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.
Abstract During pregnancy immunolglobulin 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], Pf VAR2CSA 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.
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.
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 ∼20 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.
Abstract The CYP2D6 enzyme is estimated to metabolize 25% of commonly used pharmaceuticals and is of intense pharmacogenetic interest due to the polymorphic nature of the CYP2D6 gene. Accurate allele typing of CYP2D6 has proved challenging due to frequent copy number variants (CNVs) and paralogous pseudogenes. SNP-arrays, qPCR and short-read sequencing have been employed to interrogate CYP2D6 , however these technologies are unable to capture longer range information. Long-read sequencing using the PacBio Single Molecule Real Time (SMRT) sequencing platform has yielded promising results for CYP2D6 allele typing. However, previous studies have been limited in scale and have employed nascent data processing pipelines. We present a robust data processing pipeline “PLASTER” for accurate allele typing of SMRT sequenced amplicons. We demonstrate the pipeline by typing CYP2D6 alleles in a large cohort of 377 Solomon Islanders. This pharmacogenetic method will improve drug safety and efficacy through screening prior to drug administration.
Abstract 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 .
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.
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.
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