Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane.

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.

Southeast Asian ovalocytosis in White persons.

We describe two White persons, a girl and her mother, presenting with Southeast Asian ovalocytosis. The child was evaluated for scoliosis. The red cell indices were normal but the cell counter triggered an alarm due to a high fraction of hyperdense red cells. Blood smears showed ovalocytes and ovalostomatocytes. Red cells exhibited a total lack of deformability upon osmotic gradient ektacytometry performed immediately after blood drawing. Analysis of nucleic acids and proteins ascertained a 27 nucleotide deletion, resulting in the loss of amino acids 400 to 408, and the presence in cis of the Memphis I polymorphism. The sulfate transport was diminished by more than 50%. There was no acidosis. In vitro invasion of ovalocytes by Plasmodium falciparum was decreased. The mother presented with the same hematological picture. On the whole, the condition was Southeast Asian ovalocytosis in all respects. The present kindred had ancestors who had inhabited islands in the Southwestern Indian Ocean.

Plasmodium falciparum: influence of malarial and host erythrocyte skeletal protein interactions on phosphorylation in infected erythrocytes.

Phosphorylation of components of the erythrocyte membrane skeleton has major effects on the physical properties of the membrane. Infection of red cells by the protozoan parasite Plasmodium falciparum leads to a marked increase in the level of phosphorylation of red cell protein 4.1 and the insertion into the red cell skeleton of parasite-encoded phosphoproteins, including the mature-parasite-infected erythrocyte surface antigen (MESA). Because of the tight association of MESA with protein 4.1, we set out to determine the importance of this interaction and that of other parasite-encoded skeletal-associated proteins to phosphorylation of the infected red cell membrane. Our results show that neither MESA nor protein 4.1 is required for phosphorylation of its binding partner. Further, phosphorylation of MESA and protein 4.1 occurs independently of the presence of knobs, the expression of PfHRP1, or cytoadherence phenotype. In contrast to previous studies, we were unable to detect a change in the molecular weight of protein 4.1 in erythrocytes infected with cytoadherent parasite lines. In red cells infected with parasites expressing PfHRP1 (K+), MESA and protein 4.1 are substrates for a kinase with the inhibitor profile of a casein kinase. Surprisingly, however, when we examined phosphorylation of MESA and protein 4.1 in K(-)-infected erythrocytes, we found that casein kinase I and II inhibitors had no, or greatly reduced, effectiveness, and in fact, phosphorylation of these two proteins was enhanced in some instances.

X-ray microscopic visualization of specific labeling of adhesive molecule CD36 and cytoadherence by Plasmodium falciparum infected erythrocytes.

We investigated the cytoadherence of Plasmodium falciparum infected erythrocytes to target cells that express CD36 by soft x-ray microscopy. Using immunogold beads enhanced with silver, we localized CD36 on the surface of intact melanoma cells and throughout Triton extracted melanoma cells. We examined the orientation of parasites within erythrocytes that bound to target cells, and the interactions between the red cell membrane and the target cell, and we confirmed that fibrillar structures on the surface of melanoma and endothelial cells can be involved in the association between infected erythrocytes and melanoma cells or endothelial cells.

Malaria and the erythrocyte.

In terms of global health, the most important disease involving human erythrocytes is infection by protozoan parasites of the genus Plasmodium, particularly Plasmodium falciparum. Our understanding of the complex processes of erythrocyte invasion, remodeling, and cytoadherence has advanced considerably over the past few years. Considerable advances have been made in identifying the players in each of these phenomena, although identification of the exact functional roles for many molecules is still missing. The cloning of the parasite adhesin, the development of a transfection system, and a series of new imaging and cell biology assays are recent achievements that promise to further our understanding not only of the pathogenesis of malaria, but also the functioning of erythrocytes.

Intracellular structures of normal and aberrant Plasmodium falciparum malaria parasites imaged by soft x-ray microscopy.

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 inhibitor-treated, 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.

Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy.

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 immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence 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) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence 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 MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 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.

Role of the Plasmodium falciparum mature-parasite-infected erythrocyte surface antigen (MESA/PfEMP-2) in malarial infection of erythrocytes.

During intraerythrocytic growth of Plasmodium falciparum, several parasite proteins are transported from the parasite to the erythrocyte membrane, where they bind to membrane skeletal proteins. Mature-parasite-infected erythrocyte surface antigen (MESA) has previously been shown to associate with host erythrocyte membrane skeletal protein 4.1. Using a spontaneous mutant of P falciparum that has lost the ability to synthesize MESA and 4.1-deficient erythrocytes, we examined growth of MESA(+) and MESA(-) parasites in normal and 4.1-deficient erythrocytes. Viability of MESA(+) parasites was reduced in 4.1-deficient erythrocytes as compared with that for normal erythrocytes, but MESA(-) parasites grew equally well in 4.1-deficient and normal erythrocytes. Cytoadherence of MESA(+)- and MESA (-)-parasitized normal and 4.1-deficient erythrocytes to C32 melanoma cells was similar, indicating that neither protein 4.1 nor MESA plays a major role in cytoadherence of infected erythrocytes. Localization of MESA in normal and 4.1-deficient erythrocytes was examined by confocal microscopy. MESA was diffusely distributed in the cytosol of 4.1-deficient erythrocytes but was membrane-associated in normal erythrocytes. These findings suggest that MESA binding to protein 4.1 plays a major role in intraerythrocytic parasite viability.

A Plasmodium falciparum isolate with a chromosome 9 deletion expresses a trypsin-resistant cytoadherence molecule.

Sequestration of Plasmodium falciparum infected erythrocytes in the cerebral circulation is strongly implicated in the pathogenesis of cerebral malaria. From previous studies it was postulated that genes essential for cytoadherence were located on the right arm of chromosome 9 as P. falciparum isolates with a deletion in this region lost the capacity to cytoadhere in vitro and no longer expressed Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) on the surface of the infected cells. We have selected a P. falciparum isolate from Papua New Guinea for high levels of cytoadherence to human umbilical vein endothelial cells (HUVECs) and have shown that the cloned parasite has several novel properties related to cytoadherence. The cloned parasite adheres to HUVECs, does not bind to melanoma cells, and expresses a surface molecule with most of the properties of PfEMP-1, despite a deletion in the right arm of chromosome 9. Interestingly, the surface expressed PfEMP-1 in this strain is resistant to trypsin treatment and infected cells continue to cytoadhere after trypsin digestion at a concentration of 100 micrograms ml-1. The receptor on HUVECs for the cloned parasite lines is a molecule different from any previously described, as parasitized cells do not adhere to soluble intercellular adhesion molecule 1, thrombospondin, vascular cell adhesion molecule 1, E-selectin or P-selectin, nor to CD36. Our work, taken together with the results from previous studies, suggest that the ability of parasites to cytoadhere is encoded in at least two distinct genomic locations in the parasite, and the diversity of receptor-ligand interaction is greater than previously described.

Long-term cultured human vascular endothelial cells (EC-FP5) bind Plasmodium falciparum infected erythrocytes.

We have established an endothelial cell line, EC-FP5, that binds Plasmodium falciparum infected erythrocytes after several passages in culture and multiple cryopreservations. Binding by the EC-FP5 cells, measured as the percentage that bound infected erythrocytes and the average number of infected erythrocytes bound per endothelial cell, was similar in cells cryopreserved 1, 2, or 3 times. The parasite strain of the infected erythrocytes and culture conditions, including parasitemia and pH of the erythrocyte suspension, significantly affected binding. The capability of the EC-FP5 cells to be cultured in large amounts and cryopreserved in several aliquots will provide flexibility, reduce experimental variation, and enhance the utility of the endothelial cell-dependent cytoadherence assay.

Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro.

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.

The mature erythrocyte surface antigen of Plasmodium falciparum is not required for knobs or cytoadherence.

Intraerythrocytic Plasmodium falciparum parasites at the trophozoite and schizont stages synthesize a greater than 200-kDa protein, the mature erythrocyte surface antigen (MESA), that is localized at the membrane of infected red blood cells and manifests size polymorphism and antigenic diversity among parasite isolates. Because MESA is localized in the host cell membrane, we examined parasites with differing knob and cytoadherence phenotypes to determine whether MESA expression correlated with knob formation and cytoadherence. A cloned line of P. falciparum that was cultured with repeated selection for the knobbed and cytoadherent phenotypes did not express MESA, due to at least partial deletion of the single-copy MESA gene. In contrast, parasites from the same clone that were cultured without this selection lost the knobbed and cytoadherent phenotypes, but continued to express MESA. These results indicate that MESA is apparently not required for differentiation and multiplication of erythrocyte stage P. falciparum parasites in vitro, or for knob formation and cytoadherence. We speculate that MESA may have a role in evasion of the host immune response by P. falciparum.

Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor.

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.

Cytoadherence by Plasmodium falciparum-infected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IRBCs) adhere specifically to venular endothelium and thereby evade spleen-dependent immune mechanisms. We have investigated the molecular basis of cytoadherence. We report here that the capacity for cytoadherence of IRBCs is correlated with the expression of a family of variable proteins on the surface of IRBCs. Essential to these studies was the use of in vitro techniques for modulating the cytoadherence phenotype of cloned parasites. In initial studies, we found culture-adapted parasites to be poorly cytoadherent or noncytoadherent. To select for cytoadherent parasites, we incubated knobbed IRBCs with C32 melanoma cells and cultured the adherent cells. Repeated rounds of selection produced parasites with increased cytoadherence. To select for noncytoadherent parasites, we cultured the cells that did not adhere to C32 melanoma cells. Cytoadherent IRBCs from two different cloned isolates had large (Mr greater than 2.4 x 10(5) radioiodinatable proteins that differed in size between the isolates but had in common the biochemical properties of trypsin sensitivity and insolubility with Triton X-100. The proteins were not detected with uninfected erythrocytes, indicating that they were parasite determined, nor were they detected with IRBCs containing parasites cultured for many months without selection. With continued selection for the cytoadherent phenotype, additional IRBC surface proteins with larger molecular sizes (Mr 2.9 x 10(5) and 3.2 x 10(5] appeared. A sequence of reversible changes in the cytoadherence phenotype of cloned parasites was accompanied by variation in the molecular size of the IRBC surface protein. Increased cytoadherence was correlated with expression of larger proteins and decreased cytoadherence was correlated with expression of smaller proteins; there was no change in the molecular size of two other parasite proteins associated with the IRBC membrane. The results indicate that the expression of this family of proteins is closely linked to the cytoadherence phenotype of the parasites, suggesting that the members of the protein family have a role in mediating cytoadherence between IRBCs and endothelial cells.