RESUMO
Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.
RESUMO
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.
RESUMO
Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.
Assuntos
Antimaláricos/farmacologia , Farnesiltranstransferase/biossíntese , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Ácido Risedrônico/farmacologia , Análise de Variância , Animais , Antimaláricos/análise , Western Blotting , Resistência a Medicamentos , Farnesiltranstransferase/análise , Plasmodium falciparum/crescimento & desenvolvimento , Valores de Referência , Ácido Risedrônico/análiseRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Cistatinas/farmacologia , Cisteína Proteases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Plantas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/isolamento & purificação , Cistatinas/química , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/genética , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/isolamento & purificação , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Eritrócitos/fisiologia , Hemeproteínas/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Proteínas de Plantas/química , Plasmodium falciparum/enzimologiaRESUMO
Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.
Assuntos
Animais , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Farnesiltranstransferase/biossíntese , Ácido Risedrônico/farmacologia , Antimaláricos/farmacologia , Plasmodium falciparum/crescimento & desenvolvimento , Valores de Referência , Resistência a Medicamentos , Western Blotting , Análise de Variância , Farnesiltranstransferase/análise , Ácido Risedrônico/análise , Antimaláricos/análiseRESUMO
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.
Assuntos
Cálcio/metabolismo , Imidazóis/farmacologia , Plasmodium falciparum/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Calmodulina/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Técnicas de Introdução de Genes , Humanos , Microscopia Confocal , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Tapsigargina/farmacologia , Trofozoítos/efeitos dos fármacos , Trofozoítos/metabolismoRESUMO
Malaria is a tropical disease with significant morbidity and mortality. A better understanding of the metabolism of its most important etiological agent, Plasmodium falciparum, is paramount to the development of better treatment and other mitigation measures. Farnesyldiphosphate synthase/geranylgeranyldiphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains present in many essential structures. In P. falciparum, as well as a handful of other organisms, FPPS/GGPPS has been shown to be a bifunctional enzyme. By genetic tagging and microscopy, we observed a changing localization of FPPS/GGPPS in blood stage parasites. Given the great importance of alternative splicing and other transcriptional phenomena in gene regulation and the generation of protein diversity, we have investigated the processing of the FPPS/GGPPS transcript in P. falciparum by high-throughput sequencing methods in four time-points along the intraerythrocytic cycle of P. falciparum. We have identified levels of transcript diversity an order of magnitude higher than previously observed in this organism, as well as a few stage-specific splicing events. Our data suggest that alternative splicing in P. falciparum is an important feature for gene regulation and the generation of protein diversity.
Assuntos
Processamento Alternativo/genética , Geraniltranstransferase/genética , Malária Falciparum/genética , Transcrição Gênica , Animais , Regulação Enzimológica da Expressão Gênica , Variação Genética , Geraniltranstransferase/sangue , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidadeRESUMO
The increasing resistance of malaria parasites to almost all available drugs calls for the characterization of novel targets and the identification of new compounds. Carotenoids are polyisoprenoids from plants, algae, and some bacteria, and they are biosynthesized by Plasmodium falciparum but not by mammalian cells. Biochemical and reverse genetics approaches were applied to demonstrate that phytoene synthase (PSY) is a key enzyme for carotenoid biosynthesis in P. falciparum and is essential for intraerythrocytic growth. The known PSY inhibitor squalestatin reduces biosynthesis of phytoene and kills parasites during the intraerythrocytic cycle. PSY-overexpressing parasites showed increased biosynthesis of phytoene and its derived product phytofluene and presented a squalestatin-resistant phenotype, suggesting that this enzyme is the primary target of action of this drug in the parasite.
Assuntos
Antimaláricos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carotenoides/biossíntese , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Antimaláricos/química , Compostos Bicíclicos Heterocíclicos com Pontes/químicaRESUMO
There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS) interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS) in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.
Assuntos
Malária/parasitologia , Melatonina/farmacologia , Parasitos/genética , Plasmodium falciparum/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Regulação para Cima/genética , Animais , Humanos , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin-proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co-participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT-PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation.
Assuntos
Arabidopsis/fisiologia , Ácidos Indolacéticos/metabolismo , Melatonina/metabolismo , Plasmodium falciparum/fisiologia , Triptofano/metabolismo , Ciclo Celular , Eritrócitos/parasitologia , Desenvolvimento Vegetal , Raízes de Plantas/fisiologia , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Plasmodium falciparum causes the most severe form of malaria and is responsible for the majority of deaths worldwide. The mechanism of cell cycle control within intra-erythrocytic stages has been examined as a potential means of a promising way to identifying how to stop parasite development in red blood cells. Our group determined that melatonin increases parasitemia in P. falciparum and P. chabaudi through a complex signalling cascade. In vertebrates, melatonin controls the expression of transcription factors, leading us to postulate rather that the indoleamine would affect PfNF-YB expression in human malaria parasites. We show here that PfNF-YB transcription factor is highly expressed and colocalized in the nucleus in mature parasites during intra-erythrocytic stages, thus suggesting an important role in cell division. Moreover, we demonstrate for the first time that melatonin and cAMP modulate the PfNF-YB transcription factor expression in P. falciparum at erythrocytic stages. In addition, PfNF-YB is found to be more ubiquitinated in the presence of melatonin. Finally, the proteasome inhibitor bortezomib is able to modulate PfNF-YB expression as well. Taken together, our dada reinforce the role played by melatonin in the cell cycle control of P. falciparum and point this indolamine as a target to develop new antimalarial drugs.
Assuntos
AMP Cíclico/metabolismo , Melatonina/metabolismo , Plasmodium falciparum/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antimaláricos/uso terapêutico , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless, the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real-time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase, plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7(-) parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild-type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7(-) parasites.
Assuntos
Melatonina/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Plasmodium falciparum/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Protozoários/metabolismo , Ubiquitina/metabolismo , Animais , Malária Falciparum , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Plasmodium falciparum/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ubiquitina/genéticaRESUMO
In this review, we bring together some of the approaches toward understanding the cellular and molecular biology of Plasmodium species and their interaction with their host red blood cells. Considerable impetus has come from the development of new methods of molecular genetics and bioinformatics, and it is important to evaluate the wealth of these novel data in the context of basic cell biology. We describe how these approaches are gaining valuable insights into the parasite-host cell interaction, including (1) the multistep process of red blood cell invasion by the merozoite; (2) the mechanisms by which the intracellular parasite feeds on the red blood cell and exports parasite proteins to modify its cytoadherent properties; (3) the modulation of the cell cycle by sensing the environmental tryptophan-related molecules; (4) the mechanism used to survive in a low Ca(2+) concentration inside red blood cells; (5) the activation of signal transduction machinery and the regulation of intracellular calcium; (6) transfection technology; and (7) transcriptional regulation and genome-wide mRNA studies in Plasmodium falciparum.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/genética , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/genética , Plasmodium/genética , Plasmodium/fisiologia , Animais , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica/genética , Genoma de Protozoário/genética , Biologia Molecular/tendências , Plasmodium/ultraestrutura , Transdução de Sinais/genéticaRESUMO
Serpentine receptors comprise a large family of membrane receptors distributed over diverse organisms, such as bacteria, fungi, plants and all metazoans. However, the presence of serpentine receptors in protozoan parasites is largely unknown so far. In the present study we performed a genome-wide search for proteins containing seven transmembrane domains (7-TM) in the human malaria parasite Plasmodium falciparum and identified four serpentine receptor-like proteins. These proteins, denoted PfSR1, PfSR10, PfSR12 and PfSR25, show membrane topologies that resemble those exhibited by members belonging to different families of serpentine receptors. Expression of the pfsrs genes was detected by Real Time PCR in P. falciparum intraerythrocytic stages, indicating that they potentially code for functional proteins. We also found corresponding homologues for the PfSRs in five other Plasmodium species, two primate and three rodent parasites. PfSR10 and 25 are the most conserved receptors among the different species, while PfSR1 and 12 are more divergent. Interestingly, we found that PfSR10 and PfSR12 possess similarity to orphan serpentine receptors of other organisms. The identification of potential parasite membrane receptors raises a new perspective for essential aspects of malaria parasite host cell infection.