaminergic responses), and present nuclear-distribution element-like 1 activity in a novel way, as unifying neurodevelopmental with dysfunctional dopamine response phenotypes.

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in P. falciparum.

Background Malaria represents a worldwide medical emergency affecting mainly poor areas. Plasmodium parasites during blood stages can release kinins to the extracellular space after internalization of host kininogen inside erythrocytes and these released peptides could represent an important mechanism in liver pathophysiology by activation of calcium signaling pathway in endothelial cells of vertebrate host. Receptors (B1 and B2) activated by kinins peptides are important elements for the control of haemodynamics in liver and its physiology. The aim of this study was to identify changes in the liver host responses (i.e. kinin receptors expression and localization) and the effect of ACE inhibition during malaria infection using a murine model. Methods Balb/C mice infected by Plasmodium chabaudi were treated with captopril, an angiotensin I-converting enzyme (ACE) inhibitor, used alone or in association with the anti-malarial chloroquine in order to study the effect of ACE inhibition on mice survival and the activation of liver responses involving B1R and B2R signaling pathways. The kinin receptors (B1R and B2R) expression and localization was analysed in liver by western blotting and immunolocalization in different conditions. Results It was verified that captopril treatment caused host death during the peak of malaria infection (parasitaemia about 45%). B1R expression was stimulated in endothelial cells of sinusoids and other blood vessels of mice liver infected by P. chabaudi. At the same time, it was also demonstrated that B1R knockout mice infected presented a significant reduction of survival. However, the infection did not alter the B2R levels and localization in liver blood vessels. Conclusions Thus, it was observed through in vivo studies that the vasodilation induced by plasma ACE inhibition increases mice mortality during P. chabaudi infection. Besides, it was also seen that the anti-malarial chloroquine causes changes in B1R expression in liver, even after days of parasite clearance. The differential expression of B1R and B2R in liver during malaria infection may have an important role in the disease pathophysiology and represents an issue for clinical treatments.

We have identified previously a panel of markers (C1orf24, ITM1 and PVALB) that can help to discriminate benign from malignant thyroid lesions. C1orf24 and ITM1 are specifically helpful for detecting a wide range of thyroid carcinomas, and PVALB is particularly valuable for detecting the benign Hürthle cell adenoma. Although these markers may ultimately help patient care, the current understanding of their biological functions remains largely unknown. In this article, we investigated whether PVALB is critical for the acquisition of Hürthle cell features and explored the molecular mechanism underlying the phenotypic changes. Through ectopic expression ofPVALBin thyroid carcinoma cell lines (FTC-133 and WRO), we demonstrated that PVALB sequesters free cytoplasmic Ca2+, which ultimately lowers calcium levels and precludes endoplasmic reticulum (ER) Ca2+refilling. These results were accompanied by induced expression of PERK, an ER stress marker. Additionally, forced expression of PVALB reduces Ca2+inflow in the mitochondria, which can in turn cause changes in mitochondria morphology, increase mitochondria number and alter subcellular localization. These findings share striking similarity to those observed in Hürthle cell tumors. Moreover, PVALB inhibits cell growth and induces cell death, most likely through the AKT/GSK-3β. Finally, PVALB expression coincides with Ca2+deposits in HCA tissues. Our data support the hypothesis that the loss ofPVALBplays a role in the pathogenesis of thyroid tumors.

Despite the unquestionable importance of the highly cationic feature of several small polypeptides with high content of positively charged amino acids for their biological activities, positively charged peptides do not necessarily have the capacity to cross the cell membranes. Interestingly, we found that crotamine, a positively charged amphiphilic peptide from the South American rattlesnake venom, has a unique cell-penetrating property with affinity for acidic vesicles, besides a well-characterized antimicrobial and antitumoral activities. In spite of a remarkable in vitro antifungal activity of crotamine against Candida spp., no significant effect of this peptide could be observed in the course of Candida albicans and Candida krusei infection on Caenorhabditis elegans asssed in vivo. These experiments, in which the nematode C. elegans was used as a living host, suggested, however, the potential anthelmintic activity of crotamine because of its uptake by the worms and accumulation in their acidic compartments. As described in the present work, this lysosomotropic property is consistent with a previously proposed mechanism of toxicity of crotamine on mammalian tumoral cell lines. This study also allowed us to propose the cationic peptides with lysosomotropic property, as crotamine, as a potential new class of anthelmentics with ability to overcome the challenging problems of drug resistance.

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Calcium signaling has an essential role in fundamental processes of Plasmodium life cycle, including migration, cell invasion and parasite development. Two important players in calcium homeostasis, the Histidine Triad (HIT) protein that is implicated in calcium signaling in mammalian cells and calmodulin, which is a classic calcium sensor in eukaryotes are present in Plasmodium falciparum, however theirs function is unknown in the parasite. Here, we investigated the involvement of the P. falciparum Histidine Triad protein (PfHint-1) and calmodulin (PfCaM) in calcium signaling and intracellular proteolysis. For this, we targeted PfHint-1 with a hemagglutinin tail and overexpressed both proteins. We observed that PfHint-1 is expressed throughout the erythrocytic stages and partially colocalizes to the endoplasmic reticulum. Parasites overexpressing PfHint-1 displayed lower ER Ca2+ content and a higher [Ca2+]cyt rise in the parasite cytosol upon Ca2+ addition to the extracellular medium after depletion of ER calcium store. PfCaM-overexpressing parasites exhibit a higher [Ca2+]cyt rise after challenge with the calmodulin inhibitor, calmidazolium. The calcium-dependent proteolytic activity in PfCaM- and PfHint-1-overexpressing parasites was increased and correlated to alterations in calcium homeostasis. Taken together, our results indicate the participation of these proteins in P. falciparum fundamental cellular processes and highlights promising targets for the development of antimalarial drugs.

Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQS 3D7 and CQR W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC50 values were 3.7±1.0μM, 1.1±0.2μM and 0.2±0.01μM, respectively. Using an assay performed in the presence of the ER Ca(2+)-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC50=18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.

Malaria is a disease caused by Plasmodium parasites and remains one of the most prevalent and persistent maladies, affecting hundreds of millions of people. In the present work, we evaluated the capability of Plasmodium falciparum proteases to hydrolyze the multifunctional protein plasminogen, which is implicated in angiogenesis and coagulation processes by the generation of angiostatin and plasmin, respectively. Using fluorescence microscopy, we visualized the internalization of FITC-labeled plasminogen in erythrocytes infected by P. falciparum and showed that the parasites are able to hydrolyze the protein. The cleavage of plasminogen by the P. falciparum proteases was also observed by SDS-PAGE, followed by immunoblotting with anti-angiostatin antibody. N-terminal sequencing of the main generated fragments indicated that they are comprised in the five plasminogen kringle domains, suggesting as being angiostatin-like peptides. This assumption was reinforced by the demonstration that the products of plasminogen processing mimic angiostatin functions, including the capability to inhibit angiogenesis and to stimulate calcium response in endothelial cells in vitro. However, no plasmin activity was detected after plasminogen hydrolysis by P. falciparum. Nonetheless, exogenous tissue plasminogen activator (tPA) activated plasmin in infected erythrocytes, suggesting that the uptake of plasminogen by P. falciparum may be modulated by the vertebrate host. Taken together, the data presented here provide evidence for the processing of host plasminogen by malaria parasites to generate active fragments that may modulate host physiology events during malaria infection.

The development of new antimalarial drugs is urgent to overcome the spread of resistance to the current treatment. Herein we synthesized the compound 3, a hit-to‑lead optimization of a thiazole based on the most promising 3-alkylpyridine marine alkaloid analog. Compound 3 was tested against Plasmodium falciparum and has shown to be more potent than its precursor (IC50 values of 1.55 and 14.7 μM, respectively), with higher selectivity index (74.7) for noncancerous human cell line. This compound was not mutagenic and showed genotoxicity only at concentrations four-fold higher than its IC50. Compound 3 was tested in vivo against Plasmodium berghei NK65 strain and inhibited the development of parasite at 50 mg/kg. In silico and UV–vis approaches determined that compound 3 acts impairing hemozoin crystallization and confocal microscopy experiments corroborate these findings as the compound was capable of diminishing food vacuole acidity. The assay of uptake using human intestinal Caco-2 cell line showed that compound 3 is absorbed similarly to chloroquine, a standard antimalarial agent. Therefore, we present here compound 3 as a potent new lead antimalarial compound.

Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model.

BackgroundSleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca2+) plays a critical signaling role. We performed live cell measurements of cytosolic Ca2+ mobilization to understand the changes in Ca2+ signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).MethodsAdult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72h) and Ca2+ intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca2+ mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca2+ channel gene expression was evaluatedResultsSplenocytes showed a progressive loss of intracellular Ca2+ maintenance from endoplasmic reticulum (ER) stores. Transient Ca2+ buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca2+ channels.Conclusions and general significanceThese novel data suggest that SD impairs Ca2+ signaling, most likely as a result of ER stress, leading to an insufficient Ca2+ supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.

Calcium (Ca(2+) ) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra-erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca(2+) . Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca(2+) and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca(2+) within the parasite, because buffering Ca(2+) changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca(2+) signals within the parasite, as did the application of a membrane-permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca(2+) and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca(2+) evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.

Calcium and calmodulin (CaM) are important players in eukaryote cell signaling. In the present study, by using a knockin approach, we demonstrated the expression and localization of CaM in all erythrocytic stages of Plasmodium falciparum. Under extracellular Ca(2+)-free conditions, calmidazolium (CZ), a potent CaM inhibitor, promoted a transient cytosolic calcium ([Ca(2+)]cyt) increase in isolated trophozoites, indicating that CZ mobilizes intracellular sources of calcium. In the same extracellular Ca(2+)-free conditions, the [Ca(2+)]cyt rise elicited by CZ treatment was ~3.5 fold higher when the endoplasmic reticulum (ER) calcium store was previously depleted ruling out the mobilization of calcium from the ER by CZ. The effects of the Ca(2+)/H(+) ionophore ionomycin (ION) and the Na(+)/H(+) ionophore monensin (MON) suggest that the [Ca(2+)]cyt-increasing effect of CZ is driven by the removal of Ca(2+) from at least one Ca(2+)-CaM-related (CaMR) protein as well as by the mobilization of Ca(2+) from intracellular acidic calcium stores. Moreover, we showed that the mitochondrion participates in the sequestration of the cytosolic Ca(2+) elicited by CZ. Finally, the modulation of membrane Ca(2+) channels by CZ and thapsigargin (THG) was demonstrated. The opened channels were blocked by the unspecific calcium channel blocker Co(2+) but not by 2-APB (capacitative calcium entry inhibitor) or nifedipine (L-type Ca(2+) channel inhibitor). Taken together, the results suggested that one CaMR protein is an important modulator of calcium signaling and homeostasis during the Plasmodium intraerythrocytic cell cycle, working as a relevant intracellular Ca(2+) reservoir in the parasite.

Background Parasitic diseases like malaria are a major public health problem in many countries and disrupted sleep patterns are an increasingly common part of modern life. The aim of this study was to assess the effects of paradoxical sleep deprivation (PSD) and sleep rebound (RB) on malarial parasite infection in mice. Methods After PSD, one group was immediately infected with parasites (PSD). The two other PSD rebound groups were allowed to sleep normally for either 24 h (24 h RB) or 48 h (48 h RB). After the recovery periods, mice were inoculated with parasites. Results The PSD group was the most affected by parasites presenting the higher death rate (0.02), higher number of infected cells (p < 0.01), and decrease in body weight (p < 0.04) compared to control and 48 h RB groups. The 24 h RB group was also different from control group in survival (p < 0.03), number of infected cells (p < 0.05) and body weight (p < 0.04). After 48 hours of sleep rebound animals were allowed to restore their response to parasitic infection similar to normal sleep animals. Conclusions These results suggest that PSD is damaging to the immune system and leads to an increased infection severity of malaria parasites; only 48 hours of recovery sleep was sufficient to return the mice infection response to baseline values. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0690-7) contains supplementary material, which is available to authorized users.

By using the fluorescent dye Rhod-2, we have investigated the ability of Plasmodium mitochondria to participate in cellular Ca2+ homeostasis. To this end, isolated parasites were simultaneously loaded with the mitochondrial Ca2+ probe Rhod-2 and the cytosolic Ca2+ dye Fluo-3 and their fluorescent intensities were monitored in the same cells by confocal microscopy. We here demonstrate that Ca2+ increases, as elicited by treatment of parasites with sarco-endoplasmic reticulum Ca2+ ATPase inhibitors or the hormone melatonin, induce rapid and reversible increases of the Ca2+ concentration in the mitochondria of both human and murine parasites. Pre-treatment of parasites with the mitochondrial uncoupler, FCCP, suppresses mitochondrial Ca2+ accumulation. Our data demonstrate that mitochondria of malaria parasites are able to reversibly accumulate part of the Ca2+ released in the cytoplasm by pharmacological and physiological agents and thus suggest that this organelle participate in the maintenance of Ca2+ homeostasis of Plasmodia.

Malaria is a devastating disease caused by a unicellular protozoan, Plasmodium, which affects 3.7 million people every year. Resistance of the parasite to classical treatments such as chloroquine requires the development of new drugs. To gain insight into the mechanisms that control Plasmodium cell cycle, we have examined the effects of kinase inhibitors on the blood-stage cycle of the rodent malaria parasite, Plasmodium chabaudi. In vitro incubation of red blood cells for 17 h at 37 degrees C with the inhibitors led to a decrease in the percent of infected cells, compared to control treatment, as follows: genistein (200 microM - 75%), staurosporine (1 microM - 58%), R03 (1 microM - 75%), and tyrphostins B44 (100 microM - 66%) and B46 (100 microM - 68%). All these treatments were shown to retard or prevent maturation of the intraerythrocytic parasites. The diverse concentration ranges at which these inhibitors exert their effects give a clue as to the types of signals that initiate the transitions between the different developmental stages of the parasite. The present data support our hypothesis that the maturation of the intraerythrocytic cycle of malaria parasites requires phosphorylation. In this respect, we have recently reported a high Ca2+ microenvironment surrounding the parasite within red blood cells. Several kinase activities are modulated by Ca2+. The molecular identification of the targets of these kinases could provide new strategies against malaria.

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+] (

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.

Antimalarial drug resistance remains a major obstacle in malaria control. Evidence from Southeast Asia shows that resistance to artemisinin combination therapy (ACT) is inevitable. Ethnopharmacological studies have confirmed the efficacy of curcumin against Plasmodium spp. Drug interaction assays between curcumin/piperine/chloroquine and curcumin/piperine/artemisinin combinations and the potential of drug treatment to interfere with the ubiquitin proteasome system (UPS) were analyzed. In vivo efficacy of curcumin was studied in BALB/c mice infected with Plasmodium chabaudi clones resistant to chloroquine and artemisinin, and drug interactions were analyzed by isobolograms. Subtherapeutic doses of curcumin, chloroquine, and artemisinin were administered to mice, and mRNA was collected following treatment for RT-PCR analysis of genes encoding deubiquitylating enzymes (DUBs). Curcumin was found be nontoxic in BALB/c mice. The combination of curcumin/chloroquine/piperine reduced parasitemia to 37% seven days after treatment versus the control group's 65%, and an additive interaction was revealed. Curcumin/piperine/artemisinin combination did not show a favorable drug interaction in this murine model of malaria. Treatment of mice with subtherapeutic doses of the drugs resulted in a transient increase in genes encoding DUBs indicating UPS interference. If curcumin is to join the arsenal of available antimalarial drugs, future studies exploring suitable drug partners would be of interest. publishersversion published

Antimalarial drug resistance remains a major obstacle in malaria control. Evidence from Southeast Asia shows that resistance to artemisinin combination therapy (ACT) is inevitable. Ethnopharmacological studies have confirmed the efficacy of curcumin againstPlasmodiumspp. Drug interaction assays between curcumin/piperine/chloroquine and curcumin/piperine/artemisinin combinations and the potential of drug treatment to interfere with the ubiquitin proteasome system (UPS) were analyzed.In vivoefficacy of curcumin was studied in BALB/c mice infected withPlasmodium chabaudiclones resistant to chloroquine and artemisinin, and drug interactions were analyzed by isobolograms. Subtherapeutic doses of curcumin, chloroquine, and artemisinin were administered to mice, and mRNA was collected following treatment for RT-PCR analysis of genes encoding deubiquitylating enzymes (DUBs). Curcumin was found be nontoxic in BALB/c mice. The combination of curcumin/chloroquine/piperine reduced parasitemia to 37% seven days after treatment versus the control group’s 65%, and an additive interaction was revealed. Curcumin/piperine/artemisinin combination did not show a favorable drug interaction in this murine model of malaria. Treatment of mice with subtherapeutic doses of the drugs resulted in a transient increase in genes encoding DUBs indicating UPS interference. If curcumin is to join the arsenal of available antimalarial drugs, future studies exploring suitable drug partners would be of interest.

We studied the substrate specificity requirements of recombinant cysteine peptidases from Plasmodium falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), using fluorescence resonance energy transfer (FRET) peptides as substrates. Systematic modifications were introduced in the lead sequence Abz-KLRSSKQ-EDDnp (Abz = ortho -aminobenzoic acid; EDDnp = N -[2,4-dinitrophenyl]ethylenediamine) resulting in five series assayed to map S 3 − S ′ 2 subsite specificity. Despite high sequence identity and structural similarity between FP-2 and FP-3, noteworthy differences in substrate specificity were observed. The S 1 subsite of FP-2 preferentially accommodates peptides containing the positively charged residue Arg in P 1 , while FP-3 has a clear preference for the hydrophobic residue Leu in this position. The S 2 subsite of FP-2 and FP-3 presents a strict specificity for hydrophobic residues, with Leu being the residue preferred by both enzymes. FP-2 did not show preference for the residues present at P 3 , while FP-3 hydrolysed the peptide Abz-ALRSSRQ-EDDnp, containing Ala at P 3 , with the highest catalytic efficiency of all series studied. FP-2 has high susceptibility for substrates containing hydrophobic residues in P ′ 1 , while FP-3 accommodates well peptides containing Arg in this position. The S ′ 2 subsite of both enzymes demonstrated broad specificity. In addition, radioimmunoassay experiments indicated that kinins can be generated by FP-2 and FP-3 proteolysis of high molecular weight kininogen (HK). Both enzymes excised Met-Lys-bradykinin, Lys-bradykinin and bradykinin from the fluorogenic peptide Abz-MISLMKRPPGFSPFRSSRI-NH 2 , which corresponds to the Met 375 to Ile 393 sequence of HK. The capability of FP-2 and FP-3 to release kinins suggests the involvement of these enzymes in the modulation of malaria pathophysiology.

Cellular defence against the formation of reactive oxygen species (ROS) involves a number of mechanisms in which antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) play an important role. The relation between sleep deprivation and oxidative stress has not yet been completely elucidated. Although some authors did not find evidence of this relationship, others found alterations in some oxidative stress markers in response to sleep deprivation. Thus, the objective of this study was to identify changes induced by sleep deprivation in the activity and gene expression of antioxidant enzymes in mice splenocytes, ideally corroborating a better understanding of the observed effects related to sleep deprivation, which could be triggered by oxidative imbalance. Splenocytes from mice sleep deprived for 72 h showed no significant difference in CAT and CuZnSOD gene expression compared with normal sleep mice. However, sleep-deprived mice did show higher MnSOD gene expression than the control group. Concerning enzymatic activity, CuZnSOD and MnSOD significantly increased after sleep deprivation, despite the expression in CuZnSOD remained unchanged. Moreover, CAT activity was significantly lower after sleep deprivation. The data suggest that the antioxidant system is triggered by sleep deprivation, which in turn could influence the splenocytes homoeostasis, thus interfering in physiological responses.

Background The malaria burden remains a major public health concern, especially in sub-Saharan Africa. The complex biology of Plasmodium, the apicomplexan parasite responsible for this disease, challenges efforts to develop new strategies to control the disease. Proteolysis is a fundamental process in the metabolism of malaria parasites, but roles for proteases in generating vasoactive peptides have not previously been explored. Results In the present work, it was demonstrated by mass spectrometry analysis that Plasmodium parasites (Plasmodium chabaudi and Plasmodium falciparum) internalize and process plasma kininogen, thereby releasing vasoactive kinins (Lys-BK, BK and des-Arg9-BK) that may mediate haemodynamic alterations during acute malaria. In addition, it was demonstrated that the P. falciparum cysteine proteases falcipain-2 and falcipain-3 generated kinins after incubation with human kininogen, suggesting that these enzymes have an important role in this process. The biologic activity of peptides released by Plasmodium parasites was observed by measuring ileum contraction and activation of kinin receptors (B1 and B2) in HUVEC cells; the peptides elicited an increase in intracellular calcium, measured by Fluo-3 AM fluorescence. This effect was suppressed by the specific receptor antagonists Des-Arg9[Leu8]-BK and HOE-140. Conclusions In previously undescribed means of modulating host physiology, it was demonstrated that malaria parasites can generate active kinins by proteolysis of plasma kininogen.

In this study we investigated the peptidase activity in Leishmania ( L. ) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5–9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at R A bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.

Calcium (Ca(2+) ) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra-erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca(2+) . Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca(2+) and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca(2+) within the parasite, because buffering Ca(2+) changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca(2+) signals within the parasite, as did the application of a membrane-permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca(2+) and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca(2+) evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.