Role of the Plasmodium Export Element in Trafficking Parasite Proteins to the Infected Erythrocyte

The intracellular survival of Plasmodium falciparum within human erythrocytes is dependent on export of parasite proteins that remodel the host cell. Most exported proteins require a conserved motif (RxLxE/Q/D), termed the Plasmodium export element (PEXEL) or vacuolar targeting sequence (VTS), for targeting beyond the parasitophorous vacuole membrane and into the host cell; however, the precise role of this motif in export is poorly defined. We used transgenic P. falciparum expressing chimeric proteins to investigate the function of the PEXEL motif for export. The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner. Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell. Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell. This work defines the role of each residue in the PEXEL for export into the P. falciparum -infected erythrocyte.     

Exploration of the P3 region of PEXEL peptidomimetics leads to a potent inhibitor of the Plasmodium protease,plasmepsin V

The use of arginine isosteres is a known strategy to overcome poor membrane permeability commonly associated with peptides or peptidomimetics that possess this highly polar amino acid. Here, we apply this strategy to peptidomimetics that are potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against Plasmodium parasites, and exploring the structure–activity relationship of the P₃ arginine within the S₃ pocket of plasmepsin V. Of the arginine isosteres trialled in the P₃ position, we discovered that canavanine was the ideal and that this peptidomimetic potently inhibits plasmepsin V, efficiently blocks protein export and inhibits parasite growth. Structure studies of the peptidomimetics bound to plasmepsin V provided insight into the structural basis for the enzyme activity observed in vitro and provides further evidence why plasmepsin V is highly sensitive to substrate modification.     

Export of PfSBP1 to the Plasmodium falciparum Maurer's Clefts

The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer's clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite's secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite's plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P.   falciparum -infected erythrocytes.     

Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in Plasmodium falciparum Protein Export

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.     

Protein Traffic to the Plasmodium falciparum Apicoplast: Evidence for a Sorting Branch Point at the Golgi

Plasmodium falciparum, similar to many other apicomplexan parasites, contains an apicoplast, a plastid organelle of secondary endosymbiotic origin. Nuclear‐encoded proteins are targeted to the apicoplast by a bipartite topogenic signal consisting of (i) an endoplasmic reticulum (ER)‐type N‐terminal secretory signal peptide, followed by (ii) a plant‐like transit peptide. Although the signals responsible for transport of most proteins to the apicoplast are well described, the route of trafficking from the ER to the outermost apicoplast membrane is still a matter of debate. Current models of trafficking to the apicoplast suggest that proteins destined for this organelle are, on entry into the lumen of the ER, diverted from the default secretory pathway to a specialized vesicular system which carries proteins directly from the ER to the outer apicoplast membrane. Here, we have re‐examined this trafficking pathway. By titrating wild‐type and mutant apicoplast transit peptides against different ER retrieval sequences and studying protein transport in a brefeldin A‐resistant parasite line, we generated data which suggest a direct involvement of the Golgi in traffic of soluble proteins to the P. falciparum apicoplast.     

Trafficking of the Phosphoprotein PfCRT to the Digestive Vacuolar Membrane in Plasmodium falciparum

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site‐directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane.     

New Export Pathway in Plasmodium falciparum‐Infected Erythrocytes: Role of the Parasite Group II Chaperonin,PfTRiC

The export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria parasite Plasmodium falciparum. The Maurer's clefts, membrane structures transposed by the parasite in the cytoplasm of its host erythrocyte, play the role of a marshal platform for such exported parasite proteins. We identify here the export pathway of three resident proteins of the Maurer's clefts membrane: the proteins are exported as soluble forms in the red cell cytoplasm to the Maurer's clefts membrane in association with the parasite group II chaperonin (PfTRIC), a chaperone complex known to bind and address a large spectrum of unfolded proteins to their final location. We have also located the domain of interaction with PfTRiC within the amino‐terminal domain of one of these Maurer's cleft proteins, PfSBP1. Because several Maurer's cleft membrane proteins with different export motifs seem to follow the same route, we propose a general role for PfTRiC in the trafficking of malarial parasite proteins to the host erythrocyte.      

Plasmodium falciparum apicoplast transit peptides are unstructured in vitro and during apicoplast import

Trafficking of soluble proteins to the apicoplast in Plasmodium falciparum is determined by an N-terminal transit peptide (TP) which is necessary and sufficient for apicoplast import. Apicoplast precursor proteins are synthesized at the rough endoplasmic reticulum, but are then specifically sorted from other proteins in the secretory pathway. The mechanism of TP recognition is presently unknown. Apicoplast TPs do not contain a conserved sequence motif; therefore, we asked whether they contain an essential structural motif. Using nuclear magnetic resonance to study a model TP from acyl carrier protein, we found a short, low-occupancy helix, but the TP was otherwise disordered. Using an in vivo localization assay, we blocked TP secondary structure by proline mutagenesis, but found robust apicoplast localization. Alternatively, we increased the helical content of the TP through mutation while maintaining established TP characteristics. Apicoplast import was disrupted in a helical mutant TP, but import was then restored by the further addition of a single proline. We conclude that structure in the TP interferes with apicoplast import, and therefore TPs are functionally disordered. These results provide an explanation for the amino acid bias observed in apicoplast TPs.     

Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .     

Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open

The human malaria parasite Plasmodium falciparum resides and multiplies within a membrane-bound vacuole in the cytosol of its host cell, the mature human erythrocyte. To enable the parasite to complete its intraerythrocytic life cycle, a large number of parasite proteins are synthesized and transported from the parasite to the infected cell. To gain access to the erythrocyte, parasite proteins must first cross the membrane of the parasitophorous vacuole (PVM), a process that is not well understood at the mechanistic level. Here, we review past and current literature on this topic, and make tentative predictions about the nature of the transport machinery required for transport of proteins across the PVM, and the molecular factors involved.     

Spatial organization of protein export in malaria parasite blood stages

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.      

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export,PfEMP1 Display,and Survival of Malaria Parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.     

Structure of a ribulose 5-phosphate 3-epimerase from Plasmodium falciparum

The crystal structure of Pfal009167AAA, a putative ribulose 5-phosphate 3-epimerase (PfalRPE) from Plasmodium falciparum, has been determined to 2 A resolution. RPE represents an exciting potential drug target for developing antimalarials because it is involved in the shikimate and the pentose phosphate pathways. The structure is a classic TIM-barrel fold. A coordinated Zn ion and a bound sulfate ion in the active site of the enzyme allow for a greater understanding of the mechanism of action of this enzyme. This structure is solved in the framework of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium.     

PEXEL-independent trafficking of Plasmodium falciparum SURFIN4.2 to the parasite-infected red blood cell and Maurer's clefts

SURFIN_4.2 is a parasite-infected red blood cell (iRBC) surface associated protein of Plasmodium falciparum. To analyze the region responsible for the intracellular trafficking of SURFIN_4.2 to the iRBC and Maurer's clefts, a panel of transgenic parasite lines expressing recombinant SURFIN_4.2 fused with green fluorescent protein was generated and evaluated for their localization. We found that the cytoplasmic region containing a tryptophan rich (WR) domain is not necessary for trafficking, whereas the transmembrane (TM) region was. Two PEXEL-like sequences were shown not to be responsible for the trafficking of SURFIN_4.2, demonstrating that the protein is trafficked in a PEXEL-independent manner. N-terminal replacement, deletion of the cysteine-rich domain or the variable region also did not prevent the protein from localizing at the iRBC or Maurer's clefts. A recombinant SURFIN_4.2 protein possessing 50 amino acids upstream of the TM region, TM region itself and a part of the cytoplasmic region was shown to be trafficked into the iRBC and Maurer's clefts, suggesting that there are no essential trafficking motifs in the SURFIN_4.2 extracellular region. A mini-SURFIN_4.2 protein containing WR domain was shown by Western blotting to be more abundantly detected in a Triton X-100-insoluble fraction, compared to the one without WR domain. We suggest that the cytoplasmic region containing the WR may be responsible for their difference in solubility.     

Crystal structure of S-adenosyl-L-homocysteine hydrolase from the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.     

High prevalence of Plasmodium malariae and Plasmodium ovale in co-infections with Plasmodium falciparum in asymptomatic malaria parasite carriers in southwestern Nigeria

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10–19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.     

Mutagenic exploration of the active site of lactate dehydrogenase from Plasmodium falciparum

Several site-directed mutations of residues around the active site of the lactate dehydrogenase from Plasmodium falciparum are described. These include changes to three highly, but not completely, conserved residues in the pocket of the active site and also three changes (including deletions) to the active site loop. Changes to residues in the active-site pocket resulted in little or no over-production of protein and no enzymic activity. Likewise, a five residue deletion from the active site loop gave no over-produced protein, while a two residue deletion and changes of residue type in this loop were tolerated. The results are discussed in the light of this protein being a suitable target for novel anti-malarials.