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The recent demonstration of Plasmodium spp. merozoite gliding motility changes our understanding of how the merozoite actively seeks out and potentially selects a red blood cell (RBC) to invade. The observation that motility and RBC invasion is in the direction of the merozoite's wider end will require significant changes in how merozoite invasion is represented and potentially our understanding of the biophysics of invasion compared to the current standard egg-shaped merozoite invasion model. Flipping the model to one where the wider end of the merozoite is the apex supports representing merozoites packed into a mature schizont in a wedge-shaped pattern with the wider apical end facing out (evidenced by many electron, expansion, and immunofluorescence microscopy images), instead of the egg-shaped merozoite model where the wider end is closer to the centre. A critical part of the malaria parasite's life cycle is invasion of red blood cells (RBCs) by merozoites. Inside RBCs, the parasite forms a schizont, which undergoes segmentation to produce daughter merozoites. These cells are released, establishing cycles of invasion. Traditionally, merozoites are represented as nonmotile, egg-shaped cells that invade RBCs 'narrower end' first and pack within schizonts with this narrower end facing outwards. Here, we discuss recent evidence and re-evaluate previous data which suggest that merozoites are capable of motility and have spherical or elongated-teardrop shapes. Furthermore, merozoites invade RBCs 'wider end' first and pack within schizonts with this wider end facing outwards. We encourage the field to review this revised model and consider its implications for future studies. [ABSTRACT FROM AUTHOR]

Novel antimalarial therapeutics that target multiple stages of the parasite lifecycle are urgently required to tackle the emerging problem of resistance with current drugs. Here, we describe the optimization of the 2-anilino quinazoline class as antimalarial agents. The class, identified from publicly available antimalarial screening data, was optimized to generate lead compounds that possess potent antimalarial activity against P. falciparum parasites comparable to the known antimalarials, chloroquine and mefloquine. During the optimization process, we defined the functionality necessary for activity and improved in vitro metabolism and solubility. The resultant lead compounds possess potent activity against a multidrug resistant strain of P. falciparum and arrest parasites at the ring phase of the asexual stage and also gametocytogensis. Finally, we show that the lead compounds are orally efficacious in a 4 day murine model of malaria disease burden. [ABSTRACT FROM AUTHOR]

When in their human hosts, malaria parasites spend most of their time housed within vacuoles inside erythrocytes and hepatocytes. The parasites extensively modify their host cells to obtain nutrients, prevent host cell breakdown and avoid the immune system. To perform these modifications, malaria parasites export hundreds of effector proteins into their host cells and this process is best understood in the most lethal species to infect humans, Plasmodium falciparum . The effector proteins are synthesized within the parasite and following a proteolytic cleavage event in the endoplasmic reticulum and sorting of mature proteins into the correct vesicular trafficking pathway, they are transported to the parasite surface and released into the vacuole. The effector proteins are then unfolded before extrusion across the vacuole membrane by a unique translocon complex called Plasmodium translocon of exported proteins. After gaining access to the erythrocyte cytoplasm many effector proteins continue their journey to the erythrocyte surface by utilising various membranous structures established by the parasite. This complex trafficking pathway and a large number of the effector proteins are unique to Plasmodium parasites. This pathway could, therefore, be developed as new drug targets given that protein export and the functional role of these proteins are essential for parasite survival. This review explores known and potential drug targetable steps in the protein export pathway and strategies for discovering novel drug targets. [ABSTRACT FROM AUTHOR]

Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR-Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines. [ABSTRACT FROM AUTHOR]

During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite’s life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1) an early heparin-blockable interaction which weakly deforms the erythrocyte, 2) EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite’s actin-myosin motor, 3) a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4) an AMA1–RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine. [ABSTRACT FROM AUTHOR]

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the Cterminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression. [ABSTRACT FROM AUTHOR]

Abstract: Multiple and seemingly sequential interactions between parasite ligands and their receptors on host erythrocytes are an essential precursor to invasion by the obligate intracellular pathogen, Plasmodium falciparum. Consequently, identification and characterisation of the specific effectors that facilitate these recognition events are of special interest for the development of novel therapeutic and prophylactic solutions to malaria. There have been many recent advances regarding the identification of host–parasite receptor–ligand pairs, however the precise function and temporal aspects of these interactions are far from resolved. This review provides an update on the current details of these interactions to place them in sequence and super impose them upon the known kinetic events of invasion. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The ability to genetically manipulate malaria parasites in recent times has contributed considerably to our understanding of the biology of this deadly pathogen. Epp et al. have now expanded the repertoire of molecular tools available for the transgenesis system for the human malaria parasite Plasmodium falciparum by developing a simple methodology to regulate malaria gene expression. In this article, we comment on this technique and discuss its potential applications in the study of the biology of malaria parasites. [Copyright &y& Elsevier]

Abstract: The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6s after primary contact. This period can be divided into two distinct phases. The first is an ∼11s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17s period. After invasion, a third ‘echinocytosis’ phase commences when about 36s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4s, after which the infected RBC recovered over a 5–11min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid. [Copyright &y& Elsevier]

Antibodies from malaria-exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP119) comprised of two epidermal growth factor (EGF)-like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP119‘miniproteins’ on the surface of Plasmodium falciparum merozoites. To correctly express these proteins, we determined that GPI-anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface-expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein–MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP119 domain is the rapid agglutinating of merozoites prior to erythrocyte attachment. [ABSTRACT FROM AUTHOR]

Transfection of the human malaria parasite Plasmodium falciparum has facilitated greater understanding of the biology of this devastating protozoal pathogen. However, technical limitations have restricted the options available for functional analysis. A recent study by Nkrumah and colleagues provides a powerful new transfection tool, the Bxb1 integrase system. In this article, we outline the potential of this system, describing how it enables direct site-specific integration and the rapid generation of stably transformed populations that express uniform levels of introduced transgenes. [Copyright &y& Elsevier]

The introduction of plastids into different heterotrophic protists created lineages of algae that diversified explosively, proliferated in marine and freshwater environments, and radically altered the biosphere. The origins of these secondary plastids are usually inferred from the presence of additional plastid membranes. However, two examples provide unique snapshots of secondary-endosymbiosis-in-action, because they retain a vestige of the endosymbiont nucleus known as the nucleomorph. These are chlorarachniophytes and cryptomonads, which acquired their plastids from a green and red alga respectively. To allow comparisons between them, we have sequenced the nucleomorph genome from the chiorarachniophyte Bigelowiella natans: at a mere 373,000 bp and with only 331 genes, the smallest nuclear genome known and a model for extreme reduction. The genome is eukaryotic in nature, with three linear chromosomes containing densely packed genes with numerous overlaps. The genome is replete with 852 introns, but these are the smallest introns known, being only 18, 19, 20, or 21 nt in length. These pygmy introns are shown to be miniaturized versions of normal-sized introns present in the endosymbiont at the time of capture. Seventeen nucleomorph genes encode proteins that function in the plastid. The other nucleomorph genes are housekeeping entities, presumably underpinning maintenance and expression of these plastid proteins. Chlorarachniophyte plastids are thus serviced by three different genomes (plastid, nucleomorph, and host nucleus) requiring remarkable coordination and targeting. Although originating by two independent endosymbioses, chiorarachniophyte and cryptomonad nucleomorph genomes have converged upon remarkably similar architectures but differ in many molecular details that reflect two distinct trajectories to hypercompaction and reduction. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity. [ABSTRACT FROM AUTHOR]

We compare and contrast the morphological and molecular features of six chlorarachniophyte strains, and examine their evolutionary origins. Electron microscopical studies of nucleomorphs and chloroplasts, characterization of nucleomorph karyotypes, and phylogenetic analyses of small subunit ribosomal RNA (srRNA) genes derived from the nucleomorph and host cell genomes have been used to separate the six strains into three distinct groups. One group, dubbed the ‘beast group’, contains the strains Chlorarachnion sp. 242, Chlorarachnion sp. 621, Chlorarachnion sp. 1408 and Chlorarachnion sp. 1481. Members of the beast group have a novel flagellate form and are apparently picoplanktonic. The other two groups currently contain only one species each: Chlorarachnion reptans and Lotharella sp. 240. All chlorarachniophyte nucleomorphs examined house three small linear chromosomes each furnished with telomeres and srRNA genes. [ABSTRACT FROM AUTHOR]

Studies the tiny genomes of chlorarachniophyte endosymbionts, a group of marine protists. Classification of chlorarachniophytes as amoeboflagellates; Characterization of the nucleomorph DNA; Categorization of the nucleomorph genome as the quintessence of compactness.

Presents an initial characterization and unusual organization of several nucleomorph genes found in chlorarachniophytes which are involved in the splicing of mRNAs and the translation process. Typical eukaryotic nature of the genetic structure; Polyadenylation in the mRNAs.

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors. [ABSTRACT FROM AUTHOR]

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N‐terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P2′ position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines. Author summary: Malaria is a sometimes-fatal disease caused by protozoan parasites of which Plasmodium falciparum is the deadliest species that causes hundreds of millions of infections and half a million deaths per year. A partially effective vaccine is available to block parasite forms transmitted by mosquitoes but not the subsequent blood stage which causes symptomatic disease. To fight blood stage parasites, proteins have been identified such as PfRH5, that aid parasite entry into human red blood cells (RBCs) and vaccines made from these proteins can trigger the production of antibodies that bind the parasite proteins thereby blocking RBC invasion. PfRH5 forms a complex with another parasite protein called PfCyRPA and together antibodies to PfCyRPA and PfRH5 are highly effective in reducing parasite growth. Here we investigated how antibodies to PfCyRPA and PfRH5 actually block invasion using video microscopy of live parasites. As anticipated, we found the antibodies not only stopped most parasites from invading but of those parasites that did invade, they took longer to do so, suggesting the antibodies were physically inhibiting the invasion process. One unanticipated effect of both PfRH5 antibodies and one of three PfCyRPA antibodies tested, was that they triggered the uninvaded parasites to change into cellular forms normally only seen inside RBCs. These extracellular forms are no longer competent to invade and so the PfRH5/PfCyRPA antibodies have the potential to both neutralize parasites by physically preventing RBC entry and by changing the parasites into invasion incompetent forms. [ABSTRACT FROM AUTHOR]

Examines the genome sequence of the nucleomorph of a cryptomonad alga, Guillardia theta. Comparison of the nucleomorph with the human genome; Characteristics of the nucleomorph.

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. Author summary: Plasmodium falciparum is the causative agent of the most severe form of malaria in humans, where the symptoms of the disease are derived from the continuous asexual replication of the parasite within the human red blood cells (RBCs) it infects. To survive within this niche, the parasite exports hundreds of parasite effector proteins across the vacuole it resides within and into the RBC. About a quarter of the exported proteins appear to be essential during the blood stage but the functions of these proteins largely remain uncharacterised. Protein export is facilitated by an essential protein complex termed the Plasmodium translocon of exported proteins (PTEX). Conditional depletion of PTEX's core components results in rapid parasite death presumably because essential proteins do not reach their functional destination in the RBC and their associated metabolic functions cannot be performed. To uncover what these essential metabolic functions are we knocked down a core component of PTEX, PTEX150, using a previously established conditional knockdown parasite line. Metabolic analysis of the knockdown parasites indicated that haemoglobin (Hb) digestion was inhibited resulting in a reduction of Hb derived peptides, which serve as an amino acid source for the parasite. We determined that knocking down HSP101, another PTEX core component, also disrupted the Hb digestion pathway. Furthermore, we provide evidence that reduction of Hb digestion might be due to the failure to efficiently deliver early acting Hb digesting proteases to the cytostome, a specialised location where vesicles of Hb are taken into the parasite. PTEX may therefore play a role in delivering Hb proteases to the cytostome. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a β-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1–RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1–RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines. [ABSTRACT FROM AUTHOR]

[Display omitted] • Malaria C3H1 zinc finger proteins PfCZIF1 and 2 have similar stage expression. • Both C3H1s could be truncated individually and partially in tandem. • Inability to knock-out both shows asexual parasite growth requires at least one. • Loss of PfCZIF2 led to changes in gene expression for exported proteins. Plasmodium falciparum exerts strong temporal control of gene expression across its lifecycle. Proteins expressed exclusively during late schizogony of blood stages, for example, often have a role in facilitating merozoite invasion of the host red blood cell (RBC), through merozoite development, egress, invasion or early establishment of infection in the RBC. Here, we characterise P. falciparum C3H1 zinc finger 1 (Pf CZIF1, Pf 3D7_1468400) and P. falciparum C3H1 zinc finger 2 (Pf CZIF2, Pf3D7_0818100) which we identified as the only C3H1-type zinc finger proteins with peak expression at schizogony. Previous studies reported that antibodies against Pf CZIF1 inhibit merozoite invasion, suggesting this protein may have a potential role during RBC invasion. We show using C-terminal truncations and gene knockouts of each of Pfczif1 and Pfczif2 that neither are essential for blood stage growth. However, they could not both be knocked out simultaneously, suggesting that at least one is needed for parasite growth in vitro. Immunofluorescence localisation of Pf CZIF1 and Pf CZIF2 indicated that both proteins occur in discrete foci on the periphery of the parasite's cytosol and biochemical assays suggest they are peripherally associated to a membrane. Transcriptomic analyses for the C-terminal truncation mutants reveal no significant expression perturbations with Pf CZIF1 truncation. However, modification of Pf CZIF2 appears to modify the expression for some exported proteins including Pf KAHRP. This study does not support a role for Pf CZIF1 or Pf CZIF2 in merozoite invasion of the RBC and suggests that these proteins may help regulate the expression of proteins exported into the RBC cytosol after merozoite invasion. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs—modifications which are both mediated by parasite‐derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host. [ABSTRACT FROM AUTHOR]

A highly-effective, long-lasting vaccine, targeting multiple stages of the Plasmodium falciparum life cycle, is likely to be important for the elimination of this pathogen. Key antigens of this vaccine would produce host antibodies that block the ligands required for merozoite invasion of erythrocytes, thereby curtailing the expansion of parasitemia and symptomatic disease. Recent live cell imaging of invading Plasmodium falciparum merozoites with various receptor–ligand interactions inhibited has provided new information about the function, sequence, and timing of these events, providing a rationale for a vaccine containing multiple antigens that inhibit the sequential steps of invasion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites are obligate intracellular pathogens that cause a host of human and animal diseases. These parasites have developed a universal mechanism of invasion involving formation of a ‘moving junction’ that provides a stable anchoring point through which the parasite invades host cells. The composition of the moving junction, particularly the presence of the protein Apical Membrane Antigen 1 (AMA1), has recently been the subject of some controversy. In this commentary we review findings that led to the current model of the moving junction complex and dissect the major conflicts to determine whether a substantial reassessment of the role of AMA1 is justified. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell's physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins is processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite's ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite's ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101's specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte. Author summary: Plasmodium falciparum, the most lethal species of human malaria parasite, infects erythrocytes and develops within a parasitophorous vacuole. To support rapid parasite growth and immune evasion, the parasite remodels its erythrocyte by exporting a myriad of proteins into the erythrocyte compartment. Parasite proteins destined for export are first imported into the endoplasmic reticulum (ER) and then secreted into the parasitophorous vacuole, where they are translocated across the parasitophorous vacuole membrane into the erythrocyte via a protein-conducting channel called PTEX. A missing link in the story has been how proteins destined for export are specifically guided from the ER to PTEX at the parasitophorous vacuole membrane. In this study, we found that one of the core PTEX components, HSP101, resides within the parasite's ER, in addition to its PTEX-related location at the parasitophorous vacuole. We also found that ER-located HSP101 can interact transiently with cargo proteins en route to the parasitophorous vacuole membrane. Our findings support a model in which HSP101 forms an initial interaction with exported proteins in the ER and then chaperones them to the rest of PTEX at the parasitophorous vacuole membrane for export into the erythrocyte. [ABSTRACT FROM AUTHOR]

Malaria is caused by obligate intracellular parasites, of which P lasmodium falciparum is the most lethal species. In humans, P . falciparum merozoites (invasive forms of the parasite) employ a host of parasite proteins to rapidly invade erythrocytes. One of these is the P . falciparum apical membrane antigen 1 ( PfAMA1) which forms a complex with rhoptry neck proteins at the tight junction. Here, we have placed the Pfama1 gene under conditional control using dimerizable Cre recombinase ( DiCre) in P . falciparum. DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle. This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole. These results show that PfAMA1 is an essential protein for merozoite invasion in P . falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event. [ABSTRACT FROM AUTHOR]

Symptomatic malaria is caused by the infection of human red blood cells ( RBCs) with P lasmodium parasites. The RBC is a peculiar environment for parasites to thrive in as they lack many of the normal cellular processes and resources present in other cells. Because of this, P lasmodium spp. have adapted to extensively remodel the host cell through the export of hundreds of proteins that have a range of functions, the best known of which are virulence-associated. Many exported parasite proteins are themselves involved in generating a novel trafficking system in the RBC that further promotes export. In this review we provide an overview of the parasite synthesized export machinery as well as recent developments in how different classes of exported proteins are recognized by this machinery. [ABSTRACT FROM AUTHOR]

Protein export in intra-erythrocytic Plasmodium parasites is of considerable interest in the malaria field because the process is inextricably linked to virulence and survival mechanisms in the human host. Despite many and varied functions, a common link between many exported proteins is their actual mode of export. Most exported proteins must traverse two membranes to their destination in the infected erythrocyte cytosol, the parasite plasma membrane and surrounding parasitophorous vacuole membrane (PVM). In recent years, several studies have shone light on the common molecular mechanism by which the major class of exported proteins, the so-called PEXEL/HTS motif-containing proteins, are translocated across these membranes. Roles for parasite-specific molecular processes in two distinct sites, the endoplasmic reticulum and the PVM have been revealed. [ABSTRACT FROM AUTHOR]

Background: Antibodies targeting blood stage antigens are important in protection against malaria, but the key targets and mechanisms of immunity are not well understood. Merozoite surface protein 1 (MSP1) is an abundant and essential protein. The C-terminal 19 kDa region (MSP1-19) is regarded as a promising vaccine candidate and may also be an important target of immunity. Methodology/Findings: Growth inhibitory antibodies against asexual-stage parasites and IgG to recombinant MSP1-19 were measured in plasma samples from a longitudinal cohort of 206 children in Papua New Guinea. Differential inhibition by samples of mutant P. falciparum lines that expressed either the P. falciparum or P. chabaudi form of MSP1-19 were used to quantify MSP1-19 specific growth-inhibitory antibodies. The great majority of children had detectable IgG to MSP1-19, and high levels of IgG were significantly associated with a reduced risk of symptomatic P. falciparum malaria during the 6-month follow-up period. However, there was little evidence of PfMSP1-19 specific growth inhibition by plasma samples from children. Similar results were found when testing non-dialysed or dialysed plasma, or purified antibodies, or when measuring growth inhibition in flow cytometry or microscopy-based assays. Rabbit antisera generated by immunization with recombinant MSP1-19 demonstrated strong MSP1-19 specific growth-inhibitory activity, which appeared to be due to much higher antibody levels than human samples; antibody avidity was similar between rabbit antisera and human plasma. Conclusions/Significance: These data suggest that MSP1-19 is not a major target of growth inhibitory antibodies and that the protective effects of antibodies to MSP1-19 are not due to growth inhibitory activity, but may instead be mediated by other mechanisms. Alternatively, antibodies to MSP1-19 may act as a marker of protective immunity. [ABSTRACT FROM AUTHOR]

The ability to analyze gene function in malariacausing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ringshaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1). Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA). Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion. [ABSTRACT FROM AUTHOR]

Abstract: It is somewhat paradoxical that the malaria parasite’s survival strategy involves spending almost all of its blood-stage existence residing behind a two-membrane barrier in a host red blood cell, yet giving considerable attention to exporting parasite-encoded proteins back across these membranes. These exported proteins are thought to play diverse roles and are crucial in pathogenic processes, such as re-modelling of the erythrocyte cytoskeleton and mediating the export of a major virulence protein known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), and in metabolic processes such as nutrient uptake and solute exchange. Despite these varied roles most exported proteins have at least one common link; they share a trafficking pathway that begins with entry into the endoplasmic reticulum and concludes with passage across the vacuole membrane via a proteinaceous translocon known as the Plasmodium translocon of exported proteins (PTEX). In this commentary we review recent advances in our understanding of this export pathway and suggest several models by which different aspects of the process may be interconnected. [Copyright &y& Elsevier]

The recent awarding of the Nobel prize to Andrew Fire and Craig Mello for the discovery of RNA-interference (RNAi) in plants once more demonstrated the importance of basic science in understanding biological mechanisms. Importantly, this discovery led to the establishment of powerful approaches to study gene function in a wide array of organisms. While a robust RNAi-technology remains elusive in apicomplexan parasites, other molecular genetic technologies have been introduced in recent years. Now, in the post genomic era, the task is to apply these methods to validate and functionally dissect an ever-expanding list of putative vaccine and drug candidates. The ultimate aim of such studies is to transform our knowledge of the genome to the knowledge of the phenome and ultimately new intervention strategies in these important pathogenic organisms. However, substantial limitations remain to the current repertoire of available molecular tools, which limits a comprehensive analysis of these candidates, especially of essential genes. This review summarises the methodologies available for functional gene analysis in apicomplexan parasites and discusses further needs in tool development. [Copyright &y& Elsevier]

Genetic manipulation has revolutionized research in the Apicomplexan parasite Plasmodium fakiparum, the most important causative agent of malaria. However, to date no techniques have been established that allow modifications that are deleterious to blood- stage growth, such as the disruption of essential genes or the expression of dominant-negative transgenes. The recent establishment of a screen for functional transactivators in the related parasite Toxoplasma gondii prompted us to identify transactivators in T. gondii and to examine their functionality in P. falciparum. Tetracycline-responsive minimal promoters were generated based on the characterized P. falciparum calmodulin promoter and used to assess transactivators in P. falciparum. We demonstrate that artificial tetracycline-regulated transactivators isolated in T. gondii are also functional in P. falciparum. By using the tetracycline analogue anhydrotetracycline, efficient, stage-specific gene regulation was achieved in P. falciparum. This regulatable expression technology has clear potential for the study of essential gene function in P. falciparum blood stages. On the other hand, the identified transactivators are not functional in mammalian cells, consistent with the fundamental differences in the mechanism of gene regulation between Apicomplexan parasites and their human hosts. [ABSTRACT FROM AUTHOR]

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry‐based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J‐dot complex, which overall helps clarify protein–protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane. [ABSTRACT FROM AUTHOR]

To survive inside red blood cells (RBCs), malaria parasites export many proteins to alter their host cell's physiological properties. Although most proteins of this exportome are involved in immune avoidance or in the trafficking of exported proteins to the host membrane, about 20% are essential for parasite survival in culture but little is known about their biological functions. Here, we have combined information from large-scale genetic screens and targeted gene-disruption studies to tabulate all currently known Plasmodium falciparum exported proteins according to their likelihood of being essential. We also discuss the essential functional pathways that exported proteins might be involved in to help direct research efforts towards a more comprehensive understanding of host-cell remodelling. Protein export, driven solely by the Plasmodium translocon of exported proteins (PTEX), is essential for both in vitro and in vivo growth of blood-stage parasites. Despite the large size of the Plasmodium exportome, comprehensive understanding of the function(s) of most exported protein is still lacking. Recent genome-wide screens conducted in two Plasmodium species revealed potentially crucial functions of exported proteins, including the establishment of extra-parasite trafficking networks, membrane regulation, and control of metabolic fluxes. Advanced reverse genetic tools and functional assays provide the means to validate protein functions and to advance our understanding of host-cell remodelling. [ABSTRACT FROM AUTHOR]

Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes. [Display omitted] • Combining independently regulated inducible systems is a simple way to achieve better regulation of the gene of interest. • Activation of gene expression is faster than silencing. • Regulation is more efficient when the promoter is active. • Conditional expression of Nluc allows sensitive and specific identification of fast acting antimalarials. [ABSTRACT FROM AUTHOR]

• Compounds were screened for egress or invasion inhibition in the red blood cell stage of Plasmodium falciparum. • 15 egress and 24 invasion inhibitors were identified from 400 Medicines for Malaria Venture compounds. • Most compounds were growth inhibitors that had strong effects on egress and invasion. • Three specific egress inhibitors were identified that block different egress steps. • Two specific invasion inhibitors blocked merozoite entry or arrested ring formation. With emerging resistance to frontline treatments, it is vital that new drugs are identified to target Plasmodium falciparum. One of the most critical processes during parasites asexual lifecycle is the invasion and subsequent egress of red blood cells (RBCs). Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. To identify potential inhibitors of egress and invasion, we screened the Medicines for Malaria Venture Pathogen Box, a 400 compound library against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy, and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has significant implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite. [ABSTRACT FROM AUTHOR]

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity. [ABSTRACT FROM AUTHOR]

Plasmodium spp. parasites that cause malaria disease remain a significant global-health burden. With the spread of parasites resistant to artemisinin combination therapies in Southeast Asia, there is a growing need to develop new antimalarials with novel targets. Invasion of the red blood cell by Plasmodium merozoites is essential for parasite survival and proliferation, thus representing an attractive target for therapeutic development. Red blood cell invasion requires a co-ordinated series of protein/protein interactions, protease cleavage events, intracellular signals, organelle release and engagement of an actin-myosin motor, which provide many potential targets for drug development. As these steps occur in the bloodstream, they are directly susceptible and exposed to drugs. A number of invasion inhibitors against a diverse range of parasite proteins involved in these different processes of invasion have been identified, with several showing potential to be optimised for improved drug-like properties. In this review, we discuss red blood cell invasion as a drug target and highlight a number of approaches for developing antimalarials with invasion inhibitory activity to use in future combination therapies. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The pathogenic nature of malaria infections is due in part to the export of hundreds of effector proteins that actively remodel the host erythrocyte. The Plasmodium translocon of exported proteins (PTEX) has been shown to facilitate the trafficking of proteins into the host cell, a process that is essential for the survival of the parasite. The role of the auxiliary PTEX component PTEX88 remains unclear, as previous attempts to elucidate its function through reverse genetic approaches showed that in contrast to the core components PTEX150 and HSP101, knockdown of PTEX88 did not give rise to an export phenotype. Here, we have used biochemical approaches to understand how PTEX88 assembles within the translocation machinery. Proteomic analysis of the PTEX88 interactome showed that PTEX88 interacts closely with HSP101 but has a weaker affinity with the other core constituents of PTEX. PTEX88 was also found to associate with other PV‐resident proteins, including chaperones and members of the exported protein‐interacting complex that interacts with the major virulence factor PfEMP1, the latter contributing to cytoadherence and parasite virulence. Despite being expressed for the duration of the blood‐stage life cycle, PTEX88 was only discretely observed at the parasitophorous vacuole membrane during ring stages and could not always be detected in the major high molecular weight complex that contains the other core components of PTEX, suggesting that its interaction with the PTEX complex may be dynamic. Together, these data have enabled the generation of an updated model of PTEX that now includes how PTEX88 assembles within the complex. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]