The interaction between the malaria parasite and the red cell membrane

The malaria parasite intimately interacts with the host red cell membrane throughout the cycle of invasion and intracellular development. Direct interaction between the merozoite surface and the red cell membrane involves specific binding between the surface components of both cells, which leads to the subsequent endocytotic process still incompletely understood. Intracellular development of the parasite is accompanied by various changes in the structure and function of red cell membrane components. Some changes may benefit parasite survival while others trigger host immune response. An understanding of both the direct interaction between the surface components of the parasite and the red cell during invasion, and the subsequent changes in the red cell membrane following invasion, should lead to better ways of controlling malaria.     

Inhibition of malarial parasite invasion by monoclonal antibodies against glycophorin A correlates with reduction in red cell membrane deformability

The effect of well-characterized monoclonal antibodies to red cell surface molecules on the invasion of human red cells by the malarial parasites Plasmodium falciparum and Plasmodium knowlesi was examined. Antibodies to glycophorin A (GP alpha) inhibit invasion for both parasite species, and this is highly correlated with the degree to which they decrease red cell membrane deformability as measured by ektacytometry. This effect on rigidity and invasion was also seen with monovalent Fab fragments. The closer the antibody binding site was to the membrane bilayer, the greater was its effect on inducing membrane rigidity and decreasing parasite invasion. Antibodies to the Wright determinant in particular were the most inhibitory. This differential effect of the various antibodies was not correlated with their binding affinities or the number of sites bound per cell. Antibodies to surface molecules other than GP alpha were without effect. A novel mechanism is described whereby monoclonal antibodies and their Fab fragments directed at determinants on the external surface of red cells might act to inhibit invasion by malarial parasites by altering membrane material properties.     

Malaria and the red cell membrane

Malarial parasites are primarily parasites of red cells and during infection ingest most of the haemoglobin within these cells, leaving the membrane as the only vestige of the original host cell. The red cell membrane thus plays a key role at all stages of infection with malarial parasites, and is modified in many ways during parasitisation, so that at least functionally it has little resemblance to the membrane from which it was originally derived. The highly specific and ordered process of parasite invasion of red cells is regulated at least in part by the uninfected red cell membrane. The red cell sialoglycoproteins or glycophorins of this membrane have been shown to play an important role in invasion by Plasmodium falciparum, the species of most importance to man because of it's high morbidity and mortality. Structurally, dynamic changes occur within the membrane during parasitisation, and a number of parasite proteins have been found to be associated within it, but changes on the surface of the infected cell have been more difficult to demonstrate. The membrane of the infected cell is important in the many metabolic processes of the parasite, as well as the critical cell-cell interactions that occur when cells containing mature parasites bind to endothelial cells (cytoadherence), bind to uninfected cells (rosetting), or interact with macrophages and other leucocytes. The recognition molecules on the red cell membrane involved in invasion, cytoadherence and rosetting appear to be quite distinct. Structural and functional changes have also been shown to occur in the membranes of uninfected red cells, both in infected patients, and in the presence of parasites in vitro. Interactions of the parasite P. falciparum with the red cell membrane hold the key to our understanding of the pathogenesis of severe falciparum infection in man.     

Effect of metalloprotease inhibitors on invasion of red blood cell by Plasmodium falciparum

For successful invasion, the malaria merozoite needs to attach to the red blood cell membrane, undergo reorientation, form a junction of the apical end with the host membrane, and internalize. Malaria proteases have been implicated in the invasion process, but their specific cellular functions remain unclear. To demonstrate the involvement of metalloprotease in the process of Plasmodium falciparum merozoite entry into host red blood cell, schizont-infected red blood cells and parasitophorous vacuolar membrane-enclosed merozoite structures were treated with 1,10-phenanthroline, a metal chelator, resulting in a reduction of invasion with IC50 value of 25 and 29 microM, respectively. Absence of an accumulation of schizont stages after treatment with 1,10-phenanthroline indicated that the inhibitory effect was not due to suppression of merozoite release from red blood cells, but on the invasion step. Although treatment with GM6001, a well-known inhibitor of the mammalian matrix and disintegrin metalloprotease family, was less effective, nevertheless this study points to the importance of metal-requiring protease in the process of invasion of host red blood cell by the malaria parasite.     

Preliminary characterization of the murine membrane reticulocyte proteome

The maturation from reticulocyte (immature red blood cell) to erythrocyte (mature red blood cell) includes the loss or decreased expression of cell surface molecules through exosome formation and secretion. Identifying the molecules lost and the molecular events involved is important to our understanding of this final stage of erythropoiesis and of diseases where it is deranged. Also, the presence of certain cell surface molecules is likely responsible for the invasion of certain malaria parasites into reticulocytes. Using a global proteomics approach, we identified proteins potentially lost during and/or involved in the reticulocyte maturation process. The reticulocyte proteome has not yet been published, as previous such studies have focused on the mature erythrocyte. Membrane-rich fractions were fractionated by electrophoresis followed by analysis with tandem mass spectrometry. Seven hundred forty four proteins were identified in the reticulocyte-rich membrane fraction, 192 proteins in the erythrocyte-rich membrane fraction, with 157 common to both fractions. Many of the proteins found uniquely in the reticulocyte were associated with structures known to be in reticulocytes (mitochondria, Golgi). Additional proteins detected are or may be specifically involved in vesicle trafficking, a process important in the maturation process. A number of unique plasma membrane proteins were also identified. These results provide the groundwork for future targeted studies to improve our understanding of the mechanism of reticulocyte maturation and the role of reticulocytes in disease.     

Inhibition of malarial invasion of red cells by chemical and immunochemical linking of spectrin molecules

The invasion of resealed human red cell ghosts by Plasmodium falciparum, and those from monkey cells by P. knowlesi, was strongly inhibited by anti-spectrin antibodies introduced into their cytoplasm. Univalent F(ab)1 fragments gave no such effect, but a combination of these fragments and goat anti-rabbit immunoglobulin, to restore bifunctionality, caused perceptible inhibition of invasion. Disulphide cross-links introduced between spectrin molecules in intact red cells by the membrane-permeant oxidizing agent, diamide, again led to inhibition of invasion. This effect was largely reversed by reduction of the cross-links. Gel electrophoresis was used to confirm that cross-linking was essentially confined to spectrin, and that extended covalent networks were not formed. It follows that local formation of bridges, whether by antibodies or oxidation of thiol groups, functions by inhibiting a local rearrangement of the cytoskeleton that forms a step in the invasion process.     

Preparation of a dry red blood cell Trichinella antigenic diagnosticum for indirect hemagglutination test and evaluation of its efficiency

The present paper presents the stages of a process for manufacturing a dry red blood cell Trichinella antigenic diagnosticum for indirect hemagglutination test (IHAT) and the evaluation of its diagnostic efficiency. The diagnosticum is shown to be a 3% suspension lyophilized from formalinized and tanned sheep red blood cells on which an excretory-secretory antigen of invasion Trichinella larvae was absorbed in a dose of 100 mg/ml. The rather high sensitivity (98.7%) and specificity (97.3%) of IHAT using the Trichinella diagnosticum based on the excretory-secretory antigen allow it to be considered an effective method for diagnosis of human trichinosis.     

Steroid receptor coactivator-3, a homolog of Taiman that controls cell migration in the Drosophila ovary, regulates migration of human ovarian cancer cells

Border cell migration is a process that occurs during Drosophila ovarian development in which cells derived from a simple epithelium migrate and invade neighboring tissue. This process resembles the behavior of cancerous cells that derive from the simple epithelium of the human ovary. One important regulator of border cell migration is Taiman, a homolog of steroid receptor coactivator-3 (SRC-3). Because increasing evidence indicates that similarities exist between the molecular control of migration of border cells and of cancer cells, we investigated whether SRC-3 controls ovarian cancer cell migration. Little or no SRC-3 expression was detected in normal ovarian surface epithelium, ovarian cysts and borderline ovarian tumors that lack stromal invasion. In contrast, SRC-3 was abundantly expressed in high-grade ovarian carcinomas. Inhibiting SRC-3 expression in ovarian cancer cells markedly reduced cell spreading and migration, and altered intracellular localization of focal adhesion kinase. This inhibitory effect on cell migration was independent of the estrogen receptor (ER) status of the cells. These studies reveal a novel role for SRC-3 in ovarian cancer progression by promoting cell migration, independently of its role in estrogen receptor signaling.     

The Increased Susceptibility of Young Red Cells to Invasion by the Malarial Parasite Plasmodium falciparum

S ummary The relationship between red cell age and susceptibility to invasion by the malarial parasite Plasmodium falciparum has been examined by several different methods including short-term cultures of parasitized human blood. The results indicate that reticulocytes and young red cells are more susceptible to invasion by this parasite as compared with metabolically older cell populations. This is contrary to the current belief that red cells of all ages are invaded indiscriminately by P. falciparum.
This observation has important theoretical, clinical and practical implications; its mechanism remains as yet unclear.     

Identification of an erythroid ATP-dependent aminophospholipid transporter

The asymmetric distribution of amino-containing phospholipids in plasma membranes is essential for the function and survival of mammalian cells. Phosphatidylserine (PS) is restricted to the inner leaflet of plasma membranes by an ATP-dependent transport process. Exposure of PS on the surface of cells serves as a binding site for haemostatic factors, triggers cell-cell interaction and recognition by macrophages and phospholipases. Exposure of PS on the red cell surface plays a significant role in sickle cell pathology. We report the identification of two different isoforms of the aminophospholipid translocase, Atp8a1, or flippase, in the murine red blood cell membrane.     

Therapy with hydroxyurea is associated with reduced adhesion molecule gene and protein expression in sickle red cells with a concomitant reduction in adhesive properties

Propagation of the vaso-occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady-state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4-integrin subunit genes, CD49d (alpha-subunit) and CD29 (beta-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using real-time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36.     

Microvascular rheology and hemodynamics

The goal of elucidating the biophysical and physiological basis of pressure-flow relations in the microcirculation has been a recurring theme since the first observations of capillary blood flow in living tissues. At the birth of the Microcirculatory Society, seminal observations on the heterogeneous distribution of blood cells in the microvasculature and the rheological properties of blood in small bore tubes raised many questions on the viscous properties of blood flow in the microcirculation that captured the attention of the Society's membership. It is now recognized that blood viscosity in small bore tubes may fall dramatically as shear rates are increased, and increase (dramatically with elevations in hematocrit. These relationships are strongly affected by blood cell deformability and concentration, red cell aggregation, and white cell interactions with the red cells anti endothelium. Increasing strength of red cell aggregation may result in sequestration of clumps of red cells with either reductions or increases in microvascular hematocrit dependent upon network topography. During red cell aggregation, resistance to flow may thus decrease with hematocrit reduction or increase due to redistribution of red cells. Blood cell adhesion to the microvessel wall may initiate flow reductions, as, for example, in the case of red cell adhesion to the endothelium in sickle cell disease, or leukocyte adhesion in inflammation. The endothelial glycocalyx has been shown to result from a balance of the biosynthesis of new glycans, and the enzymatic or shear-dependent alterations in its composition. Flow-dependent reductions in the endothelial surface layer may thus affect the resistance to flow and/or the adhesion of red cells and/or leukocytes to the endothelium. Thus, future studies aimed at the molecular rheology of the endothelial surface layer may provide new insights into determinants of the resistance to flow.     

Investigations on Enzyme Absorption on the Red Cell Surface

Abstract. The absorption of blood group A-decomposing enzyme from Clostridium tertium on the red cell surface was examined. The enzyme was thoroughly absorbed on the surface of red cells of blood group A. Cells which were treated for a few minutes and separated from the enzyme solution lost their A-activity more rapidly than those which were maintained in enzyme solution. Enzyme absorption occurs with biochemical and biophysical specificity; i.e. A-enzyme is absorbed solely on A-cells and it competes with anti-A agglutinin from Euphadra periomphala for their reactive sites. N-acetyl-D-galactosamine and D-galactosamine are considered to release cell-fixed A-enzyme by an elution process.     

Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells

Using an in vitro culture system, we observed the migration of malaria ookinetes on the surface of the mosquito midgut and invasion of the midgut epithelium. Ookinetes display constrictions during migration to the midgut surface and a gliding motion once on the luminal midgut surface. Invasion of a midgut cell always occurs at its lateral apical surface. Invasion is rapid and is often followed by invasion of a neighboring midgut cell by the ookinete. The morphology of the invaded cells changes dramatically after invasion, and invaded cells die rapidly. Midgut cell death is accompanied by activation of a caspase-3-like protease, suggesting cell death is apoptotic. The events occurring during invasion were identical for two different species of Plasmodium and two different genera of mosquitoes; they probably represent a universal mechanism of mosquito midgut penetration by the malaria parasite.     

Unrestricted growth of Plasmodium falciparum in microcytic erythrocytes in iron deficiency and thalassaemia

The mechanism(s) underlying the apparent resistance to malaria in certain inherited red cell disorders and iron deficiency anaemia remain poorly understood. The possibility that microcytic erythrocytes might inhibit parasite development, by physical restriction or reduced supply of nutrients, has been considered for many years, and never formally investigated. We sought to determine whether in vitro growth studies of P. falciparum could provide evidence to suggest that small red cell size contributes to malaria resistance in those red cell disorders in which microcytosis is a characteristic feature.
Invasion and development of P. falciparum in iron deficient red cells (mean values for mean cell volume [MCV] 66 fl, mean cell haemoglobin [MCH] 19 pg) and in the red cells of two gene deletion forms of α-thalassaemia (mean MCV 71 fl, MCH 22 pg) were normal, assessed both morphologically, and by ³H-hypoxanthine incorporation. Although parasite appearances were normal in all cell types, morphological abnormalities were noted in iron deficient and thalassaemic cells parasitized by mature stages of P. falciparum , notably cellular ballooning and extreme hypochromia of the red cell cytoplasm. Using electron microscopy, the red cell cytoplasm in parasitized thalassaemic cells showed reduced electron density and abnormal reticulation. Normal invasion rates were observed following schizogony in microcytic cells of both types.
Our findings indicate that whilst minor morphological abnormalities may be detected in parasitized iron deficiency and thalassaemic erythrocytes, development of P. falciparum in these conditions is not limited by small erythrocyte size.     

What do mouse gene knockouts tell us about the structure and function of the red cell membrane?

Recent development of knockout mice with targeted deletion of specific genes encoding various red cell membrane proteins has added valuable armamentarium to red cell membrane structure-function studies. In this chapter we will summarize the various recent developments regarding the structure and function of the red cell membrane derived from studies using knockout mice. In addition to being expressed in red cells, all major red cell membrane proteins are also expressed in cells of various tissues. The potential use of knockout mice to decipher the biological functions of red cell membrane proteins in non-erythroid cells is also explored.     

疟原虫入侵红细胞的相关分子及其机制

疟原虫入侵宿主红细胞具有高度的种特异性,这些特异性的分子基础是疟原虫蛋白质与宿主红细胞表面蛋白质的相互作用.寻找参与疟原虫入侵红细胞的相关分子及其入侵机制是疟疾研究领域的热点.针对疟原虫不同种和虫株的实验研究并结合生物信息学分析结果表明:裂殖子表面蛋白、网织红细胞结合蛋白家族、红细胞结合蛋白家族以及动力蛋白等是参与疟原虫入侵红细胞的重要蛋白.该文对这方面的研究进展作了系统的综述,并阐述这些蛋白质在疟原虫入侵过程不同阶段中的作用.     

Susceptibility to invasion by Plasmodium falciparum of some human erythrocytes carrying rare blood group antigens

S ummary. Tn and Cad erythrocytes which carry unusual carbohydrate moieties attached to glycophorin A and B, the main red cell membrane sialoglycoproteins, resist invasion by the malarial parasite Plasmodium falciparum. Tn red cells are defective in sialic acid and galactose whereas Cad erythrocytes are characterized by a normal sialic acid content but the presence of an additional N -acetylgalactosamine residue attached to each sialotetrasaccharide chain O-glycosidically linked to glycophorin A and B. Homozygous MgMg red cells, which are defective in the cluster of three sialotetrasaccharide chains located at positions 2, 3 and 4 of glycophorin A, are normally invaded. Erythrocytes typed McM carry a glycophorin A molecule intermediate between those from M and N (amino acid substitution at position 1 or 5) but are, like control MN red cells, susceptible to invasion. These results suggest that the primary requirement for entry of P. falciparum merozoites into human red cells is the recognition of a carbohydrate structure present on glycophorin A or B which includes sialic acid and galactose, but is not necessarily clustered at the N -terminal end of the molecule.     

Electron microscopy of the human brain in cerebral malaria

Ultrastructure of erythrocytes infected with Plasmodium falciparum in human brain, obtained 3 hours post mortem revealed gross distortion of host red cells with abnormality of the red cell surface. The superficial alterations of the parasitized cells as knob-like protrusion appear to be the sites of attachment to vascular endothelium. There was evidence of platelets sticking to the injured endothelium. The endothelial vesicular membrane is in close adhesion to the parasitized red cell, and also to the platelets involved in this mechanism. Thus, explaining the sequestration of parasitized red cell and obstruction in cerebral microcirculation, cerebral oedema and low peripheral platelet count. The was no evidence of inflammation, fibrin or thrombus formation observed in our studies.     

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia.

Spherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.