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Infections with the human malaria parasite Plasmodium falciparum are characterized by sequestration of erythrocytes infected with mature forms of the parasite. Sequestration of infected erythrocytes appears to be critical for survival of the parasite and to mediate immunopathological abnormalities in severe malaria. A leukocyte differentiation antigen (CD36) was previously suggested to have a role in sequestration of malaria-infected erythrocytes. CD36 was purified from platelets, where it is known as GPIV, and was shown to be a receptor for binding of infected erythrocytes. Infected erythrocytes adhered to CD36 immobilized on plastic; purified CD36 exhibited saturable, specific binding to infected erythrocytes; and purified CD36 or antibodies to CD36 inhibited and reversed binding of infected erythrocytes to cultured endothelial cells and melanoma cells in vitro. The portion of the CD36 molecule that reverses cytoadherence may be useful therapeutically for rapid reversal of sequestration in cerebral malaria.

Erythrocytes infected with trophozoites and schizonts ofthe human malaria parasite Plasmodium falciparum develop surface protrusions (knobs) (1) by which the infected erythrocytes (IRBCs)t adhere specifically to venular endothelium in vivo (2, 3) and to human endothelial cells (4) and some lines of melanoma cells (5) in vitro. Cytoadherence between IRBCs and venular endothelium has a critical role in the pathogenesis of falciparum malaria, since it permits the mature parasites to evade spleen-dependent immune mechanisms (6) and since the sequestered parasites may occlude blood flow, as seen in cerebral malaria (7). Antibody in immune serum reacts with a strain-specific parasite-determined antigen on IRBCs and inhibits cytoadherence in vitro (8) and in vivo (9). The inhibition of cytoadherence by antibody may protect the host from the clinical consequences of falciparum malaria.Both cytoadherence and the IRBC surface antigen were shown to be destroyed by incubating IRBCs with proteases (9, 10), suggesting that the two properties are determined by proteins on the IRBC surface. In addition, the cytoadherence phenotype ofP.falciparum parasites and the expression ofthe IRBC surface antigen were modulated together by the spleen in a monkey model of falciparum malaria (9, 10), suggesting that the two properties are linked and perhaps determined by the same protein. A family ofpotential cytoadherence proteins was identified in studies with IRBCs from Aotus monkeys (11) . The members ofthe protein family differed in antigenicity and molecular size among strains ofP.falciparum, but had in common several biochemical properties, including their accessibility to surface radioiodination, detergent solubility, and cleavage by the same concentration of trypsin that inhibited cytoadherence (11) .

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-function relationships, including elucidation of protein-protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunof luorescence antibody assays. Simultaneous dualcolor excitation and detection with NSOM provided f luorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) f luorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two f luorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA)(parasite)͞protein4.1(host) and P. falciparum histidine rich protein (PfHRP1)(parasite)͞ protein4.1(host) showed good real-space correlation for the MESA͞protein4.1 pair, but relatively poor correlation for the PfHRP1͞protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.One of the crucial aspects of current biological inquiry relates to the organization of cells and how interactions between proteins are involved in important cellular processes, such as signal transduction and receptor-ligand binding. Several experimental systems have been developed in recent years to try and identify such protein-protein interactions including the two hybrid and phage display systems (1, 2). All such attempts to identify interactions in disassembled cellular systems need to be confirmed in vivo, by approaches such as chemical cross-linking in cells or some form of colocalization study using microscopy. A common approach has been the use of confocal microscopy using fluorescent antibody probes. We report here the development of a technique based on dual-color immunofluorescence labeling together with near-field scanning optical microscopy (NSOM) (3, 4), which offers significant advances in the det...

The CD36 leukocyte differentiation antigen, recognized by MAbs OKM5 and OKM8 and found on human monocytes and endothelial cells, has been implicated as a sequestration receptor for erythrocytes infected with the human malaria parasite Plasmodium falciparum (IRBC). CD36 is also expressed on platelets and appears to be identical to platelet glycoprotein IV. We investigated receptor activation of monocytes and platelets by anti-CD36 MAbs and by IRBC. Incubation of human monocytes with anti-CD36 MAbs or IRBC resulted in stimulation of the respiratory burst as measured by reduction of nitroblue tetrazolium and generation of chemiluminescence.Incubation of human platelets with anti-CD36 MAbs resulted in platelet activation as measured by aggregation or ATP secretion. Activation of monocytes and platelets required appropriate intracellular transmembrane signaling and was inhibited by calcium antagonists or by specific inhibitors of protein kinase C or guanine nucleotide binding proteins. Soluble CD36 inhibited binding of IRBC to both monocytes and platelets, suggesting that these interactions are mediated by the CD36 receptor. Using a cytochemical electron microscopic technique, the presence of reactive oxygen intermediates was identified at the interface between human monocytes and IRBC. These data provide support for the hypothesis that reactive oxygen intermediates produced by monocytes when IRBC ligands interact with cell surface receptors may play a role in the pathophysiology of falciparum malaria.

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.

Soft x-ray microscopy is a novel approach for investigation of intracellular organisms and subcellular structures with high spatial resolution. We used x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes and in infected erythrocytes treated with cysteine protease inhibitors. Investigations in normal red blood cells enabled us to recognize anomalies in parasite structures resulting from growth under unfavorable conditions. X-ray microscopy facilitated detection of newly elaborated structures in the cytosol of fixed, unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. In cysteine protease inhibitortreated, infected erythrocytes, high concentrations of material were detected in abnormal digestive vacuoles and aggregated at the parasite plasma membrane. We have demonstrated that an abnormal host erythrocyte skeleton affects structural development of parasites and that this aberrant development can be detected in the following generation when parasites from protein 4.1-deficient red blood cells infect normal erythrocytes. This work extends our current understanding of the relationship between the host erythrocyte membrane and the intraerythrocytic malaria parasite by demonstrating for the first time that constituents of the erythrocyte membrane play a role in normal parasite structural development.

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