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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.

The malaria parasite, Plasmodium falciparum , displays the P. falciparum erythrocyte membrane protein 1 ( Pf EMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of Pf EMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct ( Pf EMP1B). We show that parasitophorous vacuole (PV)-located Pf EMP1B 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 Pf EMP1B 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 P lasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo . Plasmodium-infected red blood cells export virulence factors, such as Pf EMP1, to the cell surface. Here, the authors identify a protein complex termed EPIC that interacts with Pf EMP1 during export, and they show that knockdown of an EPIC component affects parasite virulence.

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.

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.

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.

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.

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.

Promouvoir la sante des populations a travers le monde va de pair avec la recherche-developpement responsable de la conception et de l'optimisation de produits sanitaires. Pourtant, les nouveaux produits a l'etude ne repondent pas toujours aux exigences mondiales des populations et maladies negligees. En vue de promouvoir la recherche, de favoriser les investissements et d'aligner les produits sur les besoins des utilisateurs finaux, les travaux doivent etre mieux coordonnes et leurs priorites, mieux definies. L'Organisation mondiale de la Sante (OMS) a donc elabore des profils de produits cibles qui determinent les caracteristiques requises pour les nouveaux produits sanitaires, afin qu'ils correspondent davantage aux besoins les plus criants en matiere de sante publique. Un profil de produit cible etabli par l'OMS est un document qui met en evidence un besoin et fournit des indications sur les aspects a prendre en compte pour garantir l'acces et l'equite des le depart dans le plan de recherche-developpement. LOMS a egalement publie un Repertoire des profils de produits cibles, une base de donnees en ligne consultable gratuitement qui reprend les caracteristiques employees pour decrire les produits sanitaires souhaites (medicaments, vaccins, outils diagnostiques et equipements medicaux). Dans le present document, nous detaillons le processus de developpement d'un profil de produit cible par l'OMS, mais aussi les avantages que comportent de telles indications. Nous encourageons vivement les laboratoires a partager les profils de produits qui repondent a des besoins non satisfaits en matiere de sante publique, afin de contribuer a avancer vers les objectifs mondiaux de sante et de bien-etre.

This paper demonstrates that a protein complex known as PTEX translocates all malaria parasite proteins destined for export into the cytosol of their host red blood cell. PTEX drives malarial protein export The malaria parasite, Plasmodium falciparum , infects and remodels the host's red blood cells, a process that requires the export of hundreds of proteins into the cytosol. This a considerable topological feat, as the parasite initially resides in a compartment known as the parasitophorous vacuole. A protein complex known as PTEX ( Plasmodium translocon of exported proteins) was thought to be involved in this process, but evidence of function was circumstantial and indirect. Two reports in this issue of Nature use contrasting techniques to demonstrate that PTEX is essential for the export of malaria parasite proteins into the cytoplasm of infected cells, and that such export is essential for the parasite's life cycle. Brendan Elsworth et al . generated conditional mutants of PTEX components HSP101 and PTEX150, and show that when PTEX function is perturbed the export of proteins including the major virulence factor PfEMP1 is much reduced. Josh Beck et al . use an innovative dihydrofolate reductase based destabilization domain approach to inactivate the HSP101 and show that it is needed for the secretion of all classes of exported malarial proteins. 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 1 , 2 , 3 , 4 , 5 , 6 . These proteins have a variety of roles that are essential to virulence or parasite growth 7 . There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins 8 , 9 . One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX) 10 . Although PTEX has been linked to the export of one class of exported proteins 10 , 11 , 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) 1 , 2 and PEXEL-negative exported proteins (PNEPs) 6 . 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.

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.