Henri Vial

The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion

Host cell invasion in the Apicomplexa is unique in its dependency on a parasite actin‐driven machinery and in the exclusion of most host cell membrane proteins during parasitophorous vacuole (PV) formation. This exclusion occurs at a junction between host cell and parasite plasma membranes that has been called the moving junction, a circumferential zone which forms at the apical tip of the parasite, moves backward and eventually pinches the PV from the host cell membrane. Despite having been described by electron microscopic studies 30 years ago, the molecular nature of this singular structure is still enigmatic. We have obtained a monoclonal antibody that recognizes the moving junction of invading tachyzoites of Toxoplasma gondii, in a pattern clearly distinct from those described so far for microneme and rhoptry proteins. The protein recognized by this antibody has been affinity purified. Mass spectrometry analysis showed that it is a rhoptry neck protein (RON4), a hypothetical protein with homologues restricted to Apicomplexa. Our findings reveals for the first time the participation of rhoptry neck proteins in moving junction formation and strongly suggest the conservation of this structure at the molecular level among Apicomplexa.

Synergistic role of micronemal proteins in Toxoplasma gondii virulence

Apicomplexan parasites invade cells by a unique mechanism involving discharge of secretory vesicles called micronemes. Microneme proteins (MICs) include transmembrane and soluble proteins expressing different adhesive domains. Although the transmembrane protein TRAP and its homologues are thought to bridge cell surface receptors and the parasite submembranous motor, little is known about the function of other MICs. We have addressed the role of MIC1 and MIC3, two soluble adhesins of Toxoplasma gondii, in invasion and virulence. Single deletion of the MIC1 gene decreased invasion in fibroblasts, whereas MIC3 deletion had no effect either alone or in the mic1KO context. Individual disruption of MIC1 or MIC3 genes slightly reduced virulence in the mouse, whereas doubly depleted parasites were severely impaired in virulence and conferred protection against subsequent challenge. Single substitution of two critical amino acids in the chitin binding–like (CBL) domain of MIC3 abolished MIC3 binding to cells and generated the attenuated virulence phenotype. Our findings identify the CBL domain of MIC3 as a key player in toxoplasmosis and reveal the synergistic role of MICs in virulence, supporting the idea that parasites have evolved multiple ligand–receptor interactions to ensure invasion of different cells types during the course of infection.

Phospholipids in parasitic protozoa.

Parasitic protozoa are surrounded by membrane structures that have a different lipid and protein composition relative to membranes of the host. The parasite membranes are essential structurally and also for parasite specific processes, like host cell invasion, nutrient acquisition or protection against the host immune system. Furthermore, intracellular parasites can modulate membranes of their host, and trafficking of membrane components occurs between host membranes and those of the intracellular parasite. Phospholipids are major membrane components and, although many parasites scavenge these phospholipids from their host, most parasites also synthesise phospholipids de novo, or modify a large part of the scavenged phospholipids. It was recently shown that some parasites like Plasmodium have unique phospholipid metabolic pathways. This review will focus on new developments in research on phospholipid metabolism of parasitic protozoa in relation to parasite-specific membrane structures and function, as well as on several targets for interference with the parasite phospholipid metabolism with a view to developing new anti-parasitic drugs.

Potent inhibitors of Plasmodium phospholipid metabolism with a broad spectrum of in vitro antimalarial activities.

ABSTRACT We characterized the potent in vitro antimalarial activity and biologic assessment of 13 phospholipid polar head analogs on a comparative basis. There was a positive relationship between the abilities of the drugs to inhibit parasite growth in culture and their abilities to specifically inhibit phosphatidylcholine biosynthesis of Plasmodium falciparum-infected erythrocytes. Maximal activity of G25 was observed for the trophozoite stage of the 48-h erythrocytic cycle (50% inhibitory concentration, 0.75 nM), whereas the schizont and ring stages were 12- and 213-fold less susceptible. The compounds exerted a rapid nonreversible cytotoxic effect, with complete clearance of parasitemia after 5 h of contact with the mature stages. The compounds were highly specific against P. falciparum, with much lower toxicity against three other mammalian cell lines, and the in vitro therapeutic indices ranged from 300 to 2,500,000. Finally, the monoquaternary ammonium E10 and two bis-ammonium salts, G5 and G25, were similarly active against multiresistant strains and fresh isolates of P. falciparum. This impressive selective in vitro toxicity against P. falciparum strongly highlights the clinical potential of these quaternary ammonium salts for malarial chemotherapy.

Phosphatidylinositol 3-Phosphate, an Essential Lipid in Plasmodium, Localizes to the Food Vacuole Membrane and the Apicoplast

ABSTRACT Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.

Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane

Toxoplasma gondii, as many intracellular parasites, is separated from the cytosol of its host cell by a parasitophorous vacuole membrane (PVM). This vacuole forms during host cell invasion and parasite apical organelles named rhoptries discharge proteins that associate with its membrane during this process. We report here the characterization of the rhoptry protein ROP5, which is a new member of the ROP2 family. Contrasting with what is known for other ROP2 family proteins, ROP5 is not processed during trafficking to rhoptries. We show here that ROP5 is secreted during invasion and associates with the PVM. Using differential permeabilization of infected cells, we have shown that ROP5 exposes its C‐terminus towards the host cell cytoplasm, which corresponds to a reverse topology compared with ROP2 and ROP4. Taken together with recent modelling data suggesting that the C‐terminal hydrophobic domain hitherto described as transmembrane may correspond to a hydrophobic helix buried in the catalytic domain of kinase‐related proteins, these findings call for a reappraisal of the current view of ROP2 family proteins association with the PVM.

In Vivo Antimalarial Activities of Mono- and Bis Quaternary Ammonium Salts Interfering with Plasmodium Phospholipid Metabolism

ABSTRACT We previously showed that quaternary ammonium salts have potent antimalarial activities against the blood stage of drug-resistant Plasmodium falciparum. In the present study, 13 compounds of this series were comparatively assessed in murine in vivo malarial models. Mice infected with Plasmodium berghei were successfully treated with 11 quaternary ammonium salts in a 4-day suppressive test with a once-daily intraperitoneal administration. The dose required to decrease parasitemia by 50% (ED50) ranged from 0.04 to 4.5 mg/kg of body weight. For six mono- and three bis-quaternary ammonium salts, the therapeutic indices (i.e., 50% lethal dose and ED50) were higher than 5, and at best, around 20 to 30 for five of them (E6, E8, F4, G5, and G25), which is comparable to that of chloroquine under the same conditions. Plasmodium chabaudi was significantly more susceptible to G5, G15, and G25 compounds than P. berghei. Similar therapeutic indices were obtained, regardless of the administration mode or initial parasitemia (up to 11.2%). Parasitemia clearance was complete without recrudescence. Subcutaneously administered radioactive compounds had a short elimination half-life in mice (3.5 h) with low bioavailability (17.3%), which was likely due to the permanent cationic charge of the molecule. The high in vivo therapeutic index in the P. chabaudi-infected mouse model and the absence of recrudescence highlight the enormous potential of these quaternary ammonium salts for clinical malarial treatment.

Heme Binding Contributes to Antimalarial Activity of Bis-Quaternary Ammoniums

ABSTRACT Quaternary ammonium compounds have received recent attention due to their potent in vivo antimalarial activity based on their ability to inhibit de novo phosphatidylcholine synthesis. Here we show that in addition to this, heme binding significantly contributes to the antimalarial activity of these compounds. For the study, we used a recently synthesized bis-quaternary ammonium compound, T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide), which exhibits potent antimalarial activity (50% inhibitory concentration, ∼25 nM). Accumulation assays reveal that this compound is readily concentrated several hundredfold (cellular accumulation ratio, ∼500) into parasitized erythrocytes. Approximately 80% of the drug was shown to be distributed within the parasite, ∼50% of which was located in the parasite food vacuoles. T16 uptake was affected by anion substitution (permeation increasing in the order Cl− < Br− = NO3− < I− < SCN−) and was sensitive to furosemide—properties similar to substrates of the induced new permeability pathway in infected erythrocytes. Scatchard plot analysis of in situ T16 binding revealed high-affinity and low-affinity binding sites. The high-affinity binding site Kd was similar to that measured in vitro for T16 and ferriprotoporphyrin IX (FPIX) binding. Significantly, the capacity but not the Kd of the high-affinity binding site was decreased by reducing the concentration of parasite FPIX. Decreasing the parasite FPIX pool also caused a marked antagonism of T16 antimalarial activity. In addition, T16 was also observed to associate with parasite hemozoin. Binding of T16 to FPIX in the digestive food vacuole is shown to be critical for drug accumulation and antimalarial activity. These data provide additional new mechanisms of antimalarial activity for this promising new class of antimalarial compounds.

A systematic approach to understand the mechanism of action of the bisthiazolium compound T4 on the human malaria parasite, Plasmodium falciparum.

BackgroundIn recent years, a major increase in the occurrence of drug resistant falciparum malaria has been reported. Choline analogs, such as the bisthiazolium T4, represent a novel class of compounds with strong potency against drug sensitive and resistant P. falciparum clones. Although T4 and its analogs are presumed to target the parasites lipid metabolism, their exact mechanism of action remains unknown. Here we have employed transcriptome and proteome profiling analyses to characterize the global response of P. falciparum to T4 during the intraerythrocytic cycle of this parasite.ResultsNo significant transcriptional changes were detected immediately after addition of T4 despite the drugs effect on the parasite metabolism. Using the Ontology-based Pattern Identification (OPI) algorithm with an increased T4 incubation time, we demonstrated cell cycle arrest and a general induction of genes involved in gametocytogenesis. Proteomic analysis revealed a significant decrease in the level of the choline/ethanolamine-phosphotransferase (PfCEPT), a key enzyme involved in the final step of synthesis of phosphatidylcholine (PC). This effect was further supported by metabolic studies, which showed a major alteration in the synthesis of PC from choline and ethanolamine by the compound.ConclusionOur studies demonstrate that the bisthiazolium compound T4 inhibits the pathways of synthesis of phosphatidylcholine from choline and ethanolamine in P. falciparum, and provide evidence for post-transcriptional regulations of parasite metabolism in response to external stimuli.

Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial activity by the semiautomated microdilution technique.

Phospholipid biosynthetic activity is intense in the erythrocytic stage of Plasmodium falciparum because of the parasites own enzymatic machinery. The incorporation of various labeled phospholipid precursors in comparison with the incorporation of nucleic acid and protein precursors was tested to evaluate P. falciparum growth in vitro. These precursors, namely, [3H]ethanolamine, [3H]hypoxanthine, [3H]palmitate, [14C]serine, [3H]choline, [3H]inositol, and [3H]isoleucine, were all accurate indicators of parasite growth. However, because of its high level of incorporation, [3H]ethanolamine proved to be the best tool for assessing parasite viability. When culture parameters were carefully controlled, [3H]ethanolamine incorporation into phospholipids was proportional to pulse time, precursor concentration, and initial parasitemia and was sensitive to the number of uninfected erythrocytes (hematocrit). It can be used for a wide range of infected erythrocytes, from 2 x 10(4) to 5 x 10(5). The use of [3H]ethanolamine for in vitro antimalarial drug screening is a good alternative to the method of Desjardins et al. (R. E. Desjardins, C. J. Canfield, J. D. Haynes, and J. D. Chulay, Antimicrob. Agents. Chemother. 16:710-718, 1979). The major advantage is that the culture medium can be supplemented with hypoxanthine, which results in better parasite growth. [3H]ethanolamine is also an ideal tool when compounds that interfere with DNA and/or RNA metabolism are to be investigated for their effect on Plasmodium growth.