Plasmodium falciparum histidine triad protein and calmodulin modulates calcium homeostasis and intracellular proteolysis.

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.

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4.

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.

Calmidazolium evokes high calcium fluctuations in Plasmodium falciparum.

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.

Specific calpain activity evaluation in Plasmodium parasites.

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.

Plasmodium falciparum proteases hydrolyze plasminogen, generating angiostatin-like fragments.

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.

A nature-inspired betalainic probe for live-cell imaging of Plasmodium-infected erythrocytes.

A model betalainic dye was semisynthesized from betanin, the magenta pigment of the red beet, and was effective for live-cell imaging of Plasmodium-infected red blood cells. This water-soluble fluorescent probe is photostable, excitable in the visible region and cell membrane-permeable, and its photophysical properties are not notably pH-sensitive. Fluorescence imaging microscopy of erythrocytes infected with Plasmodium falciparum, a causative agent of malaria in humans, showed that only the parasite was stained. Z-stacking analysis suggested that the probe accumulates proximal to the nucleus of the parasite. Indicaxanthin, one of the natural fluorescent betalains found in the petals of certain flowers, did not stain the parasite or the red blood cell.

Substrate specificity studies of the cysteine peptidases falcipain-2 and falcipain-3 from Plasmodium falciparum and demonstration of their kininogenase activity.

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 S3-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 S1 subsite of FP-2 preferentially accommodates peptides containing the positively charged residue Arg in P1, while FP-3 has a clear preference for the hydrophobic residue Leu in this position. The S2 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 P3, while FP-3 hydrolysed the peptide Abz-ALRSSRQ-EDDnp, containing Ala at P3, 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-NH2, 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.

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.

The role of melatonin in parasite biology.

Regarded as the circadian hormone in mammals, melatonin is a highly conserved molecule, present in nearly all species. In this review, we discuss the role of this indolamine and its precursors in the cell biology of parasites and the role of the molecule in the physiology of the host. In Plasmodium, melatonin can modulate intracellular concentrations of calcium and cAMP, which in turn can regulate kinase activity and cell cycle. In Trypanosoma infections, modulation of the immune system by melatonin is extremely important in controlling the parasite population. Melatonin also contributes to the inflammatory response to Toxoplasma gondii infection. Thus, there are a number of unique adaptations involving intricate connections between melatonin and the biology of the parasite-host relationship.

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells.

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.

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.

Calcium signaling in lizard red blood cells.

The ion calcium is a ubiquitous second messenger, present in all eukaryotic cells. It modulates a vast number of cellular events, such as cell division and differentiation, fertilization, cell volume, decodification of external stimuli. To process this variety of information, the cells display a number of calcium pools, which are capable of mobilization for signaling purposes. Here we review the calcium signaling on lizards red blood cells, an interesting model that has been receiving an increasing notice recently. These cells possess a complex machinery to regulate calcium, and display calcium responses to extracellular agonists. Interestingly, the pattern of calcium handling and response are divergent in different lizard families, which enforces the morphological data to their phylogenetic classification, and suggest the radiation of different calcium signaling models in lizards evolution.