Irwin W. Sherman

Cytoadherence and sequestration in Plasmodium falciparum: defining the ties that bind

Infected erythrocytes containing the more mature stages of the human malaria Plasmodium falciparum may adhere to endothelial cells and uninfected red cells. These phenomena, called sequestration and rosetting, respectively, are involved in both host pathogenesis and parasite survival. This review provides a critical summary of recent advances in the characterization of the molecules of the infected red blood cell involved in adhesion, i.e. parasite-encoded molecules (PfEMP1, MESA, rifins, stevor, clag 9, histidine-rich protein), a modified host membrane protein (band 3) and exofacial exposure of phosphatidylserine, as well as receptors on the endothelium, i.e. thrombospondin, CD36, ICAM-1 (intercellular adhesion molecule), and chondroitin sulfate.

Cytoadherence of Malaria-Infected Red Blood Cells Involves Exposure of Phosphatidylserine

Phosphatidylserine (PS) is a membrane phospholipid which in intact cells is exclusively localized in the inner leaflet of the lipid bilayer. However, once cells undergo apoptosis or oxidative stress, PS molecules are exposed on the external surface of the cells and this contributes to their adherence to macrophages or endothelial cells. PS exposure on Plasmodium falciparum-infected red cells was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS. Involvement of exposed PS in the adherence of malaria-infected red cells to endothelial cells was examined by in vitro cytoadherence assays. Infected cells exposed PS on their surface as the intracellular parasites matured to trophozoite and schizont stages. Adherence of malaria-infected cells to CD36, CD36-expressing Chinese hamster ovary cells, thrombospondin, and C32 amelanotic melanoma cells was inhibited by annexin V, whereas ICAM-1- and chondroitin sulfate A-mediated binding was not. Further, PS liposomes and glycerophosphorylserine, but not phosphatidylcholine liposomes and glycerophosphorylcholine, inhibited the binding of infected cells to CD36 and thrombospondin. In conclusion, these results demonstrate that PS exposed on the surface of malaria-infected red cells contributes, in part, to the adherence of P. falciparum-parasitized red cells to CD36 and thrombospondin.

Molecular approaches to malaria

Table of Contents Introduction 1. The Life of Plasmodium: an Overview, Irwin W. Sherman 2. PlasmoDB: the Plasmodium Genome Resource, Patricia L. Whetzel, Shailesh V. Date, Kobby Essien, Martin J. Fraunholz, Bindu Gajria, Gregory R. Grant, John Iodice, Jessical C. Kissinger, Philip T. Labo, Arthur J. Milgram, Christian J. Stoeckert, Jr., and David Roos 3. Making a Home for Plasmodium Post-Genomics: Ultrastructural Organization of the Blood Stages, Lawrence H. Bannister, Gabriele Margos, and John M. Hopkins 4. Genetic Manipulation of Plasmodium falciparum, Alan F. Cowman and Brendan S. Crabb 5. The Transcriptome of the Malaria Parasite Plasmodium falciparum, Karine Le Roch and Elizabeth Winzeler 6. The Plasmodium Proteome, Jeffrey R. Johnson and John R. Yates III 7. Evolutionary History and Population Genetics of Human Malaria Parasites, Martine Zilversmit and Daniel L. Hartl Invasion and Gamete Formation 8. A Mechanistic Approach to Merozoite Invasion of Red Blood Cells: Merozoite Biogenesis, Rupture, and Invasion of Erythrocytes, Mary R. Galinski, Anton R. Dluzewski, and John W. Barnwell 9. The Sporozoite, R. E. Sinden and K. Matuschewski 10. Gametocytes and Gametes, Pietro Alano and Oliver Billker Growth and Metabolism 11. Molecular Approaches to Malaria: Glycolysis in Asexual-Stage Parasites, Charles J. Woodrow and Sanjeev Krishna 12. The Mitochondrion, Akhil B. Vaidya 13. Trafficking and the Tubulovesicular Membrane Network, Kasturi Haldar, Narla Mohandas, Souvik Bhattacharjee, Travis Harrison, N. Luisa Hiller, Konstantinos Liolios, Sean Murphy, Pamela Tamez, and Christiaan van Ooij 14. The Apicoplast, Stuart A. Ralph 15. Protein Kinases Regulating Plasmodium Proliferation and Development, Christian Doerig 16. Proteases and Hemoglobin Degradation, Philip J. Rosenthal 17. Plasmodium Lipids: Metabolism and Function, Henri J. Vial and Choukri Ben Mamoun 18. Plasmodium Ribosomes and Opportunities for Drug Intervention, Indu Sharma and Thomas F.. McCutchan 19. Oxidative Stress and Antioxidant Defense in Malarial Parasites, Katja Becker, Sasa Koncarevic, and Nicolas H. Hunt 20. New Permeation Pathways, Serge L. Thomas and Stephanie Egee Immune Evasion 21. Molecular Aspects of Antigenic Variation in Plasmodium falciparum, Paul Horrocks, Susan A. Kyes, Peter C. Bull, and Kirk W. Deitsch 22. Rosetting, J. Alexandra Rowe Protection 23. Mechanisms of Antimalarial Drug Action and Resistance, Anne-Catrin Uhlemann, Yongyuth Ythavong, and David A. Fidock 24. Host Genetic Factors in Resistance and Susceptibility to Malaria, Dominic P. Kwiatkowski and Gaia Luoni 25. Progress in Development of a Vaccine To Aid Malaria Control, Vasee S. Moorthy and Filip Dubovsky Vector 26. The Anopheles gambiae Genome, Frank H. Collins and Catherine A. Hill 27. The Transcriptome of Human Malaria Vectors, Osvaldo Marinotti and Anthony A. James

Membrane structure and function of malaria parasites and the infected erythrocyte

According to the World Health Organization the global estimate of malaria is over 200 million infections, the majority of which are caused by the most life-threatening species, Plasmodium falciparum (Report of the Steering Committees of the Scientific Working Groups on Malaria, World Health Organization, June 1983). The causative agent of the disease, the malarial parasite, requires two hosts: a blood-sucking mosquito and a blood-containing vertebrate. Commonly, infection of the vertebrate begins when an infected mosquito bites a suitable vertebrate and injects minute sporozoites into the bloodstream. Within 30 mm the introduced sporozoites leave the bloodstream and enter parenchymal cells of the liver (mammals) or endothelial cells (birds). In these sites the parasite undergoes asexual multiplication (= exo-erythrocytic schizogony) producing daughter progeny called merozoites. The exo-erythrocytic merozoites are released from the tissues into the circulation where they invade red blood cells. Within an erythrocyte the merozoite undergoes asexual multiplication (= erythrocytic schizogony) producing a substantial number of merozoites. The erythrocyte lyses, merozoites are released, and invasion of another erythrocyte may then take place. The synchronous rupture of the red cell and merozoite release is marked by the periodic fever–chill cycles so characteristic of the malarial infection. Some merozoites continue to reinvade other erythrocytes and multiply by asexual means, whereas others enter erythrocytes and differentiate into sexual stages, male or female gametocytes. When a suitable mosquito feeds on an infected vertebrate gametocytes are ingested and the sexual cycle of development is initiated. In the mosquito stomach the gametocytes transform into gametes, fertilization takes place, the resultant worm-like zygote penetrates the cells of the mosquito gut and comes to lie on the outer surface of the stomach. Here each zygote forms a cyst-like body, the oocyst, within which thousands of sporozoites are produced by asexual multiplication. When the swollen oocysts burst, sporozoites are freed and these make their way to the salivary gland. At the next blood feeding the mosquito injects the infective sporozoites and the life-cycle is completed.

Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells

The cytoadherence of Plasmodium falciparum-infected erythrocytes was studied using immortalized human brain capillary endothelial cells. The immortalized cells, denoted as BB19, derived from the human brain endothelium, were transformed with the E6E7 genes of human papilloma virus and retained their endothelial nature, i.e. tubule formation occurred with Matrigel as a substratum and the cells stained positive for Factor VIII-related antigen, or vonWillebrands factor. Surface expression of ICAM-1, VCAM, E-selectin, and CD36 was demonstrated by immunofluorescence staining with monoclonal antibodies to these ligands. Exposure to cytokines (TNF, IFN gamma, IL-1 alpha, and IL-6) and lipopolysaccharide resulted in an increase in expression of ICAM-1, VCAM, E-selectin, and CD36. The BB19 cells bound P. falciparum-infected red blood cells with both the FCR-3 and the ITO4 strains. Antibodies to CD36 and ICAM-1 partially inhibited the binding of the FCR-3 and the ITO4 lines, respectively. These findings suggest that BB19 cells may be useful in the analysis of receptor-based cytoadherence and sequestration, as well as in the cell biology of microvessel formation.

Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells.

The membrane lipid composition and [3H]cholesterol exchange rate were studied in both normal human erythrocytes and those infected with the human malaria Plasmodium falciparum. The host cell membrane was separated from parasite membranes using the Affigel (731) bead method. The purity of the membrane preparation was very high, as judged by SDS-PAGE, and in several instances was estimated to be greater than 98% as determined by the activity of the parasite membrane-specific enzyme, choline phosphotransferase. No difference was found in the content of phosphatidylethanolamine and only small changes were observed for phosphatidylcholine and phosphatidylserine. The sphingomyelin content in red cell membranes of both trophozoite- and schizont-infected cells was up to 47% less than that of uninfected cells, and the cholesterol/phospholipid ratio was decreased 55%. Trophozoite- and schizont-infected cells exchanged 29 and 33% less cholesterol, respectively, than uninfected cells. These changes in lipid composition and cholesterol exchange could have a marked effect on the function of the red cell membrane of malaria-infected cells and may be responsible, in part, for the increased fluidity and permeability of P. falciparum-infected erythrocytes.

Alterations in erythrocyte membrane phospholipid organization due to the intracellular growth of the human malaria parasite, Plasmodium falciparum.

The asymmetric distribution of phospholipids in the erythrocyte membrane during the intracellular development of the human malaria parasite Plasmodium falciparum was studied. Infected cells of high parasitaemia were treated with phospholipase A2 or sphingomyelinase C, followed by isolation of the host red cell membrane using the Affigel (731) bead method. Additionally, phosphatidylserine on the surface of infected cells was probed using a phosphatidylserine-sensitive prothrombinase assay. Trophozoite-infected cells showed an increase in phosphatidylethanolamine and phosphatidylserine and a decrease in phosphatidylcholine in the outer leaflet. In addition to the changes already present in trophozoite-infected cells, schizont-infected cells showed a decrease in sphingomyelin as well as a further increase in phosphatidylserine in the outer leaflet. The results are discussed with respect to possible mechanisms and consequences of these changes.

Plasmodium lophurae: Composition and properties of hemozoin, the malarial pigment

Abstract Plasmodium lophurae hemozoin (malarial pigment) is composed of proteinaceous macromolecules bonded to iron III protoporphyrin IX by coordination bonding, van der Waals forces, and hydrophobic interactions but not by covalent bonding. Hemozoin is not composed of partially degraded globin peptides coordinated to heme, since fragments of molecular size less than that of globin monomers were not observed by SDS-PAGE. Two major polypeptides constituted the macromolecular portion of hemozoin; these had molecular weights of 21,000 and 15,000. The 21,000-molecular-weight protein is probably of parasite origin. The 15,000-molecular-weight polypeptide is believed to consist of globin monomers, and indicates the presence of irreversibly denatured hemoglobin (hemiglobin), as a constituent of hemozoin. The formation of hemozoin is hypothesized to play the following roles: protection of the parasite against molecular oxygen and compartmentation of the iron porphyrin which is a product of hemoglobin digestion by the plasmodium.

Lipids of Plasmodium lophurae, and of erythrocytes and plasmas of normal and P. lophurae-infected Pékin ducklings.

A lipid analysis was performed on the avian malaria parasite Plasmodium lophurae, freed from duckling erythrocytes by immune hemolysis, and on the erythrocytes and plasmas of normal and P. lophurae-infected ducklings. Major lipids of normal erythrocytes were: phosphatidylcholine (40% of total lipids), phosphatidylethanolamine (20%), cholesterol (20%), sphingomyelin (11%), and glycosphingolipids (5%). Major fatty acids of erythrocyte total phospholipids (74% of total lipids) were 16:0 (22%), 18:2 (n-6) (21%), 18.1 (n-7, n-9) (18%), 18:0 (9%), 20:4 (n-6) (9%), 22:6 (n-3) (5%). Erythrocyte phosphatidylcholine was greater than 90% the diacyl form, while phosphatidylethanolamine was approximately 44% alkoxy forms and phosphatidylinositol approximately 11% alkoxy forms. Major fatty aldehydes of phosphatidylethanolamine were 16:0 (47%), 18:1 (23%), 18:0 (14%), and 14:0 (12%). The lipid composition of P. lophurae (plus the parasitophorous vacuole membrane) was qualitatively and quantitatively different from that of the duckling erythrocyte in a number of respects. Major lipids were phosphatidylcholine (40%), phosphatidylethanolamine (36%), cholesterol (8%), phosphatidylinostol (4%), 1,2-diacylglycerols (3%), sphingomyelin (2%), and glycosphingolipids (2%). Diphosphatidylglycerol (approximately 1%) was also detected. The major fatty acids of parasite total phospholipids (86% of total lipids) were more saturated than those of the erythrocyte, and octadecenoic acids were notably elevated: 18:1 (33%), 16:0 (26%), 18:0 (16%), 18:2 (12%), 20:4 (3%), and 22:6 (3%). Parasite phosphatidylcholine and phosphatidylethanolamine were greater than 93% the diacyl form and phosphatidylinositol was approximately 25% alkoxy forms. Major fatty aldehydes of the phosphatidylethanolamine were 14:0 (62%), unidentified long chain forms (24%), 16:0 (7%), 18:0 (4%), 18:1 (3%). The lipid composition of the infected erythrocyte reflected the separate contributions of the erythrocyte and parasite. The major lipids of normal duckling plasma were phosphatidylcholine (33%), triacylglycerols (22%), cholesterol esters (20%), cholesterol (12%), phosphatidylethanolamine (5%), and sphingomyelin (2%). The fatty acids of plasma total lipids were 18:1 (26%), 16:0 (26%), 18:2 (12%), 20:4 (12%), 18:0 (9%), 22:6 (3%). Plasma phosphoglycerides were remarkably lower in C18 unsaturated fatty acids and higher in 20:4 than the erythrocyte phosphoglycerides. Infection of ducklings with P. lophurae caused increases in plasma unesterified fatty acids, triacylglycerols and cholesterol esters, and a notable rise in the 18:1 content of all fatty acid-containing plasma neutral lipids. These findings are compared with those reported for other species of Plasmodium infecting other avian and mammalian hosts.

Plasmodium falciparum (human malaria)-induced modifications in human erythrocyte band 3 protein

A monoclonal antibody, 1C4, was produced which recognizes a 65 kDa protein that is localized to the plasma membrane of human erythrocytes infected with Plasmodium falciparum. By immunofluorescence the antigen was visualized as dots on the surface of the infected cell. The 65 kDa protein was present in 4 strains of diverse geographical origin, and in erythrocytes infected with a knobless strain. The 65 kDa protein was insoluble in non-ionic detergents, but was partly soluble in SDS and some high (1 M) salt solutions. The 65 kDa protein is recognized by antibodies specific for the cytoplasmic domain and the N-terminal side of the membrane-spanning region of human band 3, but was not recognized by an antibody specific to the C-terminal side of the membrane-spanning region. The results of treatment of the 65 kDa protein with trypsin and chymotrypsin are consistent with the 65 kDa protein being a truncated and covalently modified band 3 molecule which consists of the first 540 amino acids of human band 3.