Plasmodium Infection Induces Dyslipidemia and a Hepatic Lipogenic State in the Host through the Inhibition of the AMPK-ACC Pathway.

Malaria is a major parasitic disease of humans and is a health public problem that affects more than 100 countries. In 2017, it caused nearly half a million deaths out of 219 million infections. Malaria is caused by the protozoan parasites of the genus Plasmodium and is transmitted by female mosquitoes of the genus Anopheles. Once in the bloodstream, Plasmodium merozoites invade erythrocytes and proliferate until the cells lyses and release new parasites that invade other erythrocytes. Remarkably, they can manipulate the vertebrate host's lipid metabolism pathways, since they cannot synthesize lipid classes that are essential for their development and replication. In this study, we show that mice infected with Plasmodium chabaudi present a completely different plasma profile from control mice, with marked hyperproteinemia, hypertriglyceridemia, hypoglycemia, and hypocholesterolemia. In addition, white adipose and hepatic tissue and analyses from infected animals revealed the accumulation of triacylglycerol in both tissues and free fatty acids and free cholesterol in the liver. Hepatic mRNA and protein expression of key enzymes and transcription factors involved in lipid metabolism were also altered by P. chabaudi infection, leading to a lipogenic state. The enzyme 5' AMP-activated protein kinase (AMPK), a master regulator of cell energetic metabolism, was also modulated by the parasite, which reduced AMPK phosphorylation levels upon infection. Pretreatment with metformin for 21 days followed by infection with P. chabaudi was effective in preventing infection of mice and also lowered the hepatic accumulation of lipids while activating AMPK. Together, these results provide new and important information on the specific molecular mechanisms induced by the malaria parasite to regulate hepatic lipid metabolism in order to facilitate its development, proliferation, and lifespan in its vertebrate host.

Intracellular proteolysis of kininogen by malaria parasites promotes release of active kinins.

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.

Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium.

Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653-3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca2+ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca2+ chelator, BAPTA. When parasites were treated with cytoplasmic Ca2+ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH2, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca2+.

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.

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 approximately 40 microM, 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.

A Plasmodium chabaudi chabaudi high molecular mass glycoprotein translocated to the host cell membrane by a non-classical secretory pathway.

We have purified and characterized a novel high molecular mass glycoprotein of P. chabaudi chabaudi (Pc550gp) that is transported to the erythrocyte membrane during the intraerythrocytic cycle. Immuno fluorescence assays with polyclonal monospecific antibodies against Pc550gp show that the protein to be localized in the periphery of young trophozoite stages i.e., on the plasma membrane or parasitophorous vacuole membrane. However, in late trophozoites and schizonts the antigen is distributed in both parasite and host cell membranes. These results were confirmed by immunoblotting of isolated parasites and infected host cell membranes at different stages of parasite development. Moreover, alkali extraction of purified infected erythrocyte membranes at mature stages of parasite development does not solubilize Pc550gp, suggesting that it is an integral membrane protein. In addition proteinase K digestion of intact infected host cells induced the disappearance of Pc550gp. Further indicating its transmembrane nature and that it presents extracellular domains susceptible to proteolysis. Brefeldin A or low temperature (15 degrees C) treatment did not affect the translocation of Pc550gp from the parasite compartments to the erythrocyte membrane, indicating that the secretion of Pc550gp does not follow the classical transport pathway described in most eukaryotic cells.

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.

Characterization of Ca2+ transport activity associated with a non-mitochondrial calcium pool in the rodent malaria parasite P. chabaudi.

Non-mitochondrial calcium deposits were investigated in the intraerythrocytic malaria parasite Plasmodium chabaudi at the trophozoite stage by means of arsenazo III in the presence of ATP and the mitochondrial poisons, antimycin and oligomycin. Addition of vanadate and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), both known to interact with SERCA pump, induced calcium release by permeabilized parasites when the medium free calcium concentration was kept at 3.5 microM. The tumor promoter thapsigargin also caused elevation of the free calcium concentration in permeabilized parasites. Our results support the view that P. chabaudi sequesters calcium in an exchangeable form and maintains its calcium homeostasis by way of an endoplasmic reticulum Ca2+ pump.

Calcium homeostasis in intraerythrocytic malaria parasites.

The fluorescent indicator, fura-2, AM, was used to measure free calcium concentrations in the intraerythrocytic malaria parasites of Plasmodium chabaudi and Plasmodium falciparum. In both species the free cytosolic calcium concentration was maintained at low levels (between 40 and 100 nM throughout the maturation process. Digital image analysis of the indicator fluorescence was performed on parasites and evaluated with the aid of a calibration of the calcium response, based on permeabilized parasites, exposed to calcium buffers. This again revealed that free calcium concentrations in the intact parasite are maintained at a predetermined level, regardless of the free calcium in the surrounding milieu. Both species of parasites are thus capable of regulating their internal free calcium levels with high precision, presumably by means of calcium pump ATPases. A small but significant elevation of the cytosolic free calcium concentration by the tumor promoter, thapsigargin, may be taken to reflect the presence of calcium stores in the endoplasmic reticulum in P. falciparum.