Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued.

The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial thin sections of parasitized erythrocytes (PE) by electron microscopy. We find that actin dynamics play multiple, important roles in the hemoglobin transport pathway, and that hemoglobin delivery to the FV via the cytostomes might be required for parasite survival. Evidence is provided for a new model, in which hemoglobin transport to the FV occurs by a vesicle-independent process.

Ribbon-like structures result when amphotericin B interacts with lipid in an aqueous environment. At high ratios of amphotericin to lipid these structures, which are lipid-stabilized amphotericin aggregates, become prevalent resulting in a dramatic attenuation of amphotericin-mediated mammalian cell, but not fungal cell, toxicity. Studies utilizing freeze-etch electron microscopy, differential scanning calorimetry, "P NMR, x-ray diffraction, and optical spectroscopy revealed that this toxicity attenuation is related to the macromolecular structure of the complexes in a definable fashion. It is likely that amphotericin in this specific form will have a much improved therapeutic utility.Amphotericin B, a polyene antibiotic, is the drug ofchoice for a variety of systemic fungal infections that were almost always fatal prior to its introduction (1). The cytotoxic mechanism of this compound has been the subject of intensive investigation over many years and is thought to reside in its ability to form membrane ion channels particularly in the presence of sterols (2). That these channels and associated lethal permeability changes occur at somewhat lower membrane concentrations in the presence of ergosterol (the predominant sterol in fungal cell membranes) rather than cholesterol (the predominant sterol in mammalian cell membranes) most likely forms the basis of the selective toxicity of this drug (3). Still, in its present dosage form, which is a deoxycholate micelle, its clinical utility is profoundly limited by host cell toxicity particularly in those cases (such as in patients with acquired immunodeficiency syndrome) where high-dose therapy is indicated.Much attention has focused on liposome suspensions containing 5-10 mol % amphotericin B because these systems have been shown to produce a significant attenuation of toxicity with little compromise to efficacy (4-7). In fact one such formulation in which amphotericin comprises 5 mol % of a 1,2-dimyristoyl-sn-glycero(3)phosphocholine/1,2-dimyristoyl-sn-glycero(3)phospho (1) PtdGro at a molar ratio of 7:3 was deposited from chloroform as a thin film on the bottom ofa 500-ml round-bottomed flask by rotoevaporation. This film was solubilized in 100 ml of amphotericin B in methanol (0.1 mg/ml) and again rotoevaporated to a thin film. When dry, the film was suspended in isotonic phosphatebuffered saline (PBS) and subjected to bath sonication for 0.5 hr or until particles exhibiting Brownian motion were observed by phase-contrast microscopy.Sucrose Density Centrifugation. Typically, 200 gl of material was layered onto a continuous sucrose gradient in 150 mM NaCI/20 mM Hepes, pH 7.4. The gradient was centrifuged for 22 hr at 22TC in a SW 60 rotor (Beckman) at 230,000 x g. After centrifugation the gradient was fractionated into 150-,ul aliquots and assayed for amphotericin B (from absorbance at 412 nm in dimethylformamide) and phospholipid (from phosphate assay after digestion).In Vivo Toxicity. LD50 values were determined by the method of Reed and Muench (12). Female...

The phospholipid and fatty acid compositions of the host infected erythrocyte plasma membrane (IEPM) have been determined for erythrocytes infected with the human malaria parasite Plasmodium falciparum. IEPM were prepared by selective lysis of the host erythrocyte (but not of the parasite membranes) with 0.1% saponin, followed by differential centrifugation. The purity of the IEPM was determined by measuring the membrane-specific enzyme markers acetylcholinesterase, glutamate dehydrogenase and lactate dehydrogenase, and by immunoelectron microscopy using monoclonal antibodies specific for human erythrocyte glycophorin A (4E7) and for a 195 kDa parasite membrane glycoprotein (Pf6 3B10.1). Both approaches demonstrated that the host erythrocyte plasma membrane preparation was free from contamination by parasite membranes. During intra-erythrocytic development of the parasite, the phospholipid composition of the erythrocyte membrane was strikingly altered. IEPM contained more phosphatidylcholine (38.7% versus 31.7%) and phosphatidylinositol (2.1% versus 0.8%) and less sphingomyelin (14.6% versus 28.0%) than normal uninfected erythrocytes. Similar alterations in phospholipid composition were determined for erythrocyte membranes of parasitized cells isolated by an alternative method utilizing polycationic polyacrylamide microbeads (Affigel 731). The total fatty acid compositions of the major phospholipids in IEPM were determined by g.l.c. The percentage of polyunsaturated fatty acids in normal erythrocyte phospholipids (39.4%) was much higher than in phospholipids from purified parasites (23.3%) or IEPM (24.0%). The unsaturation index of phospholipids in IEPM was considerably lower than in uninfected erythrocytes (107.5 versus 161.0) and was very similar to that in purified parasites (107.5 versus 98.5). Large increases in palmitic acid (C16:0) (from 21.88% to 31.21%) and in oleic acid (C18:1) (from 14.64% to 24.60%), and major decreases in arachidonic acid (C20:4) (from 17.36% to 7.85%) and in docosahexaenoic acid (C22:6) (from 4.34% to 1.8%) occurred as a result of infection. The fatty acid profiles of individual phospholipid classes from IEPM resembled in many instances the fatty acid profiles of parasite phospholipids rather than those of uninfected erythrocytes. Analysis of IEPM from P. falciparum-infected erythrocytes (trophozoite stage) revealed that, during intra-erythrocytic maturation of the parasite, the host erythrocyte phospholipid composition was markedly refashioned. These alterations were not dependent on the method used to isolate the IEPM, with similar results obtained using either a saponin-lysis method or binding to Affigel beads. Since mature erythrocytes have negligible lipid synthesis and metabolism, these alterations must occur as a result of parasite-directed metabolism of erythrocyte lipids and/or trafficking of lipids between the parasite and erythrocyte membranes.

Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents.

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Hemoglobin degradation during the asexual cycle of Plasmodium falciparum is an obligate process for parasite development and survival. It is established that hemoglobin is transported from the host erythrocyte to the parasite digestive vacuole (DV), but this biological process is not well characterized. Three-dimensional reconstructions made from serial thin-section electron micrographs of untreated, trophozoite-stage P. falciparum-infected erythrocytes (IRBC) or IRBC treated with different pharmacological agents provide new insight into the organization and regulation of the hemoglobin transport pathway. Hemoglobin internalization commences with the formation of cytostomes from localized, electron-dense collars at the interface of the parasite plasma and parasitophorous vacuolar membranes. The cytostomal collar does not function as a site of vesicle fission but rather serves to stabilize the maturing cytostome. We provide the first evidence that hemoglobin transport to the DV uses an actin-myosin motor system. Short-lived, hemoglobin-filled vesicles form from the distal end of the cytostomes through actin and dynamin-mediated processes. Results obtained with IRBC treated with N-ethylmaleimide (NEM) suggest that fusion of hemoglobin-containing vesicles with the DV may involve a soluble NEM-sensitive factor attachment protein receptor-dependent mechanism. In this report, we identify new key components of the hemoglobin transport pathway and provide a detailed characterization of its morphological organization and regulation. Malaria is a devastating disease that infects Ͼ300 million people each year, resulting in Ͼ1 million deaths, with the protozoan species Plasmodium falciparum being responsible for the majority of these deaths (1). The morbidity and mortality associated with the disease are largely the result of the parasite's asexual intraerythrocytic cycle (2). P. falciparum digests host cell hemoglobin to support parasite growth and asexual replication during the intraerythrocytic stage (3, 4). In order for the parasite to survive within the erythrocyte host, it degrades approximately 80% of the erythrocyte hemoglobin (5), with the majority of this digestion occurring during the trophozoite stage (18 to 32 h postinvasion) (3). The bulk of hemoglobin degradation occurs via a semiordered process by proteases contained within the parasite's digestive vacuole (DV) (6).The transport of hemoglobin from the host erythrocyte cytosol to the parasite DV is a long-known but poorly understood biological process. The internalization of hemoglobin is thought to occur through an unusual structure, the cytostome. A cytostome is defined as a localized invagination of the parasite's outer membranes (the parasitophorous vacuolar membrane [PVM] and the parasite plasma membrane [PPM]), with a submembranous electron-dense collar associated with the neck of the cytostome (7, 8). It has traditionally been assumed that hemoglobin transport commences with the cytostome pinching off at the neck to form a double-membrane, hemoglobin-f...

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