Leann Tilley

Quinoline antimalarials: mechanisms of action and resistance and prospects for new agents.

Quinoline-containing antimalarial drugs, such as chloroquine, quinine and mefloquine, are mainstays of chemotherapy against malaria. The molecular basis of the action of these drugs is not completely understood, but they are thought to interfere with hemoglobin digestion in the blood stages of the malaria parasites life cycle. The parasite degrades hemoglobin, in an acidic food vacuole, producing free heme and reactive oxygen species as toxic by-products. The heme moieties are neutralized by polymerisation, while the free radical species are detoxified by a vulnerable series of antioxidant mechanisms. Chloroquine, a dibasic drug, is accumulated several thousand-fold in the food vacuole. The high intravacuolar chloroquine concentration is proposed to interfere with the polymerisation of heme and/or the detoxification of the reactive oxygen species, effectively killing the parasite with its own metabolic waste. Chloroquine resistance appears to arise as a result of a decreased level of chloroquine uptake, due to an increased vacuolar pH or to changes in a chloroquine importer or receptor. The more lipophilic quinolinemethanol drugs mefloquine and quinine do not appear to be concentrated so extensively in the food vacuole and may act on alternative targets in the parasite. Resistance to the quinolinemethanols is thought to involve a plasmodial homolog of P-glycoprotein. As the malaria parasites become increasingly resistant to the quinoline antimalarials, there is an urgent need to understand the molecular mechanisms for drug action and resistance so that novel antimalarial drugs can be designed. A number of modified quinolines and bisquinoline compounds show some promise in this regard.

Malaria parasite proteins that remodel the host erythrocyte

Exported proteins of the malaria parasite Plasmodium falciparum interact with proteins of the erythrocyte membrane and induce substantial changes in the morphology, physiology and function of the host cell. These changes underlie the pathology that is responsible for the deaths of 1–2 million children every year due to malaria infections. The advent of molecular transfection technology, including the ability to generate deletion mutants and to introduce fluorescent reporter proteins that track the locations and dynamics of parasite proteins, has increased our understanding of the processes and machinery for export of proteins in P. falciparum-infected erythrocytes and has provided us with insights into the functions of the parasite protein exportome. We review these developments, focusing on parasite proteins that interact with the erythrocyte membrane skeleton or that promote delivery of the major virulence protein, PfEMP1, to the erythrocyte membrane.

Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes

The most virulent of the human malaria parasites, Plasmodium falciparum, undergoes a remarkable morphological transformation as it prepares itself for sexual reproduction and transmission via mosquitoes. Indeed P. falciparum is named for the unique falciform or crescent shape of the mature sexual stages. Once the metamorphosis is completed, the mature gametocyte releases from sequestration sites and enters the circulation, thus making it accessible to feeding mosquitoes. Early ultrastructural studies showed that gametocyte elongation is driven by the assembly of a system of flattened cisternal membrane compartments underneath the parasite plasma membrane and a supporting network of microtubules. Here we describe the molecular composition and origin of the sub-pellicular membrane complex, and show that it is analogous to the inner membrane complex, an organelle with structural and motor functions that is well conserved across the apicomplexa. We identify novel crosslinking elements that might help stabilize the inner membrane complex during gametocyte development. We show that changes in gametocyte morphology are associated with an increase in cellular deformability and postulate that this enables the gametocytes to circulate in the bloodstream without being detected and removed by the mechanical filtering mechanisms in the spleen of the host.

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob‐associated histidine‐rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite‐derived structures, known as Maurers clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua, New Guinea

SummaryMelanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.

Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion

Combination regimens that include artemisinin derivatives are recommended as first line antimalarials in most countries where malaria is endemic. However, the mechanism of action of artemisinin is not fully understood and the usefulness of this drug class is threatened by reports of decreased parasite sensitivity. We treated Plasmodium falciparum for periods of a few hours to mimic clinical exposure to the short half-life artemisinins. We found that drug treatment retards parasite growth and inhibits uptake of hemoglobin, even at sublethal concentrations. We show that potent artemisinin activity is dependent on hemoglobin digestion by the parasite. Inhibition of hemoglobinase activity with cysteine protease inhibitors, knockout of the cysteine protease falcipain-2 by gene deletion, or direct deprivation of host cell lysate, significantly decreases artemisinin sensitivity. Hemoglobin digestion is also required for artemisinin-induced exacerbation of oxidative stress in the parasite cytoplasm. Arrest of hemoglobin digestion by early stage parasites provides a mechanism for surviving short-term artemisinin exposure. These insights will help in the design of new drugs and new treatment strategies to circumvent drug resistance.

Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum, and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface‐exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurers clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O‐permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurers cleft membrane with the C‐terminal domain exposed to the erythrocyte cytoplasm, whereas the N‐terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron‐lucent extensions of the Maurers clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non‐specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6‐times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP‐depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.

Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance.

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra‐erythrocytic parasites digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil‐reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.

Food vacuole‐associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum

The malaria parasite Plasmodium falciparum induces a sixfold increase in the phospholipid content of infected erythrocytes during its intraerythrocytic growth. We have characterized the lipid environments in parasitized erythrocyte using the hydrophobic probe, Nile Red. Spectral imaging with a confocal microscope revealed heterogeneous lipid environments in parasite‐infected erythrocytes. An insight into the nature of these environments was gained by comparing these spectra with those of triacylglycerol/phospholipid emulsions and phospholipid membranes. Using this approach, we identified a population of intensely stained particles of a few hundred nanometers in size that are closely associated with the digestive vacuole of the parasite and appear to be composed of neutral lipids. Electron microscopy and isolation of food vacuoles confirmed the size of these particles and their intimate association respectively. Lipid analysis suggests that these neutral lipid bodies are composed of di‐ and triacylgycerols and may represent storage organelles for lipid intermediates that are generated during digestion of phospholipids in the food vacuole. Mono‐, di‐ and triacylglycerol suspensions promote β‐haematin formation, suggesting that these neutral lipid bodies, or their precursors, may also be involved in haem detoxification. We also characterized other compartments of the infected erythrocyte that were stained less intensely with the Nile Red probe. Both the erythrocyte membrane and the parasite membrane network exhibit red shifts compared with the neutral lipid bodies that are consistent with cholesterol‐rich and cholesterol‐poor membranes respectively. Ratiometric imaging revealed more subtle variations in the lipid environments within the parasite membrane network.