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Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival. Malaria is a life-threatening disease that affects millions of people around the world. The parasites that cause malaria have a complex life cycle that involves infecting both mosquitoes and mammals, including humans. In humans, the parasites spend part of their life cycle inside red blood cells, which causes the symptoms of the disease. In order to thrive, malaria parasites need to make the red blood cell more permeable so that they can absorb nutrients from the blood stream and get rid of toxic waste products they generate. Previous research has shown that the parasites can produce a protein that makes red blood cells more permeable to a range of nutrients. Understanding how the parasites can do this, and if they could change the permeability of their host red blood cell to prevent anti-malaria drugs from entering may help researchers to identify new approaches to starve the parasite. Counihan et al. investigated whether the parasites also use other proteins to modify red blood cells and demonstrated that a protein called RhopH2 can make the blood cells more permeable. The experiments used a genetically modified version of the parasite that lacked RhopH2. Counihan et al. show that essential nutrients and vitamins were depleted in these parasites and that the parasites were much slower to grow and reproduce. The next important step would be to identify all proteins that are involved in making red blood cells more permeable and how they achieve this, and use this knowledge to help generate anti-malarial drugs.

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.

Malaria vaccine candidate AMA1 of Plasmodium vivax complements P. falciparum AMA1 erythrocyte invasion. P. falciparum expressing PvAMA1 provides a new platform for quantifying the invasion-inhibitory capacity of antibodies and small molecules to accelerate development of vaccines and therapeutics for malaria. Abstract Plasmodium vivax and P. falciparum malaria species have diverged significantly in receptor-ligand interactions and host-cell invasion. One protein common to both is the merozoite invasion ligand AMA1. While the general structure of AMA1 is similar between species, their sequences are divergent. Surprisingly, it was possible to genetically replace PfAMA1 with PvAMA1 in P. falciparum parasites. PvAMA1 complemented PfAMA1 function and supported invasion of erythrocytes by P. falciparum. Genetically modified P. falciparum expressing PvAMA1 evaded the invasion inhibitory effects of antibodies to PfAMA1, demonstrating species specificity of functional antibodies. We generated antibodies to recombinant PvAMA1 that effectively inhibited invasion, confirming the function of PvAMA1 in genetically modified parasites. Results indicate significant molecular flexibility in AMA1 enabling conserved function despite substantial sequence divergence across species. This provides powerful new tools to quantify the inhibitory activities of antibodies or drugs targeting PvAMA1, opening new opportunities for vaccine and therapeutic development against P. vivax.

The genomes of Plasmodium parasites that cause malaria in humans, other primates, birds, and rodents all encode multiple 6-cys proteins. Distinct 6-cys protein family members reside on the surface at each extracellular life cycle stage and those on the surface of liver infective and sexual stages have been shown to play important roles in hepatocyte growth and fertilization respectively. However, 6-cys proteins associated with the blood-stage forms of the parasite have no known function. Here we investigate the biochemical nature and function of two blood-stage 6-cys proteins in Plasmodium falciparum, the most pathogenic species to afflict humans. We show that native P12 and P41 form a stable heterodimer on the infective merozoite surface and are secreted following invasion, but could find no evidence that this complex mediates erythrocyte-receptor binding. That P12 and P41 do not appear to have a major role as adhesins to erythrocyte receptors was supported by the observation that antisera to these proteins did not substantially inhibit erythrocyte invasion. To investigate other functional roles for these proteins their genes were successfully disrupted in P. falciparum, however P12 and P41 knockout parasites grew at normal rates in vitro and displayed no other obvious phenotypic changes. It now appears likely that these blood-stage 6-cys proteins operate as a pair and play redundant roles either in erythrocyte invasion or in host-immune interactions.

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.

Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is central to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here we identify in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered, and comprises heat shock protein 101 (HSP101; a ClpA/B-like ATPase from the AAA+ superfamily, of a type commonly associated with protein translocons), a novel protein termed PTEX150 and a known parasite protein, exported protein 2 (EXP2). EXP2 is the potential channel, as it is the membrane-associated component of the core PTEX complex. Two other proteins, a new protein PTEX88 and thioredoxin 2 (TRX2), were also identified as PTEX components. As a common portal for numerous crucial processes, this translocon offers a new avenue for therapeutic intervention. Malaria parasite virulence During intracellular infection of erythrocytes, malaria parasites reside in vacuoles from where they export many proteins into the host cell. These protein secretions play an important role in the virulence and viability of the Plasmodium parasite. The protein export machinery involved in this process has now been identified. Termed PTEX (for Plasmodium Translocon of EXported proteins), it is an ATP-powered complex located in the vacuole membrane, and may provide a new target for antimalarial drugs. Malaria parasites reside in vacuoles during intracellular infection of erythrocytes and export many proteins into the host cell, a process that is essential for the virulence and viability of Plasmodium . Whereas transport across the parasite membrane is known to rely on the secretory pathway, the transporter responsible for the translocation of proteins across the vacuole membrane is now identified.

Tuberculosis (TB) is ranked among the top 10 causes of death worldwide. New biomarker-based serodiagnostics and vaccines are unmet needs stalling disease control. Antigen 60 (A60) is a thermostable mycobacterial complex typically purified from Bacillus Calmette-Guérin (BCG) vaccine. A60 was historically evaluated for TB serodiagnostic and vaccine potential with variable findings. Despite containing immunogenic proteins, A60 has yet to be proteomically characterized. Here, commercial A60 was (1) trypsin-digested in-solution, analyzed by LC-MS/MS, searched against M. tuberculosis H37Rv and M.bovis BCG Uniprot databases; (2) analyzed using STRING to predict protein–protein interactions; and (3) probed with anti-TB monoclonal antibodies and patient immunoglobulin G (IgG) on Western blot to evaluate antigenicity. We detected 778 proteins in two A60 samples (440 proteins shared), including DnaK, LprG, LpqH, and GroEL1/2, reportedly present in mycobacterial extracellular vesicles (EV). Of these, 107 were also reported in EVs of M. tuberculosis, and 27 key proteins had significant protein–protein interaction, with clustering for chaperonins, ribosomal proteins, and proteins for ligand transport (LpqH and LprG). On Western blot, 7/8 TB and 1/8 non-TB sera samples had reactivity against 37–50 kDa proteins, while LpqH, GroEL2, and PstS1 were strongly detected. In conclusion, A60 comprises numerous proteins, including EV proteins, with predicted biological interactions, which may have implications on biomarker and vaccine development.

Summary 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.