A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

Complex nutrient channel phenotypes despite Mendelian inheritance in a Plasmodium falciparum genetic cross.

Malaria parasites activate a broad-selectivity ion channel on their host erythrocyte membrane to obtain essential nutrients from the bloodstream. This conserved channel, known as the plasmodial surface anion channel (PSAC), has been linked to parasite clag3 genes in P. falciparum, but epigenetic switching between the two copies of this gene hinders clear understanding of how the encoded protein determines PSAC activity. Here, we used linkage analysis in a P. falciparum cross where one parent carries a single clag3 gene to overcome the effects of switching and confirm a primary role of the clag3 product with high confidence. Despite Mendelian inheritance, CLAG3 conditional knockdown revealed remarkably preserved nutrient and solute uptake. Even more surprisingly, transport remained sensitive to a CLAG3 isoform-specific inhibitor despite quantitative knockdown, indicating that low doses of the CLAG3 transgene are sufficient to confer block. We then produced a complete CLAG3 knockout line and found it exhibits an incomplete loss of transport activity, in contrast to rhoph2 and rhoph3, two PSAC-associated genes that cannot be disrupted because nutrient uptake is abolished in their absence. Although the CLAG3 knockout did not incur a fitness cost under standard nutrient-rich culture conditions, this parasite could not be propagated in a modified medium that more closely resembles human plasma. These studies implicate oligomerization of CLAG paralogs encoded by various chromosomes in channel formation. They also reveal that CLAG3 is dispensable under standard in vitro conditions but required for propagation under physiological conditions.

Malaria parasite plasmepsins: More than just plain old degradative pepsins.

Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion, and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some (e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsin V and a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism.

Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes.

Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting membranes. Parasite Ca2+ , kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. Here, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live-cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite [Ca2+ ]i , the PV began rounding. Then after ~1.5 min, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next ~6 min, there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress programme when newly formed parasites mobilised and erythrocyte membranes permeabilised and then ruptured-a dramatic finale to the parasite cycle of replication.

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis.

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

Activation of the Plasmodium egress effector subtilisin-like protease 1 is achieved by plasmepsin X destruction of the propiece.

Following each round of replication, daughter merozoites of the malaria parasite Plasmodium falciparum escape (egress) from the infected host red blood cell (RBC) by rupturing the parasitophorous vacuole membrane (PVM) and the RBC membrane (RBCM). A proteolytic cascade orchestrated by the parasite’s serine protease, subtilisin-like protease 1 (SUB1) regulates the membrane breakdown. SUB1 activation involves primary auto-processing of the 82 kDa zymogen to a 54 kDa (p54) intermediate that remains bound to its inhibitory propiece (p31) post cleavage. A second processing step converts p54 to the terminal 47 kDa (p47) form of SUB1. Although the aspartic protease plasmepsin X (PM X) has been implicated in the activation of SUB1, the mechanism remains unknown. Here, we show that upon knockdown of PM X the inhibitory p31/p54 complex of SUB1 accumulates in the parasites. Using recombinant PM X and SUB1, we show that PM X can directly cleave both p31 and p54. We have mapped the cleavage sites on recombinant p31. Furthermore, we demonstrate that the conversion of p54 to p47 can be effected by cleavage at either a SUB1 or PM X cleavage site that are adjacent to one another. Importantly once the p31 is removed, p54 is fully functional inside the parasites suggesting that the conversion to p47 is dispensable for SUB1 activity. Relief of propiece inhibition via a heterologous protease is a novel mechanism for subtilisin activation. Significance Statement: Malaria parasites replicate inside a parasitophorous vacuole within the host red blood cells. Exit of mature progeny from the infected host cells is essential for further dissemination. Parasite exit is a highly regulated, explosive process that involves membrane breakdown. To do this, the parasite utilizes a serine protease, called the subtilisin-like protease 1 or SUB1 that proteolytically activates various effector proteins. SUB1 activity is dependent on an upstream protease, called plasmepsin X (PM X), although the mechanism was unknown. Here we describe the molecular basis for PM X mediated SUB1 activation. PM X proteolytically degrades the inhibitory segment of SUB1, thereby activating it. Involvement of a heterologous protease is a novel mechanism for subtilisin activation.

Activation of the Plasmodium Egress Effector Subtilisin-Like Protease 1 Is Mediated by Plasmepsin X Destruction of the Prodomain.

Following each round of replication, daughter merozoites of the malaria parasite Plasmodium falciparum escape (egress) from the infected host red blood cell (RBC) by rupturing the parasitophorous vacuole membrane (PVM) and the RBC membrane (RBCM). A proteolytic cascade orchestrated by a parasite serine protease, subtilisin-like protease 1 (SUB1), regulates the membrane breakdown. SUB1 activation involves primary autoprocessing of the 82-kDa zymogen to a 54-kDa (p54) intermediate that remains bound to its inhibitory propiece (p31) postcleavage. A second processing step converts p54 to the terminal 47-kDa (p47) form of SUB1. Although the aspartic protease plasmepsin X (PM X) has been implicated in the activation of SUB1, the mechanism remains unknown. Here, we show that upon knockdown of PM X, the inhibitory p31-p54 complex of SUB1 accumulates in the parasites. Using recombinant PM X and SUB1, we show that PM X can directly cleave both p31 and p54. We have mapped the cleavage sites on recombinant p31. Furthermore, we demonstrate that the conversion of p54 to p47 can be effected by cleavage at either SUB1 or PM X cleavage sites that are adjacent to one another. Importantly, once the p31 is removed, p54 is fully functional inside the parasites, suggesting that the conversion to p47 is dispensable for SUB1 activity. Relief of propiece inhibition via a heterologous protease is a novel mechanism for subtilisin activation. IMPORTANCE Malaria parasites replicate inside a parasitophorous vacuole within the host red blood cells. The exit of mature progeny from the infected host cells is essential for further dissemination. Parasite exit is a highly regulated, explosive process that involves membrane breakdown. To do this, the parasite utilizes a serine protease called SUB1 that proteolytically activates various effector proteins. SUB1 activity is dependent on an upstream protease called PM X, although the mechanism was unknown. Here, we describe the molecular basis for PM X-mediated SUB1 activation. PM X proteolytically degrades the inhibitory segment of SUB1, thereby activating it. The involvement of a heterologous protease is a novel mechanism for subtilisin activation.

Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation.

Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum.

Establishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogen's biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.

Assessment of Biological Role and Insight into Druggability of the Plasmodium falciparum Protease Plasmepsin V.

Upon infecting a red blood cell (RBC), the malaria parasite Plasmodium falciparum drastically remodels its host by exporting hundreds of proteins into the RBC cytosol. This protein export program is essential for parasite survival. Hence export-related proteins could be potential drug targets. One essential enzyme in this pathway is plasmepsin V (PMV), an aspartic protease that processes export-destined proteins in the parasite endoplasmic reticulum (ER) at the Plasmodium export element (PEXEL) motif. Despite long-standing interest in this enzyme, functional studies have been hindered by the inability of previous technologies to produce a regulatable lethal depletion of PMV. To overcome this technical barrier, we designed a system for stringent post-transcriptional regulation allowing a tightly controlled, tunable knockdown of PMV. Using this system, we found that PMV must be dramatically depleted to affect parasite growth, suggesting the parasite maintains this enzyme in substantial excess. Surprisingly, depletion of PMV arrested parasite growth immediately after RBC invasion, significantly before the death from exported protein deficit that has previously been described. The data suggest that PMV inhibitors can halt parasite growth at two distinct points in the parasite life cycle. However, overcoming the functional excess of PMV in the parasite may require inhibitor concentrations far beyond the enzyme's IC50.

4-Aryl Pyrrolidines as a Novel Class of Orally Efficacious Antimalarial Agents. Part 1: Evaluation of 4-Aryl- N-benzylpyrrolidine-3-carboxamides.

Identification of novel chemotypes with antimalarial efficacy is imperative to combat the rise of Plasmodium species resistant to current antimalarial drugs. We have used a hybrid target-phenotype approach to identify and evaluate novel chemotypes for malaria. In our search for drug-like aspartic protease inhibitors in publicly available phenotypic antimalarial databases, we identified GNF-Pf-4691, a 4-aryl- N-benzylpyrrolidine-3-carboxamide, as having a structure reminiscent of known inhibitors of aspartic proteases. Extensive profiling of the two terminal aryl rings revealed a structure-activity relationship in which relatively few substituents are tolerated at the benzylic position, but the 3-aryl position tolerates a range of hydrophobic groups and some heterocycles. Out of this effort, we identified (+)-54b (CWHM-1008) as a lead compound. 54b has EC50 values of 46 and 21 nM against drug-sensitive Plasmodium falciparum 3D7 and drug-resistant Dd2 strains, respectively. Furthermore, 54b has a long half-life in mice (4.4 h) and is orally efficacious in a mouse model of malaria (qd; ED99 ∼ 30 mg/kg/day). Thus, the 4-aryl- N-benzylpyrrolidine-3-carboxamide chemotype is a promising novel chemotype for malaria drug discovery.

Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages.

The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX.

Intraerythrocytic malaria parasites reside within a parasitophorous vacuolar membrane (PVM) generated during host cell invasion1. Erythrocyte remodelling and parasite metabolism require the export of effector proteins and transport of small molecules across this barrier between the parasite surface and host cell cytosol2,3. Protein export across the PVM is accomplished by the Plasmodium translocon of exported proteins (PTEX) consisting of three core proteins, the AAA+ ATPase HSP101 and two additional proteins known as PTEX150 and EXP24. Inactivation of HSP101 and PTEX150 arrests protein export across the PVM5,6, but the contribution of EXP2 to parasite biology is not well understood7. A nutrient permeable channel in the PVM has also been characterized electrophysiologically, but its molecular identity is unknown8,9. Here, using regulated gene expression, mutagenesis and cell-attached patch-clamp measurements, we show that EXP2, the putative membrane-spanning channel of PTEX4,10-14, serves dual roles as a protein-conducting channel in the context of PTEX and as a channel able to facilitate nutrient passage across the PVM independent of HSP101. Our data suggest a dual functionality for a channel operating in its endogenous context.

Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.

Proteases of the malaria parasite Plasmodium falciparum have long been investigated as drug targets. The P. falciparum genome encodes 10 aspartic proteases called plasmepsins, which are involved in diverse cellular processes. Most have been studied extensively but the functions of plasmepsins IX and X (PMIX and PMX) were unknown. Here we show that PMIX is essential for erythrocyte invasion, acting on rhoptry secretory organelle biogenesis. In contrast, PMX is essential for both egress and invasion, controlling maturation of the subtilisin-like serine protease SUB1 in exoneme secretory vesicles. We have identified compounds with potent antimalarial activity targeting PMX, including a compound known to have oral efficacy in a mouse model of malaria.

Synthetic RNA-protein modules integrated with native translation mechanisms to control gene expression in malaria parasites.

Synthetic posttranscriptional regulation of gene expression is important for understanding fundamental biology and programming new cellular processes in synthetic biology. Previous strategies for regulating translation in eukaryotes have focused on disrupting individual steps in translation, including initiation and mRNA cleavage. In emphasizing modularity and cross-organism functionality, these systems are designed to operate orthogonally to native control mechanisms. Here we introduce a broadly applicable strategy for robustly controlling protein translation by integrating synthetic translational control via a small-molecule-regulated RNA-protein module with native mechanisms that simultaneously regulate multiple facets of cellular RNA fate. We demonstrate that this strategy reduces 'leakiness' to improve overall expression dynamic range, and can be implemented without sacrificing modularity and cross-organism functionality. We illustrate this in Saccharomyces cerevisae and the non-model human malarial parasite, Plasmodium falciparum. Given the limited functional genetics toolkit available for P. falciparum, we establish the utility of this strategy for defining essential genes.