Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.     

The role of variant surface antigens on malaria-infected red blood cells.

It has been proposed that the primary role of variant antigens appearing on the surface of red blood cells infected with malaria parasites is to mediate cytoadherence, and that the antigenic variation they display is an adaptation to avoid immune attack. Here, Allan Saul proposes that their role is the opposite: that their primary purpose is to generate an immune response, which regulates their growth and thereby establishes a chronic infection, and that the role of cytoadherence is to ensure that parasites failing to express this flag to the immune system are destroyed by the spleen.     

Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.     

Distribution of chloroquine in normal,pronase-treated and malaria-infected red cells

Pronase treatment of mouse red cells in the presence of chloroquine leads to greatly enhanced accumulation of the drug, which after freeze-thaw or hypotonic lysis is found to be located mainly in the membrane fraction. Much lower proportions of the drug are found in the membrane fraction prepared from Plasmodium berghei-infected red cells, which also have a high capacity for chloroquine accumulation. Pronase treatment of infected cells result only in a slight enhancement of total accumulation. The membrane-bound fraction of the drug is, however, increased while the fraction in the lysate is decreased. Membranes prepared from hypotonic lysis of normal or P. berghei-infected cells have similar capacities for chloroquine binding. These results show that the distribution of chloroquine in pronase-treated and malaria-infected cells are different and that pronase treatment of both normal and infected cells followed by lysis leads to availability of potential membrane binding sites.     

Decrease of lymphoid dendritic cells in blood from malaria-infected pregnant women

Activation of dendritic cells (DCs) during malaria is poorly documented and has mainly been studied in rodent models. We conducted studies in Senegal to better understand the relationship between DC subset activation and susceptibility of pregnant women to malaria. For each woman, samples were collected at delivery from peripheral (WB), placental (PB) and cord blood (CB). The ex vivo phenotypes of DCs were assessed using flow cytometry on whole blood. The percentage of total DCs was the same for malaria-infected or non-infected pregnant women, except for PB where a decrease in DCs was observed during infection. Lymphoid dendritic cells (LDC) also decreased in the three blood compartments of infected pregnant women and less differentiated DCs (ldDCs) increased. During infection, Human Leucocyte Antigen DR (HLA-DR) expression decreased on LDCs, myeloid DCs (MDCs) and ldDCs. IL-10 increased in the three blood compartments. These data demonstrate a modulation of DC sub-populations during placental malaria. A decrease in LDCs during placental malaria could trigger major alterations in the immune response and a change in the Th1/Th2 balance. However, elevated IL-10 observed during infection substantiates a normal micro-environment triggering normal production of DCs. The decrease in LDCs could thus be due to their migration towards spleen or other lymphoid organs.     

Plasmodium lophurae: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells

Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitro-en decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm3, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm3 and 1.158 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230, 000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.     

Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability

The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging.     

Uninfected red cells from malaria-infected blood: Alteration of fatty acid composition involving a serum protein: An in vivo and in vitro study

Summary Alteration of uninfected erythrocytes, fromPlasmodium (the malaria parasite)-infected blood remained an open question. In this study we compared the in vivo fatty acid compositions of control and uninfected monkey erythrocytes. A large (40%) increase in the linoleic acid level was observed, which was recovered mostly in neutral lipids. An in vitro system was developed to study medium-mediated alterations of cultured erythrocytes byPlasmodium falciparum. The increase in the linoleate level was reproduced in vitro and was also localized in the neutral lipid fraction, especially in triacylglycerols. Studies using proteolytic digestion and heat denaturation showed that a heat-labile serum protein is indispensable for the increase in the linoleate level of red cells treated with the supernatant, ofP. falciparum cultures. Both the function and the mechanism of this modification of uninfected erythrocytes still remain unknown. This work was supported by the UNDP/World Bank/WHO special program for Research and Training in Tropical Diseases (grant T16-181-M2-15B).     

Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise

Red blood cell (RBC) susceptibility to oxidative and osmotic stress in vitro was investigated in cells from trained and untrained men before and after submaximal exercise. Whilst no significant change in peroxidative haemolysis occurred immediately after 1 h of cycling at 60% of maximal aerobic capacity ( _max), a 20% increase was found 6 h later in both groups (P<0.05). The RBC osmotic fragility decreased by 15% immediately after exercise (P<0.001) and this was maintained for 6 h (Ps<0.001). There was an associated decrease in mean cell volume (P<0.05). Training decreased RBC susceptibility to peroxidative haemolysis (P<0.025) but it did not influence any other parameter. These exercise-induced changes were smaller in magnitude but qualitatively similar to those found in haemopathological states involving haem-iron incorporation into membrane lipids and the short-circuiting of antioxidant protection. To explore this similarity, a more strenuous and mechanically stressful exercise test was used. Running at 75% _max for 45 min reduced the induction time of O₂ uptake (peroxidation), consistent with reduced antioxidation capacity, and increased the maximal rate of O2 uptake in RBC challenged with cumene hydroperoxide (P<0.001). The proportion of high-density RBC increased by 10% immediately after running (P<0.001) but no change in membrane-incorporated haem-iron occurred. In contrast, treatment of RBC with oxidants (20–50 mol·l^–1 in vitro increased cell density and membrane incorporation of haem-iron substantially. These results showed that single episodes of submaximal exercise caused significant changes in RBC susceptibility to oxidative and osmotic stress. Such responses may account for the increase in RBC turnover found in athletes undertaking strenuous endurance training.     

A recombinant peptide based on Pf EMP-1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow

During falciparum malaria infection, severe complications ensue because parasitized red blood cells (PRBCs) adhere to endothelial cells and accumulate in the microvasculature. At the molecular level, adhesion is mediated by interaction of Plasmodium falciparum erythrocyte membrane protein 1 ( Pf  EMP-1) on the PRBC surface with receptors on the surface of endothelial cells, including CD36. We have shown that a recombinant 179-residue subfragment of Pf  EMP-1 (rC1-2[1–179]), which encompasses the CD36-binding region, inhibits and reverses adhesion of PRBCs to CD36 under physiologically relevant flow conditions. rC1-2[1–179] inhibited adhesion in a concentration-dependent manner over the range 100 pM to 2 μM, with up to 99% of adhesion blocked at the highest concentration tested. The antiadhesive activity of rC1-2[1–179] was not strain specific and almost totally ablated adhesion of four different parasite lines. Furthermore, rC1-2[1–179] showed remarkable ability to progressively reverse adhesion when flowed over adherent PRBCs for 2 h. The effect of rC1-2[1–179] was, however, specific for CD36-mediated adhesion and had no effect on adhesion mediated by CSA. Interference with binding of PRBCs to the vascular endothelium using rC1-2[1–179] or smaller organic mimetics may be a useful therapeutic approach to ameliorate severe complications of falciparum malaria.