RESUMEN
A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.
Asunto(s)
Parásitos , Plasmodium falciparum , Animales , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Vacuolas/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Eritrocitos/parasitología , Parásitos/metabolismo , Péptido Hidrolasas/metabolismoRESUMEN
Research and development leading to new and improved health products is essential for achieving healthier lives for populations worldwide. However, new products in development do not always match the global need for products for neglected diseases and populations. To promote research, provide an incentive for investment and align products with the needs of end-users, research needs to be better coordinated and prioritized. The World Health Organization (WHO) has developed target product profiles that define the characteristics required in new health products to address the greatest public health needs. A WHO target product profile document presents a need and provides guidance on what to include to consider access and equity as part of the research and development plan from the outset. WHO has also set up the Target Product Profile Directory, a free-to-use online database of characteristics used to describe desired health products, including medicines, vaccines, diagnostic tools and medical equipment. Here we describe the process of developing a WHO target product profile, and the benefits of this type of guidance. We urge product developers to share product profiles addressing unmet needs in public health, to help in progress towards global targets for better health and well-being.
Promouvoir la santé des populations à travers le monde va de pair avec la recherche-développement responsable de la conception et de l'optimisation de produits sanitaires. Pourtant, les nouveaux produits à l'étude ne répondent pas toujours aux exigences mondiales des populations et maladies négligées. En vue de promouvoir la recherche, de favoriser les investissements et d'aligner les produits sur les besoins des utilisateurs finaux, les travaux doivent être mieux coordonnés et leurs priorités, mieux définies. L'Organisation mondiale de la Santé (OMS) a donc élaboré des profils de produits cibles qui déterminent les caractéristiques requises pour les nouveaux produits sanitaires, afin qu'ils correspondent davantage aux besoins les plus criants en matière de santé publique. Un profil de produit cible établi par l'OMS est un document qui met en évidence un besoin et fournit des indications sur les aspects à prendre en compte pour garantir l'accès et l'équité dès le départ dans le plan de recherche-développement. L'OMS a également publié un Répertoire des profils de produits cibles, une base de données en ligne consultable gratuitement qui reprend les caractéristiques employées pour décrire les produits sanitaires souhaités (médicaments, vaccins, outils diagnostiques et équipements médicaux). Dans le présent document, nous détaillons le processus de développement d'un profil de produit cible par l'OMS, mais aussi les avantages que comportent de telles indications. Nous encourageons vivement les laboratoires à partager les profils de produits qui répondent à des besoins non satisfaits en matière de santé publique, afin de contribuer à avancer vers les objectifs mondiaux de santé et de bien-être.
La investigación y el desarrollo de productos sanitarios nuevos y mejorados son esenciales para que las poblaciones de todo el mundo vivan más sanas. Sin embargo, los productos nuevos en desarrollo no siempre se ajustan a las necesidades mundiales de productos para enfermedades y poblaciones desatendidas. Para promover la investigación, incentivar la inversión y adaptar los productos a las necesidades de los usuarios finales, es necesario coordinar mejor la investigación y establecer prioridades. La Organización Mundial de la Salud (OMS) ha elaborado perfiles de productos específicos que definen las características que deben reunir los productos sanitarios nuevos para satisfacer las principales necesidades de salud pública. Un documento de perfil de producto específico de la OMS presenta una necesidad y ofrece orientación sobre lo que debe incluirse para tener en cuenta el acceso y la equidad como parte del plan de investigación y desarrollo desde el principio. La OMS también ha creado el Directorio de Perfiles de Productos Específicos, una base de datos en línea de uso gratuito con las características utilizadas para describir los productos sanitarios deseados, incluidos medicamentos, vacunas, herramientas de diagnóstico y equipos médicos. En el presente documento, describimos el proceso de elaboración de un perfil de producto específico de la OMS y las ventajas de este tipo de orientación. Instamos a los desarrolladores de productos a compartir perfiles de productos que aborden necesidades no cubiertas en salud pública para contribuir al avance hacia los objetivos mundiales de mejora de la salud y el bienestar.
Asunto(s)
Vacunas , Humanos , Organización Mundial de la Salud , Investigación , Estado de SaludRESUMEN
BACKGROUND: Rapid diagnostic tests (RDTs) are effective tools to diagnose and inform the treatment of malaria in adults and children. The recent development of a highly sensitive rapid diagnostic test (HS-RDT) for Plasmodium falciparum has prompted questions over whether it could improve the diagnosis of malaria in pregnancy and pregnancy outcomes in malaria endemic areas. METHODS: This landscape review collates studies addressing the clinical performance of the HS-RDT. Thirteen studies were identified comparing the HS-RDT and conventional RDT (co-RDT) to molecular methods to detect malaria in pregnancy. Using data from five completed studies, the association of epidemiological and pregnancy-related factors on the sensitivity of HS-RDT, and comparisons with co-RDT were investigated. The studies were conducted in 4 countries over a range of transmission intensities in largely asymptomatic women. RESULTS: Sensitivity of both RDTs varied widely (HS-RDT range 19.6 to 85.7%, co-RDT range 22.8 to 82.8% compared to molecular testing) yet HS-RDT detected individuals with similar parasite densities across all the studies including different geographies and transmission areas [geometric mean parasitaemia around 100 parasites per µL (p/µL)]. HS-RDTs were capable of detecting low-density parasitaemias and in one study detected around 30% of infections with parasite densities of 0-2 p/µL compared to the co-RDT in the same study which detected around 15%. CONCLUSION: The HS-RDT has a slightly higher analytical sensitivity to detect malaria infections in pregnancy than co-RDT but this mostly translates to only fractional and not statistically significant improvement in clinical performance by gravidity, trimester, geography or transmission intensity. The analysis presented here highlights the need for larger and more studies to evaluate incremental improvements in RDTs. The HS-RDT could be used in any situation where co-RDT are currently used for P. falciparum diagnosis, if storage conditions can be adhered to.
Asunto(s)
Malaria Falciparum , Malaria , Adulto , Embarazo , Niño , Humanos , Femenino , Plasmodium falciparum , Prueba de Diagnóstico Rápido , Sensibilidad y Especificidad , Malaria Falciparum/diagnóstico , Malaria Falciparum/epidemiología , Pruebas Diagnósticas de Rutina/métodos , Antígenos de Protozoos/análisisRESUMEN
Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.
Asunto(s)
Malaria Falciparum , Parásitos , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Parásitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismoRESUMEN
In order to facilitate a number of processes including nutrient acquisition and immune evasion, malaria parasites extensively remodel their host erythrocyte. This remodelling is to a large extent accomplished through protein export, a crucial process mediated by the Plasmodium translocon for exported proteins (PTEX) translocon which is comprised of three core components, HSP101, PTEX150 and EXP2. EXP2 has been structurally and electrophysiologically shown to form the pore that spans the vacuole membrane enveloping the parasite. Here, we biochemically investigate the structure and function of EXP2. By differential alkylation we provide direct evidence that cysteines C113 and C140 form an intramolecular disulphide bond, while C201 is predominantly in a reduced state. We demonstrate that EXP2 possesses a protease resistant, membrane-associated, N-terminal region of â¼20 kDa that does not project into the infected erythrocyte cytosol; however, its C-terminus does project into the vacuole space. We show that a putative transmembrane peptide derived from the N-terminal region of EXP2 is haemolytic and in a polymer-based osmotic protection assay, we demonstrate that this peptide forms a discrete haemolytic pore. This work provides further biochemical insight into the role, function and cellular arrangement of EXP2 as the pore-forming component for protein translocation.
Asunto(s)
Complejos Multiproteicos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Espectrometría de Masas , Transporte de ProteínasRESUMEN
Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.
Asunto(s)
Proteínas Portadoras , Plasmodium falciparum , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Técnicas de Silenciamiento del Gen , Humanos , Estadios del Ciclo de Vida/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM). The Plasmodium translocon for exported (PTEX) proteins is thought to span the PVM and provide a channel that unfolds and extrudes proteins across the PVM into the erythrocyte. We show that exported reporter proteins containing mouse dihydrofolate reductase domains that inducibly resist unfolding become trapped at the parasite surface partly colocalizing with PTEX. When cargo is trapped, loop-like extensions appear at the PVM containing both trapped cargo and PTEX protein EXP2, but not additional components HSP101 and PTEX150. Following removal of the block-inducing compound, export of reporter proteins only partly recovers possibly because much of the trapped cargo is spatially segregated in the loop regions away from PTEX. This suggests that parasites have the means to isolate unfoldable cargo proteins from PTEX-containing export zones to avert disruption of protein export that would reduce parasite growth.
Asunto(s)
Malaria Falciparum/parasitología , Parásitos/metabolismo , Plasmodium falciparum/metabolismo , Transporte de Proteínas/fisiología , Proteínas Protozoarias/metabolismo , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/sangre , Ratones , Vacuolas/parasitologíaRESUMEN
Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Malaria Falciparum/parasitología , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Virulencia/fisiología , Animales , Adhesión Celular/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/parasitología , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/metabolismo , Parásitos/metabolismo , Parásitos/patogenicidadRESUMEN
The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer's clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo.
Asunto(s)
Interacciones Huésped-Patógeno , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Proteínas Portadoras/metabolismo , Adhesión Celular , Femenino , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Plasmodium berghei/genética , Plasmodium falciparum/patogenicidad , Transporte de ProteínasRESUMEN
Plasmodium falciparum extensively modifies its chosen host cell, the mature human erythrocyte. This remodelling is carried out by parasite-encoded proteins that are exported into the host cell. To gain access to the human red blood cell, these proteins must cross the parasitophorous vacuole, a membrane bound compartment surrounding the parasite that is generated during the invasion process. Many exported proteins carry a so-called PEXEL/HT signal that directs their transport. We recently reported the unexpected finding of a species-restricted parasite-encoded Hsp70, termed PfHsp70x, which is exported into the host erythrocyte cytosol. PfHsp70x lacks a classical PEXEL/HT motif, and its transport appears to be mediated by a 7 amino acid motif directly following the hydrophobic N-terminal secretory signal. In this report, we analyse this short targeting sequence in detail. Surprisingly, both a reversed and scrambled version of the motif retained the capacity to confer protein export. Site directed mutagenesis of glutamate residues within this region leads to a block of protein trafficking within the lumen of the PV. In contrast to PEXEL-containing proteins, the targeting signal is not cleaved, but appears to be acetylated. Furthermore we show that, like other exported proteins, trafficking of PfHsp70x requires the vacuolar translocon, PTEX.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Secuencias de Aminoácidos , Eritrocitos/metabolismo , Eritrocitos/parasitología , Proteínas HSP70 de Choque Térmico/genética , Humanos , Plasmodium falciparum/genética , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genéticaRESUMEN
The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.
Asunto(s)
Eritrocitos/parasitología , Proteínas de Transporte de Membrana/fisiología , Plasmodium falciparum/fisiología , Proteoma/metabolismo , Proteínas Protozoarias/fisiología , Células Cultivadas , Humanos , Complejos Multiproteicos/metabolismo , Dominios Proteicos , Mapas de Interacción de Proteínas , Estabilidad Proteica , Transporte de ProteínasRESUMEN
During pregnancy immunoglobulin G (IgG) antibodies are transferred from mother to neonate across the placenta. Studies in high transmission areas have shown transfer of P. falciparum-specific IgG, but the extent and factors influencing maternal-foetal transfer in low transmission areas co-endemic for both P. falciparum and P. vivax are unknown. Pregnant women were screened weekly for Plasmodium infection. Mother-neonate paired serum samples at delivery were tested for IgG to antigens from P. falciparum, P. vivax and other infectious diseases. Antibodies to malarial and non-malarial antigens were highly correlated between maternal and neonatal samples (median [range] spearman ρ = 0.78 [0.57-0.93]), although Plasmodium spp. antibodies tended to be lower in neonates than mothers. Estimated gestational age at last P. falciparum infection, but not P. vivax infection, was positively associated with antibody levels in the neonate (P. falciparum merozoite, spearman ρ median [range] 0.42 [0.33-0.66], PfVAR2CSA 0.69; P. vivax ρ = 0.19 [0.09-0.3]). Maternal-foetal transfer of anti-malarial IgG to Plasmodium spp. antigens occurs in low transmission settings. P. vivax IgG acquisition is not associated with recent exposure unlike P. falciparum IgG, suggesting a difference in acquisition of antibodies. IgG transfer is greatest in the final weeks of pregnancy which has implications for the timing of future malaria vaccination strategies in pregnant women.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Inmunidad Materno-Adquirida , Malaria Falciparum/inmunología , Malaria Falciparum/transmisión , Malaria Vivax/inmunología , Malaria Vivax/transmisión , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunología , Adolescente , Adulto , Anticuerpos Antiprotozoarios/sangre , Estudios de Casos y Controles , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Transmisión Vertical de Enfermedad Infecciosa , Malaria Falciparum/epidemiología , Malaria Vivax/epidemiología , Embarazo , Factores de Riesgo , Estudios Seroepidemiológicos , Tailandia/epidemiología , Adulto JovenRESUMEN
Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.
Asunto(s)
Proteínas Portadoras/genética , Ensayos Analíticos de Alto Rendimiento , Luciferasas/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Trofozoítos/crecimiento & desarrollo , Animales , Transporte Biológico , Brefeldino A/farmacología , Proteínas Portadoras/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Luciérnagas/química , Luciérnagas/enzimología , Furosemida/farmacología , Genes Reporteros , Humanos , Luciferasas/metabolismo , Penaeidae/química , Penaeidae/enzimología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Ingeniería de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Protozoarias/metabolismo , Sensibilidad y Especificidad , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Trofozoítos/efectos de los fármacos , Trofozoítos/metabolismoRESUMEN
During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Malaria/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Eritrocitos/metabolismo , Eritrocitos/parasitología , Proteínas de Choque Térmico/genética , Humanos , Estadios del Ciclo de Vida/fisiología , Complejos Multiproteicos/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Vacuolas/metabolismo , Vacuolas/parasitologíaRESUMEN
Export of proteins into the infected erythrocyte is critical for malaria parasite survival. The majority of effector proteins are thought to export via a proteinaceous translocon, resident in the parasitophorous vacuole membrane surrounding the parasite. Identification of the Plasmodium translocon of exported proteins and its biochemical association with exported proteins suggests it performs this role. Direct evidence for this, however, is lacking. Here using viable purified Plasmodium falciparum merozoites and three-dimensional structured illumination microscopy, we investigate remodelling events immediately following parasite invasion. We show that multiple complexes of the Plasmodium translocon of exported proteins localize together in foci that dynamically change in clustering behaviour. Furthermore, we provide conclusive evidence of spatial association between exported proteins and exported protein 2, a core component of the Plasmodium translocon of exported proteins, during native conditions and upon generation of translocation intermediates. These data provide the most direct cellular evidence to date that protein export occurs at regions of the parasitophorous vacuole membrane housing the Plasmodium translocon of exported proteins complex.
Asunto(s)
Eritrocitos/parasitología , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Análisis por Conglomerados , Eritrocitos/metabolismo , Eritrocitos/patología , Eritrocitos/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Merozoítos/citología , Merozoítos/metabolismo , Merozoítos/ultraestructura , Modelos Biológicos , Parásitos/citología , Parásitos/metabolismo , Parásitos/ultraestructura , Plasmodium falciparum/citología , Plasmodium falciparum/ultraestructura , Estructura Terciaria de Proteína , Transporte de Proteínas , Desplegamiento Proteico , Proteínas Protozoarias/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an â¼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.