Apicomplexa are unicellular eukaryotes and obligate intracellular parasites, including Plasmodium, the causative agent of malaria and Toxoplasma, one of the most widespread zoonotic pathogens. Rhoptries, one of their specialized secretory organelles, undergo regulated exocytosis during invasion 1 . Rhoptry proteins are injected directly into the host cell to support invasion and subversion of host immune function 2 . The mechanism by which they are discharged is unclear and appears distinct from those in bacteria, yeast, animals or plants.Here we show that rhoptry secretion in Apicomplexa shares structural and genetic elements with the exocytic machinery of ciliates, their free-living relatives. Rhoptry exocytosis depends on intramembranous particles in the shape of a rosette embedded into the plasma membrane of the parasite apex. Formation of this rosette requires multiple Non-discharge (Nd) proteins conserved and restricted to Ciliata, Dinoflagellata, and Apicomplexa, that together constitute the superphylum Alveolata. We identified Nd6 at the site of exocytosis in association with an apical vesicle. Sandwiched between the rosette and the tip of the rhoptry, this vesicle appears as a central element of the rhoptry secretion machine. Our results describe a conserved secretion system that was adapted to provide defense for free-living unicellular eukaryotes and host cell injection in intracellular parasites.Apicomplexan parasites are invasive and defined by the presence of an apical complex used to recognize and gain entry into host cells. It includes two secretory organelles: micronemes and rhoptries 3 . Microneme proteins are secreted to the parasite surface and mediate motility, host cell recognition and invasion 4 . Rhoptry proteins are injected directly into the host cell 2 , where they anchor the machinery propelling the parasite into the host cell 5 , facilitate nutrient

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma , have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium . Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.

G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4’s including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.

Three new series comprising 24 novel cationic choline analogues and consisting of mono- or bis (N or C-5-duplicated) thiazolium salts have been synthesized. Bis-thiazolium salts showed potent antimalarial activity (much superior to monothiazoliums). Among them, bis-thiazolium salts 12 and 13 exhibited IC(50) values of 2.25 nM and 0.65 nM, respectively, against P. falciparum in vitro. These compounds also demonstrated good in vivo activity (ED(50)

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.Malaria is a major global health problem, with an estimated 250 to 300 million clinical cases annually and 3.3 billion people at risk, causing nearly a million deaths, mostly among children under 5 years old in sub-Saharan Africa (18, 47). The resistance of Plasmodium falciparum, the most deadly malaria parasite to most antimalarial drugs, is a major obstacle to the eradication of this disease (46). It is also of considerable concern in the light of a recent report on decreased sensitivity to artemesinin drugs in Southeast Asia (14, 28). New chemotherapeutic approaches are thus urgently needed, based on optimization of current drugs and, more importantly, on the discovery of new antimalarial drugs. The latter implies systematic screening of drug libraries, a series of natural compounds, or a structure-based drug design targeting novel targets. In all cases, in vitro evaluation of the thousands of new molecules for their antimalarial activity is an early and necessary step. This early step aims at detecting the antimalarial potential of individual or series of compounds. It is performed in vitro against P. falciparum laboratory strains and, at a later stage, against field isolates, including multidrug-resistant strains. Assays must provide a first indication on the potency of the pharmacological activity, usually expressed as the concentration required to inhibit the parasite viability ...

The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches.

Malaria is a disease caused by an intraerythrocytic protozoan parasite of the genus Plasmodium , which is transmitted by dipterans and affects vertebrates such as reptiles, birds, and mammals, including humans (see supplementary Table I). By contrast with other Apicomplexa parasite species that can infect a broad range of metazoans, Plasmodium species have a narrow specifi city range regarding insect and vertebrate hosts ( 1 ). Plasmodium falciparum is, thus, responsible for the most severe form of malaria in humans only. Other species, such as P. vivax , two P. ovale subspecies ( 2 ), P. malariae , and, according to recent reports, P. knowlesi ( 3 ) cause less complicated forms of human malaria. The host specifi city of P. knowlesi is not restricted to humans because it also infects monkeys.The evolutionary history of Plasmodium species has been highly debated, especially the position of P. falciparum , either grouped with avian parasites ( 4, 5 ) or placed as a sister species to other mammalian parasites including rodent parasites. The fi ndings of most recent analyses using three classes of rare genomic changes and mitochondrial RNA genes unambiguously support a mammalian clade and no Abstract Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium . At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identifi cation of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei , P. yoelii , and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei , and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not signifi cantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the fi rst study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The fi ndings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.

BACKGROUND AND PURPOSE Choline analogues, a new type of antimalarials, exert potent in vitro and in vivo antimalarial activity. This has given rise to albitiazolium, which is currently in phase II clinical trials to cure severe malaria. Here we dissected its mechanism of action step by step from choline entry into the infected erythrocyte to its effect on phosphatidylcholine (PC) biosynthesis. EXPERIMENTAL APPROACH We biochemically unravelled the transport and enzymatic steps that mediate de novo synthesis of PC and elucidated how albitiazolium enters the intracellular parasites and affects the PC biosynthesis. KEY RESULTS Choline entry into Plasmodium falciparum‐infected erythrocytes is achieved both by the remnant erythrocyte choline carrier and by parasite‐induced new permeability pathways (NPP), while parasite entry involves a poly‐specific cation transporter. Albitiazolium specifically prevented choline incorporation into its end‐product PC, and its antimalarial activity was strongly antagonized by choline. Albitiazolium entered the infected erythrocyte mainly via a furosemide‐sensitive NPP and was transported into the parasite by a poly‐specific cation carrier. Albitiazolium competitively inhibited choline entry via the parasite‐derived cation transporter and also, at a much higher concentration, affected each of the three enzymes conducting de novo synthesis of PC. CONCLUSIONS AND IMPLICATIONS Inhibition of choline entry into the parasite appears to be the primary mechanism by which albitiazolium exerts its potent antimalarial effect. However, the pharmacological response to albitiazolium involves molecular interactions with different steps of the de novo PC biosynthesis pathway, which would help to delay the development of resistance to this drug.