Rosetting: a new cytoadherence property of malaria-infected erythrocytes

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, "rosetting," which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.     

Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes.

Sequestration, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules, is characteristic of Plasmodium falciparum infections. We have studied two host factors, the spleen and antibody, that influence sequestration of P. falciparum in the squirrel monkey. Sequestration of trophozoite/schizont-infected erythrocytes that occurs in intact animals is reduced in splenectomized animals; in vitro, when infected blood is incubated with monolayers of human melanoma cells, trophozoite/schizont-infected erythrocytes from intact animals but not from splenectomized animals bind to the melanoma cells. The switch in cytoadherence characteristics of the infected erythrocytes from nonbinding to binding occurs with a cloned parasite. Immune serum can inhibit and reverse in vitro binding to melanoma cells of infected erythrocytes from intact animals. Similarly, antibody can reverse in vivo sequestration as shown by the appearance of trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation with immune serum. These results indicate that the spleen modulates the expression of parasite alterations of the infected erythrocyte membrane responsible for sequestration and suggest that the prevention and reversal of sequestration could be one of the effector mechanisms involved in antibody-mediated protection against P. falciparum malaria.     

Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology

The pathophysiology of severe falciparum malaria is complex, but evidence is mounting that its central feature is the old concept of a mechanical microcirculatory obstruction. Autopsy studies, but also in vivo observations of the microcirculation, demonstrate variable obstruction of the microcirculation in severe malaria. The principal cause of this is cytoadherence to the vascular endothelium of erythrocytes containing the mature forms of the parasite, leading to sequestration and obstruction of small vessels. Besides, parasitized red cells become rigid, compromising their flow through capillaries whose lumen has been reduced by sequestered erythrocytes. Adhesive forces between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) could further slow down microcirculatory flow. A more recent finding is that uninfected erythrocytes also become rigid in severe malaria. Reduction in the overall red cell deformability has a strong predictive value for a fatal outcome. Rigidity may be caused by oxidative damage to the red blood cell membrane by malaria pigment released at the moment of schizont rupture. Anti-oxidants, such as N-acetylcysteine can reverse this effect and are promising as adjunctive treatment in severe malaria.     

Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes

In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40 degrees C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37 degrees C did not. Heating to 40 degrees C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37 degrees C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance.     

Adhesive receptors on malaria-parasitized red cells

Antigenic variation, rosetting and cytoadhesion are key determinants in the survival and virulence of the malaria parasite Plasmodium falciparum. These properties reside in a multigene protein family called P. falciparum erythrocyte membrane protein I (PfEMPI), encoded by the large and diverse var gene family. PfEMPI plays a central role in the biology of P. falciparum and its interaction with the human host. The molecular mechanism and the domains involved in cytoadherence, rosetting and antigenic variation are beginning to unfold. Domains mediating rosetting and adhesion to several key host receptors have already been identified. Understanding the role of PfEMPI in the pathogenesis and survival of malaria parasites is the key for the development of anti-adhesion vaccines and therapeutics to reduce the mortality and morbidity of P. falciparum infections.     

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.     

Ultrastructural analysis of fresh Plasmodium falciparum-infected erythrocytes and their cytoadherence to human leukocytes.

Sixty fresh Plasmodium falciparum isolates obtained from Gambian children with mild or cerebral malaria were investigated by transmission electron microscopy for the expression of knob-like protrusions (K+) on the surface of the infected erythrocytes. More than six-hundred infected erythrocytes were analyzed. Knob-forming parasites were present in all 60 isolates. Although knobless parasites (K-) were found in 25 (42%) of the isolates, only 39 were K-, while 577 were K+. Nine of the 39 K- infected erythrocytes that were studied in greater detail appeared to be asexual parasites because they were either segmented or they lacked mitochondrial DNA-like filaments and cristae, which are abundant in immature gametocytes. No difference was observed in the relative frequency of K+K- infected erythrocytes in isolates from patients with mild or cerebral malaria. Binding of both knobby and knobless infected erythrocytes to autologous leukocytes including monocytes, neutrophils, lymphocytes and plasma cells was found in some of the primary in vitro cultures. By using P. falciparum laboratory strains of known phenotypes and leukocytes from healthy blood bank donors, it was established that this novel adherence phenomenon was related to that of cytoadherence to certain melanoma or endothelial cells. Cytoadherent infected erythrocytes that bind to leukocytes enhance antibody-independent phagocytosis and induce cellular aggregation, while non-cytoadherent or rosetting infected erythrocytes do not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes.

Cerebral involvement in Plasmodium falciparum malaria is associated with sequestration of infected red blood cells and occlusion of cerebral vessels. Adhesion of infected erythrocytes along the vascular endothelium as well as binding of uninfected erythrocytes to cells infected with late-stage asexual parasites (rosetting) may be important in erythrocyte sequestration. We report that the recently discovered rosetting phenomenon shares characteristics with other human cell-cell interactions (heparin sensitivity, temperature independence, Ca2+/Mg2+ and pH dependence). Mono- and polyclonal antibodies specific for PfHRP1, a histidine-rich protein present in the membrane of P. falciparum-infected erythrocytes, disrupt rosettes but do not affect attachment of infected erythrocytes to endothelial cells. The inhibitory anti-PfHRP1 antibodies reacted with rosetting parasites in indirect immunofluorescence and with P. falciparum polypeptides of Mr 28,000 and Mr 90,000 in immunoprecipitation and immunoblotting, respectively. No inhibitory effects on erythrocyte rosetting were obtained with antibodies to related histidine-rich or other antigens of P. lophurae or P. falciparum. Whether the epitope that mediates rosetting, and is recognized by the anti-PfHRP1 antibodies, is located on PfHRP1 or on a crossreactive antigen remains to be established. The results suggest that endothelial cytoadherence and erythrocyte rosetting involve different molecular mechanisms.     

Genetic analysis of circulating and sequestered populations of Plasmodium falciparum in fatal pediatric malaria

Falciparum malaria is characterized by cytoadherence of host erythrocytes containing mature asexual-stage parasites and the consequent sequestration of these forms in tissue microvasculature. A postmortem study of pediatric malaria provided us with the opportunity to compare the genetic complexity of circulating and sequestered Plasmodium falciparum populations, in patients with fatal cerebral malaria (CM) versus control subjects with incidental P. falciparum parasitemia who died of causes other than malaria. Parasite genotypes identified in peripheral blood collected at the time of admission to the hospital constituted a subset of those detected in the tissues at death. Despite a higher tissue burden of parasitized erythrocytes in patients with CM than in parasitemic control subjects, parasite populations in tissues from patients with CM were less genetically complex, and the genotypes were more homogeneously distributed throughout the body, than in patients with incidental infection. Our findings support the notion that CM is associated with the emergence of a small number of dominant genotypes in an infected individual.     

In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.     

The resistance to physiological shear stresses of the erythrocytic rosettes formed by cells infected with Plasmodium falciparum

Rosetting forces are believed to be an important contributor to the microcirculatory obstruction that occurs in malaria caused by Plasmodium falciparum. In this study, rosettes of erythrocytes from cultures of this parasite were suspended in different media and exposed to shear stresses corresponding to those encountered on the arterial and venous sides of the human circulation. The rosettes formed by infected erythrocytes in malaria culture medium containing 10% AB serum were disrupted easily (approximately 50% being broken) when exposed to very low shear stresses of < 0.5 Pa. However, use of higher concentrations of serum strengthened the rosetting binding forces considerably. Suspension of rosettes in a viscous colloid (e.g. dextran) increased the adherence forces between infected and uninfected red cells. The results indicate that rosettes do resist the physiological shear forces that are encountered in the venular side of the circulation and could thus contribute to microvascular obstruction in falciparum malaria.     

Low anticoagulant heparin disrupts Plasmodium falciparum rosettes in fresh clinical isolates

The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria.     

Alteration in cytoadherence and rosetting of Plasmodium falciparum- infected thalassemic red blood cells

Hemoglobinopathies have a protective role in malaria that appears to be related to alterations in red blood cell (RBC) properties. Thalassemic RBCs infected with Plasmodium falciparum showed greatly reduced cytoadherence and rosetting properties as well as impaired growth and multiplication. A significant decrease in the levels of falciparum antigens associated with the membrane of infected beta-thalassemic RBCs was observed at trophozoite/schizont stage, but not young ring stage. This reduction was shown when a cytoadherence inhibitory monoclonal antibody, but not a noninhibitory pooled immune serum, was used. These observations suggest that protection against malaria in thalassemia is caused by both reduced parasitemias and altered adherence properties of the infected thalassemic RBCs that promote enhanced clearance of the parasite from the circulation.     

Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by anti-idiotype antibodies.

The CD36 molecule expressed by human endothelial cells is a receptor for the adhesion of erythrocytes infected with the human malaria parasite Plasmodium falciparum. A CD36-specific monoclonal antibody, OKM8, inhibits the adhesion of malaria-infected erythrocytes (IRBC) to purified CD36 and cells expressing CD36. Monospecific polyclonal anti-idiotype (anti-Id) antibodies, raised against monoclonal antibody OKM8, expressed determinants molecularly mimicking the CD36 binding domain for the adhesion of IRBC. Purified rabbit anti-Id antibodies reacted with the surface of IRBC by immunofluorescence, directly supported the adhesion of wild-type P. falciparum malaria isolates, and inhibited IRBC cytoadherence to melanoma cells. An approximately 270-kDa protein was immunoprecipitated by the anti-Id antibodies from surface-labeled and metabolically labeled IRBC and was competitively inhibited by soluble CD36. These results support the hypothesis that CD36 is a receptor and the approximately 270-kDa protein, sequestrin, is a complementary ligand involved in the adhesion of IRBC to host-cell endothelium. Sequestrin is a candidate malaria vaccine antigen, and anti-Id antibodies that recognize this molecule may be useful for passive immunotherapy of cerebral and severe P. falciparum malaria.     

Short report: Positive correlation between rosetting and parasitemia in Plasmodium falciparum clinical isolates

Plasmodium falciparum isolates that form rosettes with uninfected red cells are associated with severe malaria in African children, although the mechanism by which rosetting contributes to severe disease is unknown. Here we have analyzed the relationship between rosetting and parasitemia in two samples of clinical isolates from children with malaria in Kilifi, Kenya. A consistent positive correlation was found between rosetting and parasitemia (Spearman's rank correlation coefficent p = 0.467, P < .001, n = 154, for 1993 study; p = .407, P < .001, n = 74, for 2000 study). Rosetting may enhance parasite growth and survival by facilitating invasion or promoting immune evasion, thus allowing higher parasitemia to develop and increasing the likelihood of severe malaria.     

Modulation of malaria virulence by determinants of Plasmodium falciparum erythrocyte membrane protein-1 display

PURPOSE OF REVIEW: Plasmodium falciparum malaria parasites carry approximately 60 var genes that encode variable adhesins termed P. falciparum erythrocyte membrane protein-1. Clonal expression of a single P. falciparum erythrocyte membrane protein-1 variant on the surface of the parasitized host erythrocyte promotes binding of the cell to blood elements (including noninfected erythrocytes, leukocytes) and walls of microvessels. These binding events enable parasitized erythrocytes to sequester and avoid clearance by the spleen, and they also contribute to disease by causing microvascular inflammation and obstruction. RECENT FINDINGS: Steps by which P. falciparum erythrocyte membrane protein-1 is exported to the parasitized erythrocyte surface have recently been elucidated. The ability of parasites to cytoadhere and cause disease depends on the variant of P. falciparum erythrocyte membrane protein-1 as well as its amount and distribution at the erythrocyte surface. An example of a host polymorphism that affects P. falciparum erythrocyte membrane protein-1 display is hemoglobin C, which may protect against malaria by impairing the parasite's ability to adhere to microvessels and induce inflammation. Interference with P. falciparum erythrocyte membrane protein-1-mediated phenomena appears to diminish cytoadherence in vivo and to protect against disease in animal models. SUMMARY: Plasmodium falciparum erythrocyte membrane protein-1-mediated sequestration of parasitized erythrocytes plays a central role in malaria pathogenesis. Clinical interventions aimed at reducing cytoadherence and microvascular inflammation may improve disease outcome.     

Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.     

Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44-45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups (odds ratio 0.34, 95% confidence interval 0.19-0.61, P < 0.0005, severe cases versus uncomplicated malaria controls). In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups (P = 0.003, Kruskal-Wallis test). Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.     

Receptor-binding residues lie in central regions of Duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites

Erythrocyte invasion by malaria parasites and cytoadherence of Plasmodium falciparum-infected erythrocytes to host capillaries are 2 key pathogenic mechanisms in malaria. The receptor-binding domains of erythrocyte-binding proteins (EBPs) such as Plasmodium falciparum EBA-175, which mediate invasion, and P falciparum erythrocyte membrane protein 1 (PfEMP-1) family members, which are encoded by var genes and mediate cytoadherence, have been mapped to conserved cysteine-rich domains referred to as Duffy-binding-like (DBL) domains. Here, we have mapped regions within DBL domains from EBPs and PfEMP-1 that contain receptor-binding residues. Using biochemical and molecular methods we demonstrate that the receptor-binding residues of parasite ligands that bind sialic acid on glycophorin A for invasion as well as complement receptor-1 and chondroitin sulfate A for cytoadherence map to central regions of DBL domains. In contrast, binding to intercellular adhesion molecule 1 (ICAM-1) requires both the central and terminal regions of DBLbetaC2 domains. Determination of functional regions within DBL domains is the first step toward understanding the structure-function bases for their interaction with diverse host receptors.     

Trafficking of the major virulence factor to the surface of transfected P. falciparum-infected erythrocytes

After invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P. falciparum-infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P. falciparum-infected RBC membrane.