RESUMEN
Malaria eradication requires the development of new drugs to combat drug-resistant parasites. We identified bisbenzylisoquinoline alkaloids isolated from Cocculus hirsutus that are active against Plasmodium falciparum blood stages. Synthesis of a library of 94 hemi-synthetic derivatives allowed to identify compound 84 that kills multi-drug resistant clinical isolates in the nanomolar range (median IC50 ranging from 35 to 88 nM). Chemical optimization led to compound 125 with significantly improved preclinical properties. 125 delays the onset of parasitemia in Plasmodium berghei infected mice and inhibits P. falciparum transmission stages in vitro (culture assays), and in vivo using membrane feeding assay in the Anopheles stephensi vector. Compound 125 also impairs P. falciparum development in sporozoite-infected hepatocytes, in the low micromolar range. Finally, by chemical pull-down strategy, we characterized the parasite interactome with trilobine derivatives, identifying protein partners belonging to metabolic pathways that are not targeted by the actual antimalarial drugs or implicated in drug-resistance mechanisms.
RESUMEN
Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.
Asunto(s)
Acuaporinas , Esporozoítos , Animales , Hepatocitos/metabolismo , Humanos , Plasmodium falciparum , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esporozoítos/metabolismo , Tetraspanina 28/metabolismoRESUMEN
Plasmodium falciparum gametocytes, the sexual stage responsible for malaria parasite transmission from humans to mosquitoes, are key targets for malaria elimination. Immature gametocytes develop in the human bone marrow parenchyma, where they accumulate around erythroblastic islands. Notably though, the interactions between gametocytes and this hematopoietic niche have not been investigated. Here, we identify late erythroblasts as a new host cell for P falciparum sexual stages and show that gametocytes can fully develop inside these nucleated cells in vitro and in vivo, leading to infectious mature gametocytes within reticulocytes. Strikingly, we found that infection of erythroblasts by gametocytes and parasite-derived extracellular vesicles delay erythroid differentiation, thereby allowing gametocyte maturation to coincide with the release of their host cell from the bone marrow. Taken together, our findings highlight new mechanisms that are pivotal for the maintenance of immature gametocytes in the bone marrow and provide further insights on how Plasmodium parasites interfere with erythropoiesis and contribute to anemia in malaria patients.
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Eritroblastos/parasitología , Eritropoyesis , Interacciones Huésped-Parásitos , Malaria Falciparum/fisiopatología , Plasmodium falciparum/fisiología , Adulto , Médula Ósea/parasitología , Médula Ósea/fisiopatología , Células Cultivadas , Eritroblastos/patología , Femenino , Humanos , Malaria Falciparum/parasitología , Adulto JovenRESUMEN
Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.
Asunto(s)
Gametogénesis , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Fosfolipasas/metabolismo , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Biología Computacional/métodos , Humanos , Estadios del Ciclo de Vida , Fosfolipasas/genética , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/genética , Eliminación de SecuenciaRESUMEN
Plasmodium falciparum is the species of human malaria parasite that causes the most severe form of the disease. Here, we used single-molecule real-time (SMRT) sequencing technology from Pacific Biosciences (PacBio) to sequence, assemble de novo, and annotate the genome of a P. falciparum NF54 clone.
RESUMEN
Malaria sporozoites must first undergo intrahepatic development before a pathogenic blood-stage infection is established. The success of infection depends on host and parasite factors. In healthy human volunteers undergoing controlled human malaria infection (CHMI), we directly compared three clinical Plasmodium falciparum isolates for their ability to infect primary human hepatocytes in vitro and to drive the production of blood-stage parasites in vivo. Our data show a correlation between the efficiency of strain-specific sporozoite invasion of human hepatocytes and the dynamics of patent parasitemia in study subjects, highlighting intrinsic differences in infectivity among P. falciparum isolates from distinct geographical locales. The observed heterogeneity in infectivity among strains underscores the value of assessing the protective efficacy of candidate malaria vaccines against heterologous strains in the CHMI model.
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Malaria Falciparum/parasitología , Parasitemia/parasitología , Esporozoítos/patogenicidad , Femenino , Hepatocitos/parasitología , Humanos , Malaria Falciparum/sangre , Parasitemia/sangre , Plasmodium falciparum/patogenicidad , Estudios Retrospectivos , VoluntariosRESUMEN
Surface-associated TRAP (thrombospondin-related anonymous protein) family proteins are conserved across the phylum of apicomplexan parasites. TRAP proteins are thought to play an integral role in parasite motility and cell invasion by linking the extracellular environment with the parasite submembrane actomyosin motor. Blood stage forms of the malaria parasite Plasmodium express a TRAP family protein called merozoite-TRAP (MTRAP) that has been implicated in erythrocyte invasion. Using MTRAP-deficient mutants of the rodent-infecting P. berghei and human-infecting P. falciparum parasites, we show that MTRAP is dispensable for erythrocyte invasion. Instead, MTRAP is essential for gamete egress from erythrocytes, where it is necessary for the disruption of the gamete-containing parasitophorous vacuole membrane, and thus for parasite transmission to mosquitoes. This indicates that motor-binding TRAP family members function not just in parasite motility and cell invasion but also in membrane disruption and cell egress.
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Eritrocitos/parasitología , Exocitosis , Merozoítos/fisiología , Plasmodium berghei/fisiología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Vacuolas/parasitología , Animales , Culicidae , Humanos , Membranas/metabolismo , RatonesRESUMEN
The development of new drugs to disrupt malaria transmission requires the establishment of an in vivo model to address the biology of Plasmodium falciparum sexual stages (gametocytes). Herein we show that chemically immune-modulated NSG mice grafted with human erythrocytes support complete sexual development of P. falciparum parasites and generate high gametocytemia. Immunohistochemistry and RT-qPCR analyses indicate an enrichment of immature gametocytes in the bone marrow and the spleen, suggesting a sequestration mechanism reminiscent to that observed in humans. Upon primaquine treatment, elimination of gametocytes from peripheral blood and from sequestration sites was observed, providing a proof of concept that these mice can be used for testing drugs. Therefore, this model allows the investigation of P. falciparum sexual commitment, gametocyte interactions with the bone marrow and spleen and provides the missing link between current in vitro assays and Phase I trials in humans for testing new malaria gametocytidal drugs.
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Antimaláricos/administración & dosificación , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Primaquina/administración & dosificación , Animales , Antimaláricos/farmacología , Médula Ósea/efectos de los fármacos , Médula Ósea/parasitología , Modelos Animales de Enfermedad , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Inyecciones Intraperitoneales , Ratones , Plasmodium falciparum/efectos de los fármacos , Primaquina/farmacología , Bazo/efectos de los fármacos , Bazo/parasitologíaRESUMEN
Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIEs) allows them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR (SubTElomeric Variable Open Reading frame) exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation, and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by protein kinase A (PKA)-mediated phosphorylation of the STEVOR C-terminal domain at a specific serine residue (S324). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterase inhibitors may block malaria parasite transmission.
Asunto(s)
Antígenos de Protozoos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Deformación Eritrocítica , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Plasmodium falciparum , Animales , Células Cultivadas , Interacciones Huésped-Parásitos , Humanos , Malaria Falciparum/sangre , Fosforilación , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismoRESUMEN
Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanized mice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Here we improve on previous protocols to achieve efficient double engraftment of TK-NOG mice by human primary hepatocytes and red blood cells. Thus, we obtain the complete hepatic development of P. falciparum, the transition to the erythrocytic stages, their subsequent multiplication, and the appearance of mature gametocytes over an extended period of observation. Furthermore, using sporozoites derived from two P. ovale-infected patients, we show that human hepatocytes engrafted in TK-NOG mice sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG mice are highly suited for in vivo observations on the Plasmodium species of humans.
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Modelos Animales de Enfermedad , Hígado/parasitología , Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium ovale/crecimiento & desarrollo , Animales , Transfusión de Eritrocitos , Femenino , Hepatocitos/trasplante , Humanos , Estadios del Ciclo de Vida , Masculino , Ratones Transgénicos , Esporozoítos/fisiologíaRESUMEN
Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.
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Deformación Eritrocítica/fisiología , Eritrocitos/parasitología , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , Transducción de Señal , Animales , Culicidae , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Malaria Falciparum/transmisiónRESUMEN
A preliminary in vitro screening of compounds belonging to various chemical families from our library revealed the thieno[3,2-d]pyrimidin-4(3H)-one scaffold displayed a promising profile against Plasmodium falciparum. Then, 120 new derivatives were synthesized and evaluated in vitro; compared to drug references, 40 showed good activity toward chloroquine sensitive (IC50 35-344 nM) and resistant (IC50 45-800 nM) P. falciparum strains. They were neither cytotoxic (CC50 15-50 µM) toward HepG2 and CHO cells, nor mutagenic. Structure-activity relationships were defined. The lead-compound also appeared active against the Plasmodium liver stages (Plasmodium yoelii IC50 = 35 nM) and a preliminary in vivo evaluation indicated the in vitro activity was preserved (45% reduction in parasitemia compared to untreated infected mice). A mechanistic study demonstrated these molecules do not involve any of the pathways described for commercial drugs and exert a specific activity on the ring and trophozoite stages.
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Antimaláricos/farmacología , Descubrimiento de Drogas , Eritrocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Malaria/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Pirimidinas/química , Animales , Antimaláricos/química , Células CHO , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Células Hep G2 , Humanos , Malaria/parasitología , Masculino , Ratones , Parasitemia/tratamiento farmacológico , Parasitemia/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Relación Estructura-Actividad , Trofozoítos/efectos de los fármacosRESUMEN
Objective: To evaluate the potential prophylactic activity of traditional polyherbal remedy against malaria. Methods: A traditional polyherbal remedy against malaria from Odisha, India was evaluated for its potential prophylactic activity using in vitro hepatic cell lines assay and the murine malaria system Plasmodium yoelii yoelii/Anopheles stephensi. Results: The polyherbal extract inhibited the Plasmodium yoelii hepatic stages in vitro (IC50 0.74 mg/mL), a therapeutic index of 9.54. In mice treated with the aqueous extract (2 000 mg/kg/day), peak parasitaemia values were 81% lower in the experimental 2.35% ± 0.14% as compared to controls 12.62% ± 0.52% (P Conclusions: The observations provide a proof of concept for a traditional malaria prophylactic remedy used by tribal populations in India.
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A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.
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Vacunas contra la Malaria/genética , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Esporozoítos/inmunología , Vacunas Atenuadas/genética , Animales , Humanos , Hígado/parasitología , Vacunas contra la Malaria/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Plasmodium falciparum/genética , Vacunas Atenuadas/inmunologíaRESUMEN
The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC) activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF), PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria.
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Apoptosis/fisiología , Encéfalo/parasitología , Células Endoteliales/parasitología , Pulmón/parasitología , Malaria Falciparum/parasitología , Plasmodium falciparum/aislamiento & purificación , Adhesión Celular/fisiología , Células Cultivadas , HumanosRESUMEN
Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi-infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.
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Hepatocitos/parasitología , Malaria/parasitología , Plasmodium cynomolgi/crecimiento & desarrollo , Esporozoítos/crecimiento & desarrollo , Animales , Línea Celular , Células Cultivadas , Epigénesis Genética , Femenino , Proteínas Fluorescentes Verdes/química , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Histonas/química , Humanos , Macaca/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Recurrencia , Factores de TiempoRESUMEN
The Plasmodium-infected hepatocyte has been considered necessary to prime the immune responses leading to sterile protection after vaccination with attenuated sporozoites. However, it has recently been demonstrated that priming also occurs in the skin. We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes. To this end, we developed a subcutaneous (s.c.) immunization protocol where few, possibly none, of the immunizing irradiated Plasmodium yoelii sporozoites infect hepatocytes, and also used CD81-deficient mice non-permissive to productive hepatocyte infections. We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver. Using sterile immunity as a primary read-out, we exploited an inhibitor of T-cell migration, transgenic mice with conditional depletion of dendritic cells and adoptive transfers of draining lymph node-derived T cells, to provide evidence that responses leading to sterile immunity can be primed in the skin-draining lymph nodes with little, if any, contribution from the infected hepatocyte.