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Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome
Abstract
Plasmodium falciparum-infected erythrocytes sequester from blood circulation by binding host endothelium. A large family of variant proteins mediates cytoadherence and their binding specificity determines parasite sequestration patterns and potential for disease. The aim of the present study was to understand how binding properties are encoded into family members and to develop sequence algorithms for predicting binding. To accomplish these goals computational approaches and a binding assay were used to characterize adhesion across Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins in the 3D7 parasite genome. We report that most family members encode the capacity to bind CD36 in the protein's semi-conserved head structure and describe the sequence characteristics of a group of PfEMP1 proteins that do not. Structural and functional grouping of PfEMP1 proteins based upon head structure and additional domain architectural properties provide new insights into the protein family. These can be used to investigate the role of proteins in malaria pathogenesis and potentially tailor vaccines to recognize particular binding variants.
... A subset of chimeric B-type proteins (group B/A, also known as DC8-containing proteins) has a DBLα2 domain (chimeric DBLα0/1 domain) and an EPCRbinding CIDRα1.1 or CIDRα1.8 domain typically attached to a complement component C1q receptor (C1qR)-binding DBLβ12 domain [27][28][29][30][31][32][33][34]. Thus, the head structure confers mutually exclusive binding properties to either EPCR (14%), CD36 (72%), CSA (3%), or to one or more unknown ECRs via the CIDRβ/γ/δ domains (10%) or VAR3 (1%) [35]. ...
... known as DC8-containing proteins) has a DBLα2 domain (chimeric DBLα0/1 domain an EPCR-binding CIDRα1.1 or CIDRα1.8 domain typically attached to a comple component C1q receptor (C1qR)-binding DBLβ12 domain [27][28][29][30][31][32][33][34]. Thus, the head ture confers mutually exclusive binding properties to either EPCR (14%), CD36 ( CSA (3%), or to one or more unknown ECRs via the CIDRβ/γ/δ domains (10%) or (1%) [35]. ...
... At least 24 ECRs were described as binding partners for PfIEs. These include EPCR, gC1qR, ICAM-1, and CD36, mentioned above, as well as platelet endothelial cell adhesion molecule-1 (PECAM-1), CSA (adhesion to placental epithelium) [49], heparan sulphate, hyaluronic acid, neuronal cell adhesion molecule (NCAM), P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), thrombospondin, fractalkine, ανβ3-and αVβ6-integrin, fibronectin, CD9, CD151, multidrug resistance protein 1 and 2, erythropoietin receptor 1, and tumour necrosis factor receptor (TNFR) 1 and 2 [6, 33,37,48,[50][51][52][53]. To date, only a few ECRs have been shown to interact via PfEMP1, and PfEMP1 binding domains have only been identified for CD36, ICAM-1, EPCR, PECAM-1, and gC1qR [18,27,32,33,54]. ...
Plasmodium falciparum-infected erythrocytes (PfIEs) present P. falciparum erythrocyte membrane protein 1 proteins (PfEMP1s) on the cell surface, via which they cytoadhere to various endothelial cell receptors (ECRs) on the walls of human blood vessels. This prevents the parasite from passing through the spleen, which would lead to its elimination. Each P. falciparum isolate has about 60 different PfEMP1s acting as ligands, and at least 24 ECRs have been identified as interaction partners. Interestingly, in every parasite genome sequenced to date, at least 75% of the encoded PfEMP1s have a binding domain for the scavenger receptor CD36 widely distributed on host endothelial cells and many other cell types. Here, we discuss why the interaction between PfIEs and CD36 is optimal to maintain a finely regulated equilibrium that allows the parasite to multiply and spread while causing minimal harm to the host in most infections.
... Receptor for infected erythrocyte placental sequestration in pregnancy malaria (Fried and Duffy, 1996) Soluble CS did not disrupt rosettes (Rogerson et al., 1994;Rowe et al., 1994) Chondroitinase treatment reduced rosetting in one parasite line only (Barragan et al., 1999) No evidence that CS is present on mature RBC Minimal evidence for a role in rosetting CD36 Widely distributed membrane protein and scavenger receptor (Silverstein and Febbraio, 2009) Deficiency is common in Africa but not associated with severe malaria (Fry et al., 2009) Antibodies disrupt rosettes in single culture-adapted line only PfEMP1 variants that mediate rosetting are group A types that do not bind CD36 (Robinson et al., 2003) Minimal evidence for a widespread role in rosetting Glycophorin C (GYPC) ...
... It is expressed on a variety of cell types including monocytes, macrophages, platelets, microvascular endothelial cells and adipocytes (Silverstein and Febbraio, 2009), and at low levels on erythrocytes . The binding of PfEMP1 (group B and C variants) to CD36 on microvascular endothelial cells plays a major role in P. falciparum sequestration (Baruch et al., 1996;Robinson et al., 2003). Almost all P. falciparum isolates bind to CD36, and increased CD36 binding Ochola et al., 2011) and predominant expression of group B and C PfEMP1 (Kraemer and Smith, 2006;Kyriacou et al., 2006) are associated with uncomplicated malaria. ...
... Anti-CD36 mAbs are capable of disrupting rosettes in a single culture-adapted parasite line, Malayan Camp , but not in a wide range of other laboratory lines or clinical isolates Rowe et al., 2000;Niang et al., 2014). The PfEMP1 variants identified as parasite rosetting ligands (Rowe et al., 1997;Vigan-Womas et al., 2011;Ghumra et al., 2012) are mostly of the group A type, which do not bind to CD36 (Robinson et al., 2003). ...
Malaria remains a major cause of mortality in African children, with no adjunctive treatments currently available to ameliorate the severe clinical forms of the disease. Rosetting, the adhesion of infected erythrocytes (IEs) to uninfected erythrocytes, is a parasite phenotype strongly associated with severe malaria, and hence is a potential therapeutic target. However, the molecular mechanisms of rosetting are complex and involve multiple distinct receptor-ligand interactions, with some similarities to the diverse pathways involved in P. falciparum erythrocyte invasion. This review summarizes the current understanding of the molecular interactions that lead to rosette formation, with a particular focus on host uninfected erythrocyte receptors including the A and B blood group trisaccharides, complement receptor one, heparan sulphate, glycophorin A and glycophorin C. There is strong evidence supporting blood group A trisaccharides as rosetting receptors, but evidence for other molecules is incomplete and requires further study. It is likely that additional host erythrocyte rosetting receptors remain to be discovered. A rosette-disrupting low anti-coagulant heparin derivative is being investigated as an adjunctive therapy for severe malaria, and further research into the receptor-ligand interactions underlying rosetting may reveal additional therapeutic approaches to reduce the unacceptably high mortality rate of severe malaria.
... Each P. falciparum genome harbors up to 60 distinct var genes with high levels of sequence diversity both within and between genomes (16). The Duffy Binding Like alpha (DBLα) domains present within the relatively conserved head structure of almost all var genes have been used as a marker for var gene diversity and gene expression studies (14,(23)(24)(25)(26)(27). In order to extrapolate these findings to full length var genes with diverse functions, short (150-200 amino acid residue) DBLα sequence tags have been classified into six subgroups based on the number of cysteine residues (Cys2, Cys4, CysX) and specific sequence motifs (MFK and REY) in positions of limited variability (Cys/PoLV, (17,18)), whilst full length DBLα domains have been classified into 3 subgroups (DBLα0, 1, 2) based on evolutionary relationships (19). ...
... Group 1-3 DBLα tags (Cys2) are found in group A var genes and are expressed at high levels in parasites infecting young children with limited immunity and with severe disease (20)(21)(22). DBLα groups 4-6 (Cys4/X) account for the majority of the sequences from non-group A var genes (23). Expression of group 1 DBLα (Cys2 MFK+REY-) is associated with impaired consciousness and cerebral malaria (21,22), whilst expression of group 2 DBLα (Cys2 MFK-REY+) and group 5 DBLα (Cys4 MFK-REY+) has been associated with rosetting and severe malarial anaemia (17,24). ...
... Given that different PfEMP1 subgroups are expressed during clinical, uncomplicated malaria (22,23), high levels of antibodies against virulence-associated variants may also be associated with reduced risk of uncomplicated malaria. Only 16 of the 456 variants were associated with a 30-40% reduction in the risk of clinical malaria, and 14 variants All rights reserved. ...
Extreme diversity of the major surface antigen and virulence determinant of the malaria parasite Plasmodium falciparum, Erythrocyte Membrane Protein-1 (PfEMP1), poses a major barrier to identifying targets of protective immunity. To overcome this problem, we developed a PfEMP1 protein microarray containing 456 DBLα domains, which was used to characterize the immunome of a cohort of semi-immune children and to identify variants associated with protective immune responses. Children with high mean antibody levels to DBLα group 2 had a 26-36% reduced risk of uncomplicated (clinical) malaria, however only 8 diverse DBLα variants were weakly associated with protection from clinical malaria and had low predictive accuracy. On the other hand, children with high mean antibodies to DBLα groups 1 and 2 (which are markers for pathogenic Type A PfEMP1) and elevated antibodies to 85 (18.6%) of individual DBLα variants had a 70 -100% reduced risk of severe malaria. Of the top 20 predictive variants for severe disease protection, 17 were strongly associated with protection (86 - 100% reduction in risk of severe malaria) and had high predictive accuracy for severe disease risk. Many variants were conserved and had highly correlated antibody responses, including the three highest-ranking variants, which were linked to EPCR-binding CIDR domains. The results suggest that while immunity to uncomplicated malaria is characterised by antibodies to a diverse repertoire of PfEMP1, immunity to severe malaria requires antibodies to a limited subset of antigenically conserved variants. These findings provide new insights into antimalarial immunity and potential biomarkers for tracking disease risk.
... Some characteristics of the platelet-mediated clumping ligand are similar to PfEMP-1, the adhesion for other IE adhesion phenotypes including rosetting (Rowe et al., 1994), CD36 (Robinson et al., 2003), P-Selectin (Senczuk, 2001), PECAM-1 (Fernandez et al., 1998). Furthermore, two of the known clumping ligand receptors, CD36 and P-Selectin are known receptors of PfEMP-1 (Senczuk, 2001;Robinson et al., 2003). ...
... Some characteristics of the platelet-mediated clumping ligand are similar to PfEMP-1, the adhesion for other IE adhesion phenotypes including rosetting (Rowe et al., 1994), CD36 (Robinson et al., 2003), P-Selectin (Senczuk, 2001), PECAM-1 (Fernandez et al., 1998). Furthermore, two of the known clumping ligand receptors, CD36 and P-Selectin are known receptors of PfEMP-1 (Senczuk, 2001;Robinson et al., 2003). Platelets also express PECAM-1, another receptor for PfEMP-1. ...
... An interesting paradox exists in the relationship of the platelet-mediated clumping phenotype and severe disease. On one hand, platelet mediated clumping is associated with severe disease while on the other hand platelet-mediated clumping is dependent on CD36, which on the endothelium is associated with non-severe malaria causing IEs (Pain et al., 2001;Robinson et al., 2003;Ghumra et al., 2012;Pleass, 2009). Furthermore, virtually all field isolates bind to CD36 whereas not all of them exhibit the platelet-mediated clumping phenotype. ...
... In other words, receptor binding is often associated with a single adhesive domain, or a combination of adhesive domains found in domain cassettes (Table 3). For example, binding to CD36 is due to the common DBLα/CIDRα head group [78,123]. This explains the large number of PfEMP1 variants that bind CD36 since more than 75% of PfEMP1 variants contain this common head group. ...
... DBLα/CIDRα head group [78], CIDRα2-6 [123,124] Endothelial protein C receptor CIDRα1 associated with DC8 or DC13 [97] Intercellular adhesion molecule-1 DBLβ [125][126][127], DBLβ3 in DC4 [128] Platelet endothelial cell adhesion molecule-1 DBLα/CIDRα head group or DBLδ2 [78], DC5 [129] gC1qR ...
Plasmodium falciparum can cause a severe disease with high mortality. A major factor contributing to the increased virulence of P. falciparum, as compared to other human malarial parasites, is the sequestration of infected erythrocytes in the capillary beds of organs and tissues. This sequestration is due to the cytoadherence of infected erythrocytes to endothelial cells. Cytoadherence is primarily mediated by a parasite protein expressed on the surface of the infected erythrocyte called P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is embedded in electron-dense protuberances on the surface of the infected erythrocytes called knobs. These knobs are assembled on the erythrocyte membrane via exported parasite proteins, and the knobs function as focal points for the cytoadherence of infected erythrocytes to endothelial cells. PfEMP1 is a member of the var gene family, and there are approximately 60 antigenically distinct PfEMP1 alleles per parasite genome. Var gene expression exhibits allelic exclusion, with only a single allele being expressed by an individual parasite. This results in sequential waves of antigenically distinct infected erythrocytes and this antigenic variation allows the parasite to establish long-term chronic infections. A wide range of endothelial cell receptors can bind to the various PfEMP1 alleles, and thus, antigenic variation also results in a change in the cytoadherence phenotype. The cytoadherence phenotype may result in infected erythrocytes sequestering in different tissues and this difference in sequestration may explain the wide range of possible clinical manifestations associated with severe falciparum malaria.
... PfEMP1 proteins encode multiple adhesion domains, called Duffy binding-like (DBL) and cysteine-rich interdomain region (CIDR), which confer different binding properties . For instance, different subsets of PfEMP1 proteins encode binding activity for CD36 (Robinson et al., 2003;Hsieh et al., 2016), endothelial protein C receptor (EPCR) (Turner et al., 2013;Lau et al., 2015), and intercellular adhesion molecule 1 (ICAM-1) (Smith et al., 2000;Lennartz et al., 2019). Whereas the EPCR binders comprise only a small minority of the var gene repertoire (~10% of genes) (Rask et al., 2010), this subset is transcriptionally elevated in severe malaria infections and both the DC8 and Group A PfEMP1 variants are linked to severe malaria complications (Lavstsen et al., 2012;Turner et al., 2013;Bernabeu et al., 2016;Kessler et al., 2017;Lennartz et al., 2017;Mkumbaye et al., 2017;Sahu et al., 2021;Wichers et al., 2021). ...
... To address this knowledge gap, we studied whether parasite lines with affinity for primary human brain endothelial cells would bind to primary microvascular endothelial cells from kidney and gut. Although CD36 binding is the most common of the PfEMP1 adhesion traits and is encoded by up to 80-85% of proteins in each parasite's var gene repertoire (Robinson et al., 2003;Rask et al., 2010;Smith et al., 2013), this receptor was expressed at very low or negligible levels on brain, intestinal, and peritubular kidney endothelial cells. From histology, CD36 is strongly expressed on liver, spleen, lung, and muscle blood vessels, but is low or absent on brain and kidney endothelial cells (Turner , 1994). ...
Cytoadhesion of Plasmodium falciparum-infected red blood cells is a virulence determinant associated with microvascular obstruction and organ complications. The gastrointestinal tract is a major site of sequestration in fatal cerebral malaria cases and kidney complications are common in severe malaria, but parasite interactions with these microvascular sites are poorly characterized. To study parasite tropism for different microvascular sites, we investigated binding of parasite lines to primary human microvascular endothelial cells from intestine (HIMEC) and peritubular kidney (HKMEC) sites. Of the three major host receptors for P. falciparum, CD36 had low or negligible expression; endothelial protein C receptor (EPCR) had the broadest constitutive expression; and intercellular adhesion molecule 1 (ICAM-1) was weakly expressed on resting cells and was strongly upregulated by TNF-α on primary endothelial cells from the brain, intestine, and peritubular kidney sites. By studying parasite lines expressing var genes linked to severe malaria, we provide evidence that both the DC8 and Group A EPCR-binding subsets of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encodes binding affinity for brain, intestinal, and peritubular kidney endothelial cells, and that DC8 parasite adhesion was partially dependent on EPCR. Collectively, these findings raise the possibility of a brain-gut-kidney binding axis contributing to multi-organ complications in severe malaria.
... Elle ne dépend donc pas de la présence d'Ac et, en ce sens, pourrait être primordiale chez les sujets non immuns comme les enfants. De plus, contrairement à la phagocytose opsonique (190), elle n'induit pas la production de cytokines pro-inflammatoires reconnues pour être associées aux formes graves de paludisme (247,257,258 (81,245,247,(258)(259)(260)(261)(262)(263)(264)(265). ...
... Ces résultats soulignent l'importance du récepteur CD36 dans la lutte contre l'infection palustre et son rôle protecteur vis-à-vis du neuropaludisme(513). En effet, comme cela a déjà été discuté, CD36 possède de nombreuses fonctions lorsqu'il est exprimé à la surface des cellules monocytaires telles que la phagocytose non-opsonique des GRi et des parasites(81,245,247,(257)(258)(259)(260)(261)(262)(263)(264)(265), mais également l'élimination des cellules apoptotiques, lipoprotéines LDL oxydées (oxLDL) et autres débris(245,247,253,266,468,469). Ainsi, la diminution de son expression à la surface des monocytes traduit une capacité limitée à promouvoir l'élimination de ces éléments inflammatoires, processus nécessaire à la résolution de l'inflammation palustre. ...
En 2020, le paludisme reste un problème majeur de santé publique. Malgré des avancées scientifiques capitales, il est encore responsable de plus de 400 000 décès chaque année, principalement chez les enfants en Afrique Sub-saharienne. Le neuropaludisme est l'une des formes les plus sévères du paludisme. Sa physiopathologie est complexe, impliquant l'adhésion des érythrocytes infectés par Plasmodium falciparum à l'endothélium vasculaire dans le cerveau. Ce mécanisme conduit à une obstruction du flux sanguin, une inflammation locale, une altération de la barrière hémato-encéphalique et à un éventail de réponses cellulaires visant à résoudre le processus de neuroinflammation. Parmi ces cellules, les monocytes/macrophages jouent un rôle clé de par leur capacité à s'adapter à leur microenvironnement en fonction des différents signaux qu'ils perçoivent, modulant ainsi les balances pro/anti-inflammatoire et pro/antioxydante, essentielles à la résolution du neuropaludisme. Les monocytes sont composés de trois sous-populations dont le rôle dans la physiopathologie du neuropaludisme a été peu étudié et reste à éclaircir. Pour compléter ces connaissances, une approche combinant modèle murin et études chez des patients béninois a été menée. Une première étude a été conduite chez des enfants béninois présentant un paludisme simple, une anémie sévère palustre, ou un neuropaludisme dans le but de déterminer si les paramètres monocytaires constituent des facteurs de sévérité et/ou de risque de décès au cours d'un paludisme grave. Les pourcentages et phénotypes des sous-populations monocytaires obtenus chez les trois groupes d'enfants lors de leur admission dans les centres de santé (J0) ont été comparés. Les résultats ont permis de mettre en lumière l'implication des monocytes non classiques dans la protection vis-à-vis des formes sévères de paludisme et de confirmer l'importance du récepteur CD36 des monocytes comme facteur protecteur de sévérité et de survenue de décès. Pour aller plus loin, un travail a ensuite été mené sur un modèle murin de neuropaludisme mis en place pour se rapprocher des conditions réelles, à savoir un traitement tardif, et débouchant sur une résolution de l'infection. Des souris C57BL/6 ont été infectées par P. berghei ANKA (J0) et traitées tardivement à la chloroquine. La cinétique d'évolution des sous-populations monocytaires a été suivie en termes de pourcentage et d'expression protéique et génique, à la fois dans le sang, la rate et le cerveau, jusqu'à la résolution de l'infection (J12). Les résultats suggèrent une implication des monocytes classiques et intermédiaires dans la neuroinflammation, au contraire des monocytes non classiques. A J12 était observé un nouvel afflux de monocytes intermédiaires vers le cerveau, suggérant également un rôle de cette sous-population dans la résolution tardive de la neuroinflammation. Les analyses protéique et génique ont mis en évidence une implication favorable de Nrf2, CD36, HO-1, CMH-II, COX-2 et de la 12/15-LOX dans la résolution de l'infection. Une deuxième étude a ensuite été menée chez des enfants béninois, avec un plus grand nombre de patients et un suivi à J3 et J30. Deux groupes de sujets ont été inclus, présentant un paludisme simple (n = 94) ou un neuropaludisme (n = 65). Dans l'ensemble, les résultats indiquent que les monocytes non classiques et intermédiaires étaient respectivement impliqués dans l'amélioration clinique et la survie des patients. L'expression de CD14, CD16, CD36 et HLA-DR était un facteur protecteur vis-à-vis du neuropaludisme. De manière intéressante, nos résultats montrent également une association entre une réponse pro-oxydante moins active, la sévérité du paludisme, et la survenue de décès. L'ensemble de ces résultats apporte un nouvel éclairage sur l'implication des monocytes/macrophages au cours du neuropaludisme.
... Indexed to these ATS domain counts, we then analyzed expression of N-terminal var gene head structure domains which predict or directly bind the major PfEMP1 host binding partners: CD36, EPCR, or CSA (Aggregated N-terminal domain raw read counts summarized in S1 Table). Because specific DBLα and CIDRα domain types are found together in PfEMP1's head structure [11,25], we analyzed the correlation of DBLα and CIDRα domain expression stratified by head structure type. The expression (normalized read counts) of DBL and CIDR domains of the same head structure were highly correlated across the patient samples (R 2 = 0.9, Table 1 Fig 5C). ...
... It is the most common host interaction partner for PfEMP1s: 80% of the var gene repertoire contains CD36-binding CIDRα2-6 domains, while only 10% of var genes contain EPCR-binding CIDRα1 domains. [11,25]. Var genes encoding CD36-binding PfEMP1s have been shown to dominate in uncomplicated malaria in children [15]. ...
Sickle-trait hemoglobin protects against severe Plasmodium falciparum malaria. Severe malaria is governed in part by the expression of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that are encoded by var genes, specifically those variants that bind Endothelial Protein C Receptor (EPCR). In this study, we investigate the effect of sickle-trait on parasite var gene expression and function in vitro and in field-collected parasites. We mapped var gene reads generated from RNA sequencing in parasite cultures in normal and sickle-cell trait blood throughout the asexual lifecycle. We investigated sickle-trait effect on PfEMP1 interactions with host receptors CD36 and EPCR using static adhesion assays and flow cytometry. Var expression in vivo was compared by assembling var domains sequenced from total RNA in parasites infecting Malian children with HbAA and HbAS. Sickle-trait did not alter the abundance or type of var gene transcripts in vitro , nor the abundance of overall transcripts or of var functional domains in vivo . In adhesion assays using recombinant host receptors, sickle-trait reduced adhesion by 73–86% to CD36 and 83% to EPCR. Similarly, sickle-trait reduced the surface expression of EPCR-binding PfEMP1. In conclusion, Sickle-cell trait does not directly affect var gene transcription but does reduce the surface expression and function of PfEMP1. This provides a direct mechanism for protection against severe malaria conferred by sickle-trait hemoglobin.
Trial Registration: ClinicalTrials.gov Identifier: NCT02645604 .
... Notably, however, the VAR2CSA PfEMP1 variants do not contain typical CIDR domains and bind placental chondroitin sulfate A via specialized DBL domains (14,15). PfEMP1 has diversified to bind the endothelial protein C receptor (EPCR) (10), the scavenger receptor CD36 (16), or yet undermined receptors via head structure CIDR domains. These phenotypes are maintained by the chromosomal organization of the var genes (17). ...
... PfEMP1 variants containing domains of the CIDRα1 class generally bind to EPCR on endothelial cells and are associated with severe malaria (10), whereas variants containing domains of the CIDRα2-6 classes bind to CD36 present on several host cell types, including microvascular endothelial cells, mononuclear phagocytes, and platelets (16,37). Antibodies targeting these PfEMP1 domains can potentially disrupt adhesion of IEs to host receptors but can also facilitate IE clearance via opsonization and phagocytosis or antibody-mediated cytotoxicity (10,38,39). ...
Background:
Malaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum infected erythrocytes to the microvasculature mediated via specific interactions between PfEMP1 variant domains to host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria.
Methods:
We evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission.
Results:
Using longitudinal data, we found that IgG to the pathogenic domain variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG to CIDRγ3 was associated with reduced prospective risk of febrile malaria and recurrent malaria episodes.
Conclusion:
This study provides evidence that acquisition of IgG antibodies to PfEMP1 variants is ordered and demonstrates that antibodies to CIDRα1 domains are acquired the earliest in children residing in an area of intense, seasonal malaria transmission. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant-specific antibodies.
Funding:
Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health.
... n = 13) and B/A (CIDRα1.1 and CIDRα1.8; n = 6); those binding CD36 [19] (CIDRα2-6; n = 12); and those binding neither EPCR nor CD36 [29] (CIDRδ [n = 3] and CIDRγ [n = 1]). Mean antibody levels to these proteins were compared among and within the different sample groups (severe and uncomplicated acute stage, severe and uncomplicated convalescent stage, and community controls), across different age groups and against each of the CIDR domain types. ...
... PfEMP1 molecules can be grouped into mutually exclusive binding phenotypes determined by their N-terminal CIDR domains. These mediate adhesion to EPCR [11] or CD36 [19] or have unknown functions possibly associated with rosetting [20]. Using acute and convalescent plasma from children from PNG, we found malaria syndrome-specific and age-dependent differences in antibody recognition of different types of CIDR domains. ...
Background:
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration in postcapillary venules in P. falciparum malaria. PfEMP1 types can be classified based on their cysteine-rich interdomain region (CIDR) domains. Antibodies to different PfEMP1 types develop gradually after repeated infections as children age, and antibodies to specific CIDR types may confer protection.
Methods:
Levels of immunoglobulin G to 35 recombinant CIDR domains were measured by means of Luminex assay in acute-stage (baseline) and convalescent-stage plasma samples from Papua New Guinean children with severe or uncomplicated malaria and in healthy age-matched community controls.
Results:
At baseline, antibody levels were similar across the 3 groups. After infection, children with severe malaria had higher antibody levels than those with uncomplicated malaria against the endothelial protein C receptor (EPCR) binding CIDRα1 domains, and this difference was largely confined to older children. Antibodies to EPCR-binding domains increased from presentation to follow-up in severe malaria, but not in uncomplicated malaria.
Conclusions:
The acquisition of antibodies against EPCR-binding CIDRα1 domains of PfEMP1 after a severe malaria episode suggest that EPCR-binding PfEMP1 may have a role in the pathogenesis of severe malaria in Papua New Guinea.
... Increased cytoadherence may result in higher parasitemia and increased malaria severity [5]. This brings to the fore the potential role of VSAs; antibodies against these proteins have been shown to select against parasites with high binding ability [50,51], hence inadvertently selecting for parasites with low binding ability in semi-immune asymptomatic carriers. These parasites with lower binding ability will predominate and circulate for a longer time in the bloodstream, increasing the splenic clearance of infected erythrocytes [5] and reducing parasitemia. ...
Background
Cumulative malaria parasite exposure in endemic regions often results in the acquisition of partial immunity and asymptomatic infections. There is limited information on how host-parasite interactions mediate the maintenance of chronic symptomless infections that sustain malaria transmission.
Methods
Here, we determined the gene expression profiles of the parasite population and the corresponding host peripheral blood mononuclear cells (PBMCs) from 21 children (< 15 years). We compared children who were defined as uninfected, asymptomatic and those with febrile malaria.
Results
Children with asymptomatic infections had a parasite transcriptional profile characterized by a bias toward trophozoite stage (~ 12 h-post invasion) parasites and low parasite levels, while early ring stage parasites were characteristic of febrile malaria. The host response of asymptomatic children was characterized by downregulated transcription of genes associated with inflammatory responses, compared with children with febrile malaria,. Interestingly, the host responses during febrile infections that followed an asymptomatic infection featured stronger inflammatory responses, whereas the febrile host responses from previously uninfected children featured increased humoral immune responses.
Conclusions
The priming effect of prior asymptomatic infection may explain the blunted acquisition of antibody responses seen to malaria antigens following natural exposure or vaccination in malaria endemic areas.
... Increased cytoadherence may result in higher parasitemia and increased malaria severity (5). This brings to the fore the potential role of VSAs, antibodies against these proteins have been shown to select against parasites with high binding ability (31,32), hence inadvertently selecting for parasites with low binding ability in semi-immune asymptomatic carriers. These parasites with lower binding ability will predominate and circulate for a longer time in the bloodstream, increasing the splenic clearance of infected erythrocytes (5) and reducing parasitemia. ...
Cumulative malaria parasite exposure in endemic regions often results in the acquisition of partial immunity and asymptomatic infections. There is limited information on how host-parasite interactions mediate maintenance of chronic symptomless infections that sustain malaria transmission. Here, we have determined the gene expression profiles of the parasite population and the corresponding host peripheral blood mononuclear cells (PBMCs) from 21 children (< 15 years). We compared children who were defined as uninfected, asymptomatic and those with febrile malaria. Children with asymptomatic infections had a parasite transcriptional profile characterized by a bias toward trophozoite stage (~ 12 hours-post invasion) parasites and low parasite levels, while earlier ring stage parasites were characteristic of febrile malaria. The host response of asymptomatic children was characterized by downregulated transcription of genes associated with inflammatory responses, compared with children with febrile malaria, which may lead to less cytoadherence of more mature parasite stages. Interestingly, the host responses during febrile infections that followed an asymptomatic infection featured stronger inflammatory responses, whereas the febrile host responses from previously uninfected children featured increased humoral immune responses. The priming effect of prior asymptomatic infection may explain the blunted acquisition of antibody responses seen to malaria antigens following natural exposure or vaccination in malaria endemic areas.
... Three major PfEMP1 families (A, B, and C) have been identified based on their conserved upstream sequences and genomic locations. Group B and C variants bind to the endothelial protein and scavenger receptor CD36 and are mainly implicated in uncomplicated malaria [6], while group A variants often bind to endothelial protein C receptor (EPCR) [7], and sometimes intercellular adhesion molecule 1, and are associated with severe malaria [8]. Domain cassettes (DCs) are evolutionarily conserved arrangements of Duffy binding-like (DBL) domains and cysteine-rich interdomain regions (CIDRs) within PfEMP1, and increased expression of DC4, DC8, DC13, and DC5 have been associated with severe malaria in young children [9]. ...
Background:
Antibodies to variant surface antigens (VSA) such as Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) may vary with malaria severity. The influence of ABO blood group on antibody development is not understood.
Method:
Immunoglobulin G antibodies to VSA in Papua New Guinean children with severe (N=41) or uncomplicated malaria (N= 30) were measured by flow cytometry using homologous P. falciparum isolates. Isolates were incubated with ABO-matched homologous and heterologous acute and convalescent plasma. RNA was used to assess var gene transcription.
Results:
Antibodies to homologous, but not heterologous, isolates were boosted in convalescence. The relationship between antibody and severity varied by blood group. Antibodies to VSA were similar in severe and uncomplicated malaria at presentation, higher in severe than uncomplicated malaria in convalescence, and higher in children with blood group O than other children. Six var gene transcripts best distinguished severe from uncomplicated malaria, including UpsA and two CIDRα1 domains6.
Conclusion:
ABO blood group may influence antibody acquisition to VSA and susceptibility to severe malaria. Children in PNG showed little evidence of acquisition of cross-reactive antibodies following malaria. Var gene transcripts in PNG children with severe malaria were similar to those reported from Africa.
... Following growth in vivo, parasites switched away from primarily expressing a single group C var gene at baseline (inoculation), to elevated expression of most of the var repertoire in two subjects (subject 5 and subject 6) and a less broad but still fairly diverse pattern of expression in subject 7 ( Fig. 6 and Additional File 3: Text S8). The most abundantly transcribed genes differed between the subjects and none had a published association with virulence (Additional File 3: Table S2) [44]. Wild-type P. falciparum 3D7 parasites from a CHMI study using the IBSM model [45] were analyzed as a control. ...
Background
There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes.
Methods
The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 10 ⁵ (2 subjects), or 3 × 10 ⁶ viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression).
Results
None of the subjects who were administered with 1800 or 1.8 × 10 ⁵ parasites developed parasitemia; 3/4 subjects administered 3× 10 ⁶ parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo.
Conclusions
This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp – strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses.
Trial registration
Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).
... The CIDRα domains of some PfEMP1s bind CD36, the most common target receptor of PfEMP1s [11][12][13][14] . CD36 is a leukocyte differentiation antigen and scavenger receptor involved in fatty acid metabolism, phagocytosis, and angiogenesis [15][16][17] . PfEMP1s binding CD36 via CIDRα2 domains have been associated with uncomplicated or asymptomatic malaria 18,19 , whereas PfEMP1s binding the endothelial protein C receptor (EPCR) 20 via CIDRα1 domains and PfEMP1s binding the intercellular adhesion molecule-1 (ICAM-1) via DBLβ domains have been associated with severe malaria [21][22][23][24][25][26][27] . ...
Plasmodium falciparum erythrocyte membrane protein-1s (PfEMP1s), diverse malaria proteins expressed on the infected erythrocyte surface, play an important role in pathogenesis, mediating adhesion to host vascular endothelium. Antibodies to particular non-CD36-binding PfEMP1s are associated with protection against severe disease. We hypothesized that given lifelong P. falciparum exposure, Malian adults would have broad PfEMP1 serorecognition and high seroreactivity levels during follow-up, particularly to non-CD36-binding PfEMP1s such as those that attach to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1). Using a protein microarray, we determined serologic responses to 166 reference PfEMP1 fragments during a dry and subsequent malaria transmission season in Malian adults. Malian adult sera had PfEMP1 serologic responses throughout the year, with decreased reactivity to a small subset of PfEMP1 fragments during the dry season and increases in reactivity to a different subset of PfEMP1 fragments during the subsequent peak malaria transmission season, especially for intracellular PfEMP1 domains. For some individuals, PfEMP1 serologic responses increased after the dry season, suggesting antigenic switching during asymptomatic infection. Adults were more likely to experience variable serorecognition of CD36-binding PfEMP1s than non-CD36-binding PfEMP1s that bind EPCR or ICAM-1, which remained serorecognized throughout the year. Sustained seroreactivity to non-CD36-binding PfEMP1s throughout adulthood amid seasonal fluctuation patterns may reflect underlying protective severe malaria immunity and merits further investigation.
... Western blotting was used to confirm that protein became expressed after treatment end (Supplementary Figure 4). After two panning assays, the most expressed var genes were PF3D7_1240600 and PF3D7_0412400, which encode CD36 binding PfEMP1s (28,29), and PF3D7_0420900 ( Figure 3). Under PfACS knockdown ( Figure 3A) and iACS treatment ( Figure 3B), a very similar var transcript profile for the two different treatments was observed, however, the transcripts were observed in relatively smaller quantities. ...
The malaria parasite Plasmodium falciparum possesses a unique Acetyl-CoA Synthetase (PfACS) which provides acetyl moieties for different metabolic and regulatory cellular pathways. We characterized PfACS and studied its role focusing on epigenetic modifications using the var gene family as reporter genes. For this, mutant lines to modulate plasmodial ACS expression by degron-mediated protein degradation or ribozyme induced transcript decay were created. Additionally, an ACS inhibitor was tested for its effectiveness and specificity in interfering with PfACS. The knockdown of PfACS or its inhibition led to impaired parasite growth. Decreased levels of PfACS also led to differential histone acetylation patterns, altered variant gene expression and concomitantly decreased cytoadherence of infected red blood cells containing knocked-down parasites. Further, ChIP analysis revealed the presence of PfACS in many loci in ring stage parasites, underscoring its involvement in the regulation of chromatin. Due to its significant differences to human ACS, PfACS seems an interesting target for drug development.
... Cloning and expression of PfEMP-1 antigen domains PF08_0106 CIDR1⍺3.1 and PF11_0521 DBL2β3 in COS-7, and PFL0030c DBL3X and DBL5ε in E.coli expression systems and their purification/immobilization on BioPlex beads has been described in detail previously [39,40]. PF08_0106 CIDR1⍺3.1 binds to CD36 [41], PF11_0521 DBL2β3 binds to ICAM-1 [42], and both PFL0030c DBL3X and DBL5ε bind to CSA receptors [40,43,44]. Phycoerythrin (PE)-labeled molecules (Jackson Immunoresearch) were used for detection of the human receptors bound to the Bio-Plex bead-immobilized domains as described earlier [40,45]. ...
Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2β3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment.
Biological significance
Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems.
We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies.
This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.
... Numerous studies have linked EPCR-binding parasites, or parasites expressing CIDRα1-PfEMP1, with development of severe malaria in including when it is presenting as cerebral malaria and severe anemia. No other PfEMP1 domain is consistently associated with severe malaria pathology [19][20][21]. However, CIDRα1-PfEMP1 are large multi-domain molecules, and it is likely that endothelial receptor-interactions of some accompanying domains act in concert with EPCR-binding to promote parasite survival. ...
Background:
During the erythrocytic cycle, Plasmodium falciparum malaria parasites express P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that anchor the infected erythrocytes (IE) to the vascular lining of the host. The CIDRα1 domain of PfEMP1 is responsible for binding host endothelial protein C receptor (EPCR), and increasing evidence support that this interaction triggers severe malaria, accounting for the majority of malaria-related deaths. In high transmission regions, children develop immunity to severe malaria after the first few infections. This immunity is believed to be mediated by antibodies targeting and inhibiting PfEMP1, causing infected erythrocytes to circulate and be cleared in the spleen. The development of immunity to malaria coincides with acquisition of broad antibody reactivity across the CIDRα1 protein family. Altogether, this identifies CIDRα1 as an important vaccine target. However, the antigenic diversity of the CIDRα1 domain family is a challenge for vaccine development.
Methods:
Immune responses in mice vaccinated with Virus-Like Particles (VLP) presenting CIDRα1 antigens were investigated. Antibody reactivity was tested to a panel of recombinant CIDRα1 domains, and the antibodies ability to inhibit EPCR binding by the recombinant CIDRα1 domains was tested in Luminex-based multiplex assays.
Results:
VLP-presented CIDRα1.4 antigens induced a rapid and strong IgG response capable of inhibiting EPCR-binding of multiple CIDRα1 domains mainly within the group A CIDRα1.4-7 subgroups.
Conclusions:
The study observations mirror those from previous CIDRα1 vaccine studies using other vaccine constructs and platforms. This suggests that broad CIDRα1 antibody reactivity may be achieved through vaccination with a limited number of CIDRα1 variants. In addition, this study suggest that this may be achieved through vaccination with a human compatible VLP vaccine platform.
... www.nature.com/scientificreports/ 60 PfEMP1 family members contain a CD36-binding domain (CIDRα2-6) 35,43,44 . This supports the theory that PfEMP1-CD36 interactions are essential for survival of the parasite in its host. ...
Characterizing the adhesive dynamics of Plasmodium falciparum infected erythrocytes (IEs) to different endothelial cell receptors (ECRs) in flow is a big challenge considering available methods. This study investigated the adhesive dynamics of IEs to five ECRs (CD36, ICAM-1, P-selectin, CD9, CSA) using simulations of in vivo-like flow and febrile conditions. To characterize the interactions between ECRs and knobby and knobless IEs of two laboratory-adapted P. falciplarum isolates, cytoadhesion analysis over time was performed using a new tracking bioinformatics method. The results revealed that IEs performed rolling adhesion exclusively over CD36, but exhibited stationary binding to the other four ECRs. The absence of knobs affected rolling adhesion both with respect to the distance travelled by IEs and their velocity. Knobs played a critical role at febrile temperatures by stabilizing the binding interaction. Our results clearly underline the complexity of the IE-receptor interaction and the importance of knobs for the survival of the parasite at fever temperatures, and lead us to propose a new hypothesis that could open up new strategies for the treatment of malaria.
... 32-34 CIDRa 1 domains from group A bind to EPCR 35,36 and CIDRa 2-6 domains of groups B and C bind to CD36. 37,38 Recent studies have attempted to establish a link between CM and subtypes of PfEMP1 and their domains. In this context, the ability of certain domains to bind multiple endothelial receptors has been investigated. ...
There is currently a global effort to reduce malaria morbidity and mortality. However, malaria still results in the deaths of thousands of people every year. Malaria is caused by Plasmodium spp., parasites transmitted through the bite of an infected female Anopheles mosquito. Treatment timing plays a decisive role in reducing mortality and sequelae associated with the severe forms of the disease such as cerebral malaria (CM). The available antimalarial therapy is considered effective but parasite resistance to these drugs has been observed in some countries. Antimalarial drugs act by increasing parasite lysis, especially through targeting oxidative stress pathways. Here we discuss the roles of reactive oxygen species and reactive nitrogen intermediates in CM as a result of host-parasite interactions. We also present evidence of the potential contribution of oxidative and nitrosative stress-based antimalarial drugs to disease treatment and control.
... Over 70% of IT4 PfEMP1 proteins contain CIDRα2-6 domains, which are involved in CD36 binding [62]. Interestingly, the enrichment of IEs to CD36 at 40 °C did not lead to selection of knobby IEs. ...
Changes in the erythrocyte membrane induced by Plasmodium falciparum invasion allow cytoadhesion of infected erythrocytes (IEs) to the host endothelium, which can lead to severe complications. Binding to endothelial cell receptors (ECRs) is mainly mediated by members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, encoded by var genes. Malaria infection causes several common symptoms, with fever being the most apparent. In this study, the effects of febrile conditions on cytoadhesion of predominately knobless erythrocytes infected with the laboratory isolate IT4 to chondroitin-4-sulfate A (CSA), intercellular adhesion molecule 1 (ICAM-1), and CD36 were investigated. IEs enriched for binding to CSA at 40 °C exhibited significantly increased binding capacity relative to parasites enriched at 37 °C. This interaction was due to increased var2csa expression and trafficking of the corresponding PfEMP1 to the IE surface as well as to a selection of knobby IEs. Furthermore, the enrichment of IEs to ICAM-1 at 40 °C also led to selection of knobby IEs over knobless IEs, whereas enrichment on CD36 did not lead to a selection. In summary, these findings demonstrate that knobs are crucial for parasitic survival in the host, especially during fever episodes, and thus, that selection pressure on the formation of knobs could be controlled by the host.
... In P. falciparum, var genes have diverged to encode proteins that bind the endothelial receptors CD36 and EPCR 8,[32][33][34] . Molecular analysis performed with recombinant CIDR domains from P. reichenowi revealed a similar interaction with the human receptors 24 , providing evidence that the CD36 and EPCR binding phenotypes evolved in an ancestral clade B parasite, and has been maintained as a key functional molecular interface in great apes and human hosts. ...
VAR2CSA is a leading candidate for developing a placental malaria (PM) vaccine that would protect pregnant women living in malaria endemic areas against placental infections and improve birth outcomes. Two VAR2CSA-based PM vaccines are currently under clinical trials, but it is still unclear if the use of a single VAR2CSA variant will be sufficient to induce a broad enough humoral response in humans to cross-react with genetically diverse parasite populations. Additional immuno-focusing vaccine strategies may therefore be required to identify functionally conserved antibody epitopes in VAR2CSA. We explored the possibility that conserved epitopes could exist between VAR2CSA from the chimpanzee parasite Plasmodium reichenowi and Plasmodium falciparum sequences. Making use of VAR2CSA recombinant proteins originating from both species, we showed that VAR2CSA from P. reichenowi (Pr-VAR2CSA) binds to the placental receptor CSA with high specificity and affinity. Antibodies raised against Pr-VAR2CSA were able to recognize native VAR2CSA from different P. falciparum genotypes and to inhibit the interaction between CSA and P. falciparum-infected erythrocytes expressing different VAR2CSA variants. Our work revealed the existence of cross-species inhibitory epitopes in VAR2CSA and calls for pre-clinical studies assessing the efficacy of novel VAR2CSA-based cross-species boosting regimens.
... PfEMP1 bind to a variety of human ligands (11,12), the most common of which are cluster of differentiation 36 (CD36), endothelial protein C receptor (EPCR), and intercellular adhesion molecule 1 (ICAM-1) (13)(14)(15). Group B and C (BC) PfEMP1 that contain cysteine-rich interdomain region (CIDR) α2 to α6 domains bind to CD36 and are associated with parasites which cause mild or uncomplicated malaria (16,17) while the A-type PfEMP1 that contain CIDRα1 domains that bind to EPCR are associated with severe childhood malaria (14,(18)(19)(20)(21). However, finding a correlation between expression of PfEMP1 that bind to ICAM-1 and malaria outcome has been much more challenging, with conflicting results about whether ICAM-1 binding is linked to cerebral malaria (22)(23)(24)(25). ...
A major determinant of pathogenicity in malaria caused by Plasmodium falciparum is the adhesion of parasite-infected erythrocytes to the vasculature or tissues of infected individuals. This occludes blood flow, leads to inflammation, and increases parasitemia by reducing spleen-mediated clearance of the parasite. This adhesion is mediated by PfEMP1, a multivariant family of around 60 proteins per parasite genome which interact with specific host receptors. One of the most common of these receptors is intracellular adhesion molecule-1 (ICAM-1), which is bound by 2 distinct groups of PfEMP1, A-type and B or C (BC)-type. Here, we present the structure of a domain from a B-type PfEMP1 bound to ICAM-1, revealing a complex binding site. Comparison with the existing structure of an A-type PfEMP1 bound to ICAM-1 shows that the 2 complexes share a globally similar architecture. However, while the A-type PfEMP1 bind ICAM-1 through a highly conserved binding surface, the BC-type PfEMP1 use a binding site that is more diverse in sequence, similar to how PfEMP1 interact with other human receptors. We also show that A- and BC-type PfEMP1 present ICAM-1 at different angles, perhaps influencing the ability of neighboring PfEMP1 domains to bind additional receptors. This illustrates the deep diversity of the PfEMP1 and demonstrates how variations in a single domain architecture can modulate binding to a specific ligand to control function and facilitate immune evasion.
... Nearly all PfEMP1 have a semi-conserved N-terminal head structure consisting of an NTS and a DBLα-CIDR tandem domain [5]. Different types of the N-terminal CIDR domains confer mutually exclusive receptor binding phenotypes [6]. CIDRα1 domains, with the exception of those found in the var1 pseudogene, bind EPCR [7] and this interaction has been linked in particular to cerebral malaria [8,9]. ...
The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity.
... Individual domains bind to diverse receptors, including CD36, ICAM-1 (intercellular adhesion molecule 1), and EPCR (endothelial protein C receptor) (8). PfEMP1 proteins have diverged in binding properties, such that group A and DC8 proteins bind to EPCR (CIDR␣1 domains) (9, 10), while group B and C proteins bind to CD36 (CIDR␣2 to -6 domains) (11,12). In addition, the ICAM-1 binding subset has diverged into PfEMP1 with dual binding activity for EPCR and ICAM-1 (found in group A) (13,14) or CD36 and ICAM-1 (found in group B) (15,16). ...
Cerebral malaria research has been hindered by the inaccessibility of the brain. Here, we have developed an engineered 3D human brain microvessel model that mimics the blood flow rates and architecture of small blood vessels to study how P. falciparum - infected human erythrocytes attach to brain endothelial cells. By studying parasite lines with different adhesive properties, we show that the malaria parasite binding rate is heterogeneous and strongly influenced by physiological differences in flow and whether the endothelium has been previously activated by TNF-α, a proinflammatory cytokine that is linked to malaria disease severity. We also show the importance of human EPCR and ICAM-1 in parasite binding. Our model sheds new light on how P. falciparum binds within brain microvessels and provides a powerful method for future investigations of recruitment of human brain pathogens to the blood vessel lining of the brain.
... Molecular insight into PfEMP1 function has been gained by sequence classification of the extracellular Duffy binding-like (DBL/////) and cysteine-rich interdomain region (CIDR///) adhesion domains (13,14). CIDR1 domains bind endothelial protein C receptor (EPCR) (15,16), CIDR2-6 domains bind to CD36 (17)(18)(19), and CIDR// domains bind neither receptor (reviewed in reference 20). Additionally, PfEMP1 can simultaneously bind to other coreceptors, as some DBL1/3 (group A) and DBL5 (group B and C) domains mediate binding to ICAM-1 (21)(22)(23)(24)(25). PfEMP1 also includes sets of domains typically found tandemly arrayed in the same protein, termed domain cassettes (DCs). ...
P. falciparum malaria can cause multiple disease complications that differ by patient age. Previous studies have attempted to address the roles of parasite adhesion and biomass in disease severity; however, these studies have been limited to single geographical sites, and there is limited understanding of how parasite adhesion and biomass interact to influence disease manifestations. In this meta-analysis, we compared parasite disease determinants in African children and Indian adults. This study demonstrates that parasite biomass and specific subsets of var genes are independently associated with detrimental outcomes in both childhood and adult malaria. We also explored how parasite var adhesion types and biomass play different roles in the development of specific severe malaria pathologies, including childhood cerebral malaria and multiorgan complications in adults. This work represents the largest study to date of the role of both var adhesion types and biomass in severe malaria.
... As PFL2665c also con- tains CD36-binding CIDRα2.3_D3 domain 24 ( Fig. 2A inset), this line binds to CD36 and αVβ3 integrin but not to CSA, ICAM1, or control protein (BSA) ( Fig. 2A). Binding of E9 to αVβ3 is inhibited by soluble αVβ3 integrin ( Supplementary Fig. 4), and binding to CD36 is inhibited by soluble CD36 and by binding-inhibitory anti-CD36 mAb FA6-152 25,26 (Supplementary Fig. 5), confirming specificity of receptor binding. ...
Abstract Major complications and mortality from Plasmodium falciparum malaria are associated with cytoadhesion of parasite-infected erythrocytes (IE). The main parasite ligands for cytoadhesion are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. Interactions of different host receptor-ligand pairs may lead to various pathological outcomes, like placental or cerebral malaria. It has been shown previously that IE can bind integrin αVβ3. Using bead-immobilized PfEMP1 constructs, we have identified that the PFL2665c DBLδ1_D4 domain binds to αVβ3 and αVβ6. A parasite line expressing PFL2665c binds to surface-immobilized αVβ3 and αVβ6; both are RGD motif-binding integrins. Interactions can be inhibited by cyloRGDFV peptide, an antagonist of RGD-binding integrins. This is a first, to the best of our knowledge, implication of a specific PfEMP1 domain for binding to integrins. These host receptors have important physiological functions in endothelial and immune cells; therefore, these results will contribute to future studies and a better understanding, at the molecular level, of the physiological outcome of interactions between IE and integrin receptors on the surface of host cells.
... The BOEC analysed in this study did not express any CD36 (Fig 3), however CD36 expression is tissue specific and in many cases, CD36 is expressed by a sub-population of BOEC cells at different levels [17]. Whilst CD36 is indeed a common parasite adhesion receptor [34], it is not associated with severe malaria in Africa [35][36][37]. Access to clinically relevant tissue has until now relied upon biopsy or post-mortem samples, which are costly, time-consuming, and require technical expertise that is not always available. ...
The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from a pool of donors (e.g., HBEC-5i). Here we demonstrate, for the first time, the successful isolation of blood outgrowth endothelial cells (BOECs) from frozen stabilates of peripheral blood mononuclear cells obtained from small-volume peripheral blood samples from paediatric malaria patients. BOECs are a sub-population of human endothelial cells, found within the peripheral blood. We demonstrate that these cells express receptors such as Intercellular Adhesion Molecule 1 (ICAM-1/CD54), Endothelial Protein C Receptor (EPCR/CD201), platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), Thrombomodulin (CD141), and support adhesion of P. falciparum IEs.
... The domains, which sometimes exist in combination as short tandem domain cassettes (DCs) of particular subtypes, mediate interactions with a variety of human cell surface receptors [5]. For instance, DBLb domains of PfEMP1 in groups A, B, and C bind intercellular adhesion molecule 1 (ICAM-1) [7][8][9]; group A PfEMP1 CIDRa domains bind endothelial protein C receptor (EPCR) [10,11]; and some group B and C PfEMP1 contain CIDRa domains that bind CD36 [12,13]. ...
Acquired antibodies directed towards antigens expressed on the surface of merozoites and infected erythrocytes play an important role in protective immunity to Plasmodium falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major parasite component of the infected erythrocyte surface, has been implicated in malaria pathology, parasite sequestration and host immune evasion. However, the extent to which unique PfEMP1 domains interact with host immune response remains largely unknown. In this study, we sought to comprehensively understand the naturally acquired antibody responses targeting different Duffy binding-like (DBL), and Cysteine-rich interdomain region (CIDR) domains in a Ugandan cohort. Consequently, we created a protein library consisting of full-length DBL (n = 163) and CIDR (n = 108) domains derived from 62-var genes based on 3D7 genome. The proteins were expressed by a wheat germ cell-free system; a system that yields plasmodial proteins that are comparatively soluble, intact, biologically active and immunoreactive to human sera. Our findings suggest that all PfEMP1 DBL and CIDR domains, regardless of PfEMP1 group, are targets of naturally acquired immunity. The breadth of the immune response expands with children's age. We concurrently identified 10 DBL and 8 CIDR domains whose antibody responses were associated with reduced risk to symptomatic malaria in the Ugandan children cohort. This study highlights that only a restricted set of specific domains are essential for eliciting naturally acquired protective immunity in malaria. In light of current data, tandem domains in PfEMP1s PF3D7_0700100 and PF3D7_0425800 (DC4) are recommended for extensive evaluation in larger population cohorts to further assess their potential as alternative targets for malaria vaccine development.
... Antigenic variation is advantageous for the parasite because it prolongs the length of infection within the host and thus increases the chances of transmission to new hosts[68]. The infection would be cleared once the host immune response detects and controls all variants The features of PfEMP-1 structure include the extracellular exposed Nterminal composed of the NTS followed by a variable number of polymorphic DBL domains and CIDR domains, and the TM region, encoded by the highly variable exon I. Adhesion of some DBL and CIDR domains to host receptors such as CD36 by CIDR1α domain[70][71][72][73], ICAM-1 by DBLβC2[74], CSA by DBLγ[75] ...
To avoid clearance by the spleen, P. falciparum-infected erythrocytes adhere to the microvascular endothelial cells lining the blood vessels and sequester in the microvasculature of vital organs. Cytoadhesion results in reduced blood perfusion and inflammatory endothelial cell activation. While the parasite matures during the intraerythrocytic cycle, the infected erythrocyte undergoes a series of modifications including altered morphology, reduced deformability and increased adhesiveness. These properties govern the cytoadherence process and determine the dynamic adhesion behavior under physiological flow conditions. Several red blood cell polymorphisms, including sickle hemoglobin and hemoglobin C, have been associated with protection against severe malaria and malaria-related death. The mechanisms underlying this protection are poorly understood but it might be conferred, in part, by the reduced binding capability of infected erythrocytes to microvascular endothelial cells. Here, we quantitatively compared the adhesion dynamics of infected wild-type and sickle cell trait erythrocytes, at different parasite developmental stages, using flow chamber assays. Differences in the dynamic adhesion behavior were observed for trophozoite and schizont-stages. While a discoid shape in early stage caused flipping of the infected cell, an almost spherical cell at the late stage of the intraerythrocytic cycle results in a regular rolling motion. We further showed that changes in mechanical and adhesive properties of infected sickle cell trait erythrocytes resulted in substantial differences in the flipping and rolling dynamics, relative to infected wild-type erythrocytes, which led to a reduced contact time and predicted contact area to the endothelial cells as well as a reduced firm adherence. As a consequence of the differential firm and dynamic adhesion behavior, infected sickle trait-erythrocytes were less likely to activate microvascular endothelial cells, which in turn, might reduce the pathology observed in sickle cell trait individuals infected with P. falciparum. Overall, our findings improve the understanding of the protection mechanism against severe malaria conferred by sickle hemoglobin.
... Sequence classification has revealed conserved tandem arrangements of domains known as domain cassettes (DCs) (Rask et al., 2010;Smith et al., 2000) and provided molecular insight into protein diversification. The N-terminal DBL-CIDR head structure has diverged between var groups, such that group B and C PfEMP1 encode CD36-binding properties (CIDRa2-6 domains) (Hsieh et al., 2016;Robinson et al., 2003), whereas group A proteins have diversified into those that bind endothelial protein C receptor (EPCR) (CIDRa1 domains) or non-EPCR binders (CIDRb/g/d domains) (Lau et al., 2015;Turner et al., 2013). The latter group A subset is less well characterized, although some mediate rosetting of pRBCs with unparasitized RBCs (Ghumra et al., 2012). ...
Brain swelling is a major predictor of mortality in pediatric cerebral malaria (CM). However, the mechanisms leading to swelling remain poorly defined. Here, we combined neuroimaging, parasite transcript profiling, and laboratory blood profiles to develop machine-learning models of malarial retinopathy and brain swelling. We found that parasite var transcripts encoding endothelial protein C receptor (EPCR)-binding domains, in combination with high parasite biomass and low platelet levels, are strong indicators of CM cases with malarial retinopathy. Swelling cases presented low platelet levels and increased transcript abundance of parasite PfEMP1 DC8 and group A EPCR-binding domains. Remarkably, the dominant transcript in 50% of swelling cases encoded PfEMP1 group A CIDRα1.7 domains. Furthermore, a recombinant CIDRα1.7 domain from a pediatric CM brain autopsy inhibited the barrier-protective properties of EPCR in human brain endothelial cells in vitro. Together, these findings suggest a detrimental role for EPCR-binding CIDRα1 domains in brain swelling. Brain swelling is associated with cerebral malaria mortality, but the parasite and host factors responsible for development of brain swelling are unknown. Kessler et al. demonstrate an association of low platelet count and EPCR-binding PfEMP1 with brain swelling in children with cerebral malaria.
... Group A var genes encode PfEMP1 with CIDRα1 domains shown to bind endothelial protein C receptor (EPCR) [24] or a set of more diverse CIDRβ/γ/δ domains of unknown function, but potentially associated with rosetting [16]. Groups B and C var genes encode cluster of differentiation 36 (CD36)-binding PfEMP1 [25]. One exception to this rule is the so-called conserved tandem arrangements known as domain cassette 8 (DC8) PfEMP1 [21], which is a group A-like EPCR-binding PfEMP1, recombined into a group B var gene location. ...
Background:
Expression of group A and the A-like subset of group B Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is associated with severe malaria (SM). The diversity of var sequences combined with the challenges of distinct classification of patient pathologies has made studying the role of distinct PfEMP1 variants on malaria disease severity challenging. The application of retinopathy in the recent years has provided a further method to clinically evaluate children with cerebral malaria (CM). The question of whether children with clinical CM but no retinopathy represent a completely different disease process or a subgroup within the spectrum of CM remains an important question in malaria. In the current study, we use newly designed primer sets with the best coverage to date in a large cohort of children with SM to determine the role of var genes in malaria disease severity and especially CM as discriminated by retinopathy.
Methods:
We performed qRT-PCR targeting the different subsets of these var genes on samples from Ugandan children with CM (n = 98, of whom 50 had malarial retinopathy [RP] and 47 did not [RN]), severe malarial anemia (SMA, n = 47), and asymptomatic parasitemia (AP, n = 14). The primers used in this study were designed based on var sequences from 226 Illumina whole genome sequenced P. falciparum field isolates.
Results:
Increasing severity of illness was associated with increasing levels of endothelial protein C receptor (EPCR)-binding PfEMP1. EPCR-binding PfEMP1 transcript levels were highest in children with combined CM and SMA and then decreased by level of disease severity: RP CM > RN CM > SMA > AP.
Conclusions:
The study findings indicate that PfEMP1 binding to EPCR is important in the pathogenesis of SM, including RN CM, and suggest that increased expression of EPCR-binding PfEMP1 is associated with progressively more severe disease. Agents that block EPCR-binding of PfEMP1 could provide novel interventions to prevent or decrease disease severity in malaria.
... PfEMP1 are variant surface antigens which owing to their hypervariability, enable the parasite to evade host immune system. [1] PfEMP1 also bind to various host-receptors such as CD36, [2] ICAM, [3] thrombospondin, [4] chondroitin sulphate A, [5] endothelial protein C receptor, [6] complementreceptor CR1, [7] etc., thereby causing iRBC to cytoadhere. Cytoadhesion to endothelial cells enables iRBC to get sequestered in deep microvasculature, thereby, avoiding splenic clearance; and cytoadhesion to uninfected erythrocytes, also called rosetting, [8] makes it easy for merozoites to quickly invade new host cells. ...
Purpose:
The cell membrane of the erythrocytes infected with the malaria parasite Plasmodium falciparum undergoes several changes during the course of parasite life cycle and forms protrusions known as 'knobs' on its surface during the mature trophozoite and schizont stages. The structural organization of knob components especially PfEMP1 on the iRBC surface is the main determinant for the cytoadhesive and rosetting capacity of the iRBC by binding to various host receptors as well as for the variable antigenicity, which is crucial for immunoevasion. Although several studies report individual interactions among knob constituents, a comprehensive identification of the knob proteome is lacking.
Experimental design:
The detergent-resistant membrane (DRM) rafts are isolated from the infected erythrocyte membrane and knob (KAHRP) positive fractions are subjected to proteomics analysis. In addition, structures of various knob components are modeled and assembled ab initio based on experimentally established protein interactions.
Results:
Proteins of various functional classes are found to be present in the knobs including the newly identified knob constituents which include host Hsp70, elongation factor 1A, acyl CoA synthetase, and some hypothetical proteins. Ab initio structural prediction of PfEMP1, KHARP, PfEMP2, PfEMP3, and PHIST shows that these proteins are intrinsically disordered and can have varying number of protein-protein interactions depending on their lowest energy structure. Further in silico mathematical modeling of a single repeat unit of PfEMP1-PHIST is present 63-112 times along the periphery of a single knob.
Conclusions and clinical relevance:
This study provides structural insight into the organization of the core knob components and uncovers novel proteins as knob components. This structural information can be used for the development of better vaccine design strategies or drug design to destabilize the knob structure, which is a major virulence determinant in P. falciparum malaria.
The var multigene family encodes the P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is important in host-parasite interaction as a virulence factor and major surface antigen of the blood stages of the parasite, responsible for maintaining chronic infection. Whilst important in the biology of P. falciparum, these genes (50 to 60 genes per parasite genome) are routinely excluded from whole genome analyses due to their hyper-diversity, achieved primarily through recombination. The PfEMP1 head structure almost always consists of a DBLα-CIDR tandem. Categorised into different groups (upsA, upsB, upsC), different head structures have been associated with different ligand-binding affinities and disease severities. We study how conserved individual DBLα types are at the country, regional, and local scales in Sub-Saharan Africa. Using publicly-available sequence datasets and a novel ups classification algorithm, cUps, we performed an in silico exploration of DBLα conservation through time and space in Africa. In all three ups groups, the population structure of DBLα types in Africa consists of variants occurring at rare, moderate, and high frequencies. Non-rare variants were found to be temporally stable in a local area in endemic Ghana. When inspected across different geographical scales, we report different levels of conservation; while some DBLα types were consistently found in high frequencies in multiple African countries, others were conserved only locally, signifying local preservation of specific types. Underlying this population pattern is the composition of DBLα types within each isolate repertoire, revealed to consist of a mix of types found at rare, low, moderate, and high frequencies in the population. We further discuss the adaptive forces and balancing selection, including host genetic factors, potentially shaping the evolution and diversity of DBLα types in Africa.
Infections with Plasmodium falciparum and Plasmodium vivax cause over 600,000 deaths each year, concentrated in Africa and in young children, but much of the world’s population remain at risk of infection. In this article, we review the latest developments in the immunogenicity and pathogenesis of malaria, with a particular focus on P. falciparum, the leading malaria killer. Pathogenic factors include parasite-derived toxins and variant surface antigens on infected erythrocytes that mediate sequestration in the deep vasculature. Host response to parasite toxins and to variant antigens is an important determinant of disease severity. Understanding how parasites sequester, and how antibody to variant antigens could prevent sequestration, may lead to new approaches to treat and prevent disease. Difficulties in malaria diagnosis, drug resistance, and specific challenges of treating P. vivax pose challenges to malaria elimination, but vaccines and other preventive strategies may offer improved disease control.
Plasmodium falciparum-infected erythrocytes (PfIEs) adhere to endothelial cell receptors (ECRs) of blood vessels mainly via PfEMP1 proteins to escape elimination via the spleen. Evidence suggests that P. vivax-infected reticulocytes (PvIRs) also bind to ECRs, presumably enabled by VIR proteins, as shown by inhibition experiments and studies with transgenic P. falciparum expressing vir genes. To test this hypothesis, our study investigated the involvement of VIR proteins in cytoadhesion using vir gene-expressing P. falciparum transfectants. Those VIR proteins with a putative transmembrane domain were present in Maurer’s clefts, and some were also present in the erythrocyte membrane. The VIR protein without a transmembrane domain (PVX_050690) was not exported. Five of the transgenic P. falciparum cell lines, including the one expressing PVX_050690, showed binding to CD36. We observed highly increased expression of specific var genes encoding PfEMP1s in all CD36-binding transfectants. These results suggest that ectopic vir expression regulates var expression through a yet unknown mechanism. In conclusion, the observed cytoadhesion of P. falciparum expressing vir genes depended on PfEMP1s, making this experimental unsuitable for characterizing VIR proteins.
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLβ13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.
Malaria, a deadly disease caused by pathogen Plasmodium, is a global problem. After discovery of Plasmodium as a causative agent of malaria, the understanding of the malaria biology using conventional techniques was at slower pace as compared to the variation in the parasite, e.g., drug resistance. In the era of omics technologies, e.g., proteomics, genomics, and metabolomics, newer strategies to combat this infection have evolved. Also, these different technologies have helped in better understanding of Plasmodium parasite biology in detail. Omics tools involving high-throughput technologies, automation, and data mining have helped in better understanding of the pathways and key proteins required in the life cycle of the parasite and hence pathogenesis of this disease in faster, reliable, and affordable mode. In the recent times, advance in the field of omics displayed high potential in accelerating malaria research to fight the diseases. This chapter will highlight the role of omics tools in deciphering mysteries of malaria parasite biology and their applications in diagnosis, treatment, and eradication of the disease.
MicroRNAs (miRNAs) are short endogenous (~ 22 nucleotides long) noncoding RNAs synthesized by RNA polymerase class II enzyme in the nucleus. The exploration of miRNAs has become an emerging field of research due to their epigenetic regulation associated with a wide array of human diseases including cholera, hepatitis, malaria, and leishmaniasis. The miRNAs that are involved in a disease can be used as a biomarker due to their upregulated or downregulated expression level. miRNAs regulate the expression of mRNA through complementary base pairing with its 3-prime untranslated regions (3′ UTRs). miRNAs are categorized according to its processes of precise formation demarcated as the canonical and noncanonical biogenic pathway. Since a huge amount of mRNA and miRNA data have been generated from the past researches, computational methods are needed to provide experimental validation with statistically significant outcomes. Computational approach is considered as one of the robust methods for miRNA target prediction.
RNA-Seq techniques have added to associate, in treatment, growth of the knowledge distinct to the natural and cell measures needed throughout diseases. Meta-investigation of RNA-Seq tests was performed to rethink the previous proof for the higher comprehension of the pathological process. Moreover, the gene ontology and Reactome investigation performed upheld the discretionary suspicion that the antiviral pathways unremarkably moved in lightweight of intense infective agent contamination that was accountable for a debilitated growing cell. Within the meta-examination measures, it had been recognized that there are a unit 3 potential novel competition qualities inescapably enclosed throughout this antiviral response. In general, RNA-Seq may be a tremendous technique permitting depiction of cell nonuniformity among a cell community. The relationship of transcriptome identification with rapid cell aggregates might encourage clearly expressed proteins or biomarkers of interest distressed in the infectious diseases. Future specialized upgrades might conceivably beat this constraint, a minimum of halfway. Improvement of novel conventions permitting co-occurring investigation of various subatomic particles, as an example, DNA, compound, and proteins, among indistinguishable singular cell would give an extra development toward an extra careful “single-cell integrome,” which may probably be instrumental to extend our perception of uncontrollable disease.
A plethora of studies have shown several types of chromosomal mutations (i.e., deletion, insertion, and substitution) being present for α -CoVs, β -CoVs, γ-CoVs, and δ-CoVs. The current pandemic is caused by the β-CoV and SARS-CoV-2. The attributes of a virus are associated with its genomic composition. Mutations can cause changes in the viral genome that can lead to the crossing of the animal-human barrier or result in a more virulent strain that can increase transmission and pathogenicity for coronaviruses in general and SARS-CoV-2 in particular. Additionally, these mutations may result into new genomic properties. Some mutations have caused changes in the structure of amino acids, and this can be a potential explanation for failure in antiviral therapies. Through genome mapping, we can focus on conserved regions in a viral genome so that even if the virus undergoes a chromosomal mutation into a more virulent strain, the vaccine will still work. One of the prime targets for antiviral therapies against SARS-CoV-2 includes spike protein. Using genomic mapping we can better understand, monitor, and treat a viral infection. With the worldwide spread of Sars-CoV-2, it is ever so important to have a greater understanding of the genomic as well as the proteomic landscapes for the virus, which can guide in the future development of antiviral therapies.
Malaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum infected erythrocytes to the microvasculature mediated via specific interactions between PfEMP1 variant domains to host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria. We evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission. Using longitudinal data, we found that IgG to the pathogenic domain variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG to CIDRγ3 was associated with reduced prospective risk of febrile malaria and recurrent malaria episodes. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant-specific antibodies.
Malaria, a mosquito‐borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium‐parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti‐malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto‐adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti‐disease malaria vaccine. Here we review the role of ICAM‐1 and EPCR receptor adhering falciparum‐parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant sub-families. ICAM-1-binding Group A PfEMP1 proteins also bind EPCR and have been associated with cerebral malaria in children. IgG to these PfEMP1 is acquired later in life than to Group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to Group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here.
Gene sequences encoding Groups B and C PfEMP1 with DBLβ domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBLβ domains from Group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria.
Seven new ICAM-1-binding DBLβ domains from Group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBLβ domains from Group A than from Group B and C. However, the opposite pattern was found in children with malaria, particularly among young patients.
Acquisition of IgG specific for DBLβ domains binding ICAM-1 differs between PfEMP1 groups.
PfEMP1 are variant parasite antigens that are inserted on the surface of Plasmodium falciparum infected erythrocytes (IE). Through interactions with various host molecules, PfEMP1 mediate IE sequestration in tissues and play a key role in the pathology of severe malaria. PfEMP1 is encoded by a diverse multi-gene family called var. Previous studies have shown that that expression of specific subsets of var genes are associated with low levels of host immunity and severe malaria. However, in most clinical studies to date, full-length var gene sequences were unavailable and various approaches have been used to make comparisons between var gene expression profiles in different parasite isolates using limited information. Several studies have relied on the classification of a 300 – 500 base-pair “DBLα tag” region in the DBLα domain located at the 5’ end of most var genes.
We assessed the relationship between various DBLα tag classification methods, and sequence features that are only fully assessable through full-length var gene sequences. We compared these different sequence features in full-length var gene from six fully sequenced laboratory isolates.
These comparisons show that despite a long history of recombination, DBLα sequence tag classification can provide functional information on important features of full-length var genes. Notably, a specific subset of DBLα tags previously defined as “group A-like” is associated with CIDRα1 domains proposed to bind to endothelial protein C receptor.
This analysis helps to bring together different sources of data that have been used to assess var gene expression in clinical parasite isolates.
Erythrocytes infected with mature forms of Plasmodium falciparum do not circulate but are withdrawn from the peripheral circulation; they are bound to the endothelial lining and to uninfected erythrocytes in the microvasculature. Blockage of the blood flow, hampered oxygen delivery, and severe malaria may follow if binding is excessive. The NH2-terminal head structure (Duffy binding–like domain 1 [DBL1α]–cysteine-rich interdomain region [CIDR1α]) of a single species of P. falciparum erythrocyte membrane protein 1 (PfEMP1) is here shown to mediate adherence to multiple host receptors including platelet-endothelial cell adhesion molecule 1 (PECAM-1)/CD31, the blood group A antigen, normal nonimmune immunoglobulin M, three virulence-associated receptor proteins, a heparan sulfate–like glucosaminoglycan, and CD36. DBL2δ was found to mediate additional binding to PECAM-1/CD31. The exceptional binding activity of the PfEMP1 head structure and its relatively conserved nature argues that it holds an important role in erythrocyte sequestration and therefore in the virulence of the malaria parasite.
Adhesion of mature Plasmodium falciparum parasitized erythrocytes to microvascular endothelial cells or to placenta contributes directly to the virulence and severe pathology of P falciparum malaria. Whereas CD36 is the major endothelial receptor for microvasculature sequestration, infected erythrocytes adhering in the placenta bind chondroitin sulfate A (CSA) but not CD36. Binding to both receptors is mediated by different members of the large and diverse protein family P falciparum erythrocyte membrane protein-1 (PfEMP-1) and involves different regions of the molecule. The PfEMP-1–binding domain for CD36 resides in the cysteine-rich interdomain region 1 (CIDR-1). To explore why CSA-binding parasites do not bind CD36, CIDR-1 domains from CD36- or CSA-binding parasites were expressed in mammalian cells and tested for adhesion. Although CIDR-1 domains from CD36-adherent strains strongly bound CD36, those from CSA-adherent parasites did not. The CIDR-1 domain has also been reported to bind CSA. However, none of the CIDR-1 domains tested bound CSA. Chimeric proteins between CIDR-1 domains that bind or do not bind CD36 and mutagenesis experiments revealed that modifications in the minimal CD36-binding region (M2 region) are responsible for the inability of CSA-selected parasites to bind CD36. One of these modifications, mapped to a 3–amino acid substitution in the M2 region, ablated binding in one variant and largely reduced binding of another. These findings provide a molecular explanation for the inability of placental sequestered parasites to bind CD36 and provide additional insight into critical residues for the CIDR-1/CD36 interaction.
The var gene family of Plasmodium falciparum encodes the protein PfEMP1 which is located on the surface of infected erythrocytes and is the receptor that mediates binding to ligands on endothelial cells. This family of proteins is responsible for antigenic variation and differences in binding phenotype to ligands such as CD36 and ICAM1. We have compared the organization of the var gene family in three in vitro cloned lines of P. falciparum and show that most var genes are located in the subtelomeric region of each chromosome closely linked to the repetitive sequence rep20. While most chromosomes possess var genes in the subtelomeric region, in each in vitro cloned line there are some chromosomes that have deleted subtelomeric repetitive regions which include var genes. Comparison of the location of var genes in a field isolate showed that it does not have any detectable subtelomeric deletions as all chromosomes contain var genes and rep20 sequences. We have detected three chromosomes (4, 7 and 12) that contain var gene loci in more stable central regions and the position of these genes on chromosome 4 in the cloned lines analysed is conserved. The location of most of the var gene family in the subtelomeric region of the genome of P. falciparum has important implications for the generation of antigenic diversity of the PfEMP1 protein.
Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.
Chondroitin sulfate A (CSA) is an important receptor for the sequestration of Plasmodium falciparum in the placenta, but the parasite ligand involved in adhesion has not previously been identified. Here we report the identification of a var gene transcribed in association with binding to CSA and present evidence that the P. falciparum erythrocyte membrane protein 1 product of the gene is the parasite ligand mediating CSA binding. Description of this gene and the implication of P. falciparum erythrocyte membrane protein 1 as the parasite ligand paves the way to a more detailed understanding of the pathogenesis of placental infection and potential therapeutic strategies targeting the interaction.
Adherence of mature parasitized erythrocytes (PE) of Plasmodium falciparum to microvascular endothelial cells contributes directly to the virulence and pathology of this human malaria. The malarial variant antigen, P falciparum erythrocyte membrane protein 1 (PfEMP1), has been implicated as the PE receptor for CD36 on endothelial cells. We identified the region of PfEMP1 that mediates adherence of PE to CD36 and showed that a recombinant protein fragment from this region blocked and reversed adherence of antigenically different parasites. Sequence variation was evident in the CD36 binding domain of different PfEMP1 genes, yet many highly conserved residues, particularly cysteine residues, are evident. This suggests a highly conserved shape that mediates adherence to CD36. Immunization with the CD36-binding domain elicited sera that are cross-reactive with the different recombinant proteins but are strain-specific for the PE surface. Novel anti-adherence therapeutics and a malaria vaccine may derived from exploitation of the structure of the CD36 binding domain of PfEMP1.
One important factor in the virulence of infections with Plasmodium falciparum is the adherence of infected erythrocytes to small vessel endothelium. In infections that lead to serious, life-threatening disease accumulation of large numbers of infected cells in particular organs is thought to lead to organ dysfunction or failure. This is of particular relevance when the affected organ is the brain, leading to the development of cerebral malaria. Many different endothelial receptors for infected red blood cells have been identified. Some receptors such as CD36 and thrombospondin are used by all parasite isolates, whereas others such as intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule (VCAM) are used by a subset of field and laboratory isolates. While it has been speculated that the ability to bind or affinity of binding to a particular endothelial receptor may be related to the pattern of disease, only studies with limited numbers of patients have been carried out to date and these have been in general inconclusive. Here we have taken parasite isolates from 150 patients with defined clinical syndromes as well as isolates from 50 healthy but parasitized community controls and quantitatively assessed their binding to purified endothelial receptors in vitro. Our results show that disregarding the level of adhesion, all parasites bind to CD36, most bind to ICAM-1, few bind to VCAM, and almost none bind to E-selectin. In assessing the degree of binding we show that 1) binding to all receptors was reduced in parasites taken from severely anemic patients; 2) binding to CD36 is identical in parasites from cerebral malaria patients and community controls but slightly elevated in parasites from nonsevere cases; and 3) binding to ICAM-1 is highest in cerebral malaria patients. Because rosette formation by uninfected cells has also been a phenotype associated with disease severity and one that may interfere in vitro with receptor binding, we also assessed rosette formation in all isolates. In this study the highest level of rosette-forming parasites was found in the anemic group and not the cerebral malaria group. Stratifying the data for the frequency of rosette formation showed that the above results were not significantly altered by this phenomenon. Our data are not consistent with a role for binding to CD36 in the development of severe disease but show an association between the degree of binding to ICAM-1 and clinical illness in nonanemic patients.
Chromosome 2 of Plasmodium falciparum was sequenced; this sequence contains 947,103 base pairs and encodes 210 predicted genes. In comparison with the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, introns are more frequent, and proteins are markedly enriched in nonglobular domains. A family of surface proteins, rifins, that may play a role in antigenic variation was identified. The complete sequencing of chromosome 2 has shown that sequencing of the A+T-rich P. falciparum genome is technically feasible.
Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages.
Plasmodium falciparum malaria during pregnancy is an important cause of maternal and infant morbidity and mortality. Accumulation of large numbers
of P. falciparum—infected erythrocytes in the maternal blood spaces of the placenta may be mediated by adhesion of infected erythrocytes to molecules
presented on the syncytiotrophoblast surface. In this study, isolates from placentas and peripheral blood of infected pregnant
women and from children were tested for binding to purified receptors and for agglutination with adult sera. Results suggest
that adhesion to chondroitin sulfate A may be involved in placental parasite sequestration in most cases, but other factors
are also likely to be important. Agglutination assay results suggest that parasites infecting pregnant women are antigenically
distinct from those common in childhood disease. The prevalence of agglutinating antibodies to pregnancy isolates was generally
low, but it was highest in multigravidae who are likely to have had the greatest exposure.
The malaria parasite Plasmodium falciparum is one of the most successful human pathogens. Specific virulence factors remain poorly defined, although the adhesion of infected erythrocytes to the venular endothelium has been associated with some of the syndromes of severe disease. Immune responses cannot prevent the development of symptomatic infections throughout life, and clinical immunity to the disease develops only slowly during childhood. An understanding of the obstacles to the development of protective immunity is crucial for developing rational approaches to prevent the disease. Here we show that intact malaria-infected erythrocytes adhere to dendritic cells, inhibit the maturation of dendritic cells and subsequently reduce their capacity to stimulate T cells. These data demonstrate both a novel mechanism by which malaria parasites induce immune dysregulation and a functional role beyond endothelial adhesion for the adhesive phenotypes expressed at the surface of infected erythrocytes.
Analysis of Plasmodium falciparum chromosome 3, and comparison with chromosome 2, highlights novel features of chromosome organization and gene structure. The sub-telomeric regions of chromosome 3 show a conserved order of features, including repetitive DNA sequences, members of multigene families involved in pathogenesis and antigenic variation, a number of conserved pseudogenes, and several genes of unknown function. A putative centromere has been identified that has a core region of about 2 kilobases with an extremely high (adenine + thymidine) composition and arrays of tandem repeats. We have predicted 215 protein-coding genes and two transfer RNA genes in the 1,060,106-base-pair chromosome sequence. The predicted protein-coding genes can be divided into three main classes: 52.6% are not spliced, 45.1% have a large exon with short additional 5' or 3' exons, and 2.3% have a multiple exon structure more typical of higher eukaryotes.
Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.
Accumulation of Plasmodium falciparum-infected erythrocytes in the placenta is a key feature of maternal malaria. This process is mediated in part by the parasite
ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the surface of the infected erythrocyte interacting with the host receptor chondroitin
sulfate A (CSA) on the placental lining. We have localized CSA binding activity to two adjacent domains in PfEMP1 of an adherent
parasite line and shown the presence of at least three active glycosaminoglycan binding sites. A putative CSA binding sequence
was identified in one domain, but nonlinear binding motifs are also likely to be present, since binding activity in the region
was shown to be dependent on conformation. Characterization of this binding region provides an opportunity to investigate
further its potential as a target for antiadhesion therapy.
In areas of intense Plasmodium falciparum transmission, clinical immunity is acquired during childhood, and adults enjoy substantial protection against malaria. An exception to this rule is pregnant women, in whom malaria is both more prevalent and severe than in nonpregnant women. Pregnancy-associated malaria (PAM) in endemic areas is concentrated in the first few pregnancies, indicating that protective immunity to PAM is a function of parity. The placenta is often heavily infected in PAM, and placental parasites show a striking preference for chondroitin sulfate A (CSA) as an adhesion receptor. Plasma Abs from malaria-exposed multiparous women are able to interfere with binding of P. falciparum parasites to CSA in vitro, and acquisition of Abs interfering with CSA-specific parasite sequestration thus appears to be a critical element in acquired protection against PAM. Here we show that adults from an area of hyperendemic P. falciparum transmission generally possessed low levels of Abs specifically recognizing surface Ags expressed by a CSA-adhering parasite isolate, while unselected isolates were well recognized. In marked contrast, most third-trimester pregnant women from that area had very high plasma levels of such Abs. Plasma levels of Abs specifically recognizing the CSA-adhering isolate strongly depended on parity, whereas recognition of CSA-nonadhering isolates did not. Finally, we demonstrate a clear correlation between plasma levels of Abs recognizing the CSA-specific isolate and the ability to interfere with its sequestration to CSA in vitro. Our study supports the hypothesis that Abs inhibiting CSA-specific parasite sequestration are important in acquisition of protection against PAM.
Plasmodium falciparum infection during pregnancy results in the accumulation of infected red blood cells (IRBCs) in the placenta, leading to poor pregnancy outcome. In the preceding paper (Achur, R. N., Valiyaveettil, M., Alkhalil, A., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40344-40356), we reported that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta mediate the IRBC adherence. In this study, we report the structural requirements for the adherence and the minimum chondroitin 4-sulfate (C4S) structural motif that supports IRBC adherence. Partially sulfated C4Ss with varying sulfate contents were prepared by solvolytic desulfation of a fully sulfated C4S. These and other nonmodified C4Ss, with different proportions of 4-, 6-, and nonsulfated disaccharide repeats, were analyzed for inhibition of IRBC adherence to the placental CSPG. C4Ss containing 30-50% 4-sulfated and 50-70% nonsulfated disaccharide repeats efficiently inhibited IRBC adherence; C6S had no inhibitory activity. Oligosaccharides of varying sizes were prepared by the partial depolymerization of C4Ss containing varying levels of 4-sulfation, and their ability to inhibit the IRBC adherence was studied. Oligosaccharides with six or more disaccharide repeats inhibited IRBC adherence to the same level as that of the intact C4Ss, indicating that a dodecasaccharide is the minimum structural motif required for optimal IRBC adherence. Of the C4S dodecasaccharides, only those with two or three sulfate groups per molecule showed maximum IRBC inhibition. These data define the structural requirements for the IRBC adherence to placental CSPGs with implications for the development of therapeutics for maternal malaria.
In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) selectively accumulate in the intervillous spaces of placenta, leading to poor fetal outcome and severe health complications in the mother. Although chondroitin 4-sulfate is known to mediate IRBC adherence to placenta, the natural receptor has not been identified. In the present study, the chondroitin sulfate proteoglycans (CSPGs) of human placenta were purified and structurally characterized, and adherence of IRBCs to these CSPGs investigated. The data indicate that the placenta contains three distinct types of CSPGs: significant quantities of uniquely low sulfated, extracellular CSPGs localized in the intervillous spaces, minor amounts of two cell-associated CSPGs, and major amounts of dermatan sulfate-like CSPGs of the fibrous tissue. Of the various CSPGs isolated from the placenta, the low sulfated CSPGs of the intervillous spaces most efficiently bind IRBCs. Based on IRBC adherence capacities and localization patterns of various CSPGs, we conclude that the CSPGs of the intervillous spaces are the receptors for placental IRBC adherence. The identification and characterization of these CSPGs provide a valuable tool for understanding the precise molecular interactions involved in placental IRBC adherence and for the development of therapeutic strategies for maternal malaria. In the accompanying paper (Alkhalil, A., Achur, R. N., Valiyaveettil, M., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40357-40364), we report the structural requirements for the IRBC adherence.
Plasmodium falciparum-infected erythrocytes roll on and/or adhere to CD36, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and P-selectin under shear conditions in vitro. However, the lack of an adequate animal model has made it difficult to determine whether infected erythrocytes do indeed interact in vivo in microvessels. Therefore, we made use of an established model of human skin grafted onto severe combined immunodeficient (SCID) mice to directly visualize the human microvasculature by epifluorescence intravital microscopy. In all grafts examined, infected erythrocytes were observed to roll and/or adhere in not just postcapillary venules but also in arterioles. In contrast, occlusion of capillaries by infected erythrocytes was noted only in approximately half of the experiments. Administration of an anti-CD36 antibody resulted in a rapid reduction of rolling and adhesion. More importantly, already adherent cells quickly detached. The residual rolling after anti-CD36 treatment was largely inhibited by an anti-ICAM-1 antibody. Anti-ICAM-1 alone reduced the ability of infected erythrocytes to sustain rolling and subsequent adhesion. These findings provide conclusive evidence that infected erythrocytes interact within the human microvasculature in vivo by a multistep adhesive cascade that mimics the process of leukocyte recruitment.
Plasmodium falciparum is the most lethal form of malaria and is increasing both in incidence and in its resistance to antimalarial agents. An improved understanding of the mechanisms of malarial clearance may facilitate the development of new therapeutic interventions. We postulated that the scavenger receptor CD36, an important factor in cytoadherence of P falciparum-parasitized erythrocytes (PEs), might also play a role in monocyte- and macrophage-mediated malarial clearance. Exposure of nonopsonized PEs to Fc receptor-blocked monocytes resulted in significant PE phagocytosis, accompanied by intense clustering of CD36 around the PEs. Phagocytosis was blocked 60% to 70% by monocyte pretreatment with monoclonal anti-CD36 antibodies but not by antibodies to alpha(v)beta(3), thrombospondin, intercellular adhesion molecule-1, or platelet/endothelial cell adhesion molecule-1. Antibody-induced CD36 cross-linking did result in the early increase of surface CD11b expression, but there was no increase in, or priming for, tumor necrosis factor (TNF)-alpha secretion following either CD36 cross-linking or PE phagocytosis. CD36 clustering does support intracellular signaling: Antibody-induced cross-linking initiated intracellular tyrosine phosphorylation as well as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Both broad-spectrum tyrosine kinase inhibition (genistein) and selective ERK and p38 MAPK inhibition (PD98059 and SB203580, respectively) reduced PE uptake to almost the same extent as CD36 blockade. Thus, CD36-dependent binding and signaling appears to be crucial for the nonopsonic clearance of PEs and does not appear to contribute to the increase in TNF-alpha that is prognostic of poor outcome in clinical malaria.
Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.
Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.
Adhesion of mature Plasmodium falciparum parasitized erythrocytes to microvascular endothelial cells or to placenta contributes directly to the virulence and severe pathology of P falciparum malaria. Whereas CD36 is the major endothelial receptor for microvasculature sequestration, infected erythrocytes adhering in the placenta bind chondroitin sulfate A (CSA) but not CD36. Binding to both receptors is mediated by different members of the large and diverse protein family P falciparum erythrocyte membrane protein-1 (PfEMP-1) and involves different regions of the molecule. The PfEMP-1-binding domain for CD36 resides in the cysteine-rich interdomain region 1 (CIDR-1). To explore why CSA-binding parasites do not bind CD36, CIDR-1 domains from CD36- or CSA-binding parasites were expressed in mammalian cells and tested for adhesion. Although CIDR-1 domains from CD36-adherent strains strongly bound CD36, those from CSA-adherent parasites did not. The CIDR-1 domain has also been reported to bind CSA. However, none of the CIDR-1 domains tested bound CSA. Chimeric proteins between CIDR-1 domains that bind or do not bind CD36 and mutagenesis experiments revealed that modifications in the minimal CD36-binding region (M2 region) are responsible for the inability of CSA-selected parasites to bind CD36. One of these modifications, mapped to a 3-amino acid substitution in the M2 region, ablated binding in one variant and largely reduced binding of another. These findings provide a molecular explanation for the inability of placental sequestered parasites to bind CD36 and provide additional insight into critical residues for the CIDR-1/CD36 interaction.
Infections with Plasmodium falciparum during pregnancy lead to the accumulation of parasitized red blood cells (infected erythrocytes, IEs) in the placenta. IEs of P. falciparum isolates that infect the human placenta were found to bind immunoglobulin G (IgG). A strain of P. falciparum cloned for IgG binding adhered massively to placental syncytiotrophoblasts in a pattern similar to that of natural infections. Adherence was inhibited by IgG-binding proteins, but not by glycosaminoglycans or enzymatic digestion of chondroitin sulfate A or hyaluronic acid. Normal, nonimmune IgG that is bound to a duffy binding-like domain beta of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) might at the IE surface act as a bridge to neonatal Fc receptors of the placenta.
Plasmodium falciparum is the most lethal form of malaria and is increasing both in incidence and in its resistance to antimalarial agents. An improved understanding of the mechanisms of malarial clearance may facilitate the development of new therapeutic interventions. We postulated that the scavenger receptor CD36, an important factor in cytoadherence of P falciparum–parasitized erythrocytes (PEs), might also play a role in monocyte- and macrophage-mediated malarial clearance. Exposure of nonopsonized PEs to Fc receptor–blocked monocytes resulted in significant PE phagocytosis, accompanied by intense clustering of CD36 around the PEs. Phagocytosis was blocked 60% to 70% by monocyte pretreatment with monoclonal anti-CD36 antibodies but not by antibodies to αvβ3, thrombospondin, intercellular adhesion molecule-1, or platelet/endothelial cell adhesion molecule-1. Antibody-induced CD36 cross-linking did result in the early increase of surface CD11b expression, but there was no increase in, or priming for, tumor necrosis factor (TNF)-α secretion following either CD36 cross-linking or PE phagocytosis. CD36 clustering does support intracellular signaling: Antibody-induced cross-linking initiated intracellular tyrosine phosphorylation as well as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Both broad-spectrum tyrosine kinase inhibition (genistein) and selective ERK and p38 MAPK inhibition (PD98059 and SB203580, respectively) reduced PE uptake to almost the same extent as CD36 blockade. Thus, CD36-dependent binding and signaling appears to be crucial for the nonopsonic clearance of PEs and does not appear to contribute to the increase in TNF-α that is prognostic of poor outcome in clinical malaria.
A critical step in infection by Plasmodium falciparum, the microorganism
that causes the most severe form of malaria, is the adhesion of parasitized
red blood cells to capillary endothelium. The human protein CD36 is a major
receptor for P. falciparum-infected red blood cells1, 2
and may contribute to the disease by sequestering infected red blood cells1, 2 and inhibiting the immune response to the parasite3.
We have found that African populations contain an exceptionally high frequency
of mutations in CD36. Unexpectedly, these mutations that cause CD36 deficiency
are associated with susceptibility to severe malaria, suggesting that the
presence of distinct CD36 mutations in Africans and Asians4, 5, 6
is due to some selection pressure other than malaria.
A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.
The propensity of isolates of the malaria parasite Plasmodium falciparum to delete a segment of chromosome 9 has provided positional information that has allowed us to identify a gene necessary for cytoadherence. It has been termed the cytoadherence-linked asexual gene (clag9). clag9 encodes at least nine exons and is expressed in blood stages. The hydrophobicity profile of the predicted CLAG9 protein identifies up to four transmembrane domains. We show here that targeted gene disruption of clag9 ablated cytoadherence to C32 melanoma cells and purified CD36. DNA-induced antibodies to the clag9 gene product reacted with a polypeptide of 220 kDa in the parental malaria clone but not in clones with a disrupted clag9 gene.
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.
The CD36 and ICAM-l glycoproteins on vascular endothelial cells have been implicated as cytoadherence receptors for Plasmodium falciparum-infected erythrocytes (IRBC).Adhesion of IRBC from Thai patients with uncomplicated and severe falciparum malaria to purified
CD36 or ICAM-l and to C32 melanoma cells was compared. All malaria isolates bound to solid phase-adsorbed CD36 and to fluid-phase
125I-labeled CD36. IRBC adhesion to purified ICAM-l varied widely, and no correlation with clinical severity of disease was observed.The
cytoadherent phenotype of IRBC was modulated by selective panning on plates coated with purified CD36 or ICAM-1. IRBC selected
by panning on CD36+, ICAM-1+ melanoma cells bound to cells that express surface CD36 but not to CD36-deficient cells, indicating that CD36 exerts a strong
selective pressure on the IRBC cytoadherent phenotype. IRBC adhesion to CD36 and ICAM-l suggests that P. falciparum parasites may use these receptors in vivo to promote parasite survival and immune evasion.
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.
Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. We show here that monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocytes, and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an 125I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.
The cytoadherent behavior of two Plasmodium falciparum (human malaria) cell lines, FCR-3 and ITO4 (a cell line with elevated ICAM-1 adherence), was studied using CHO cells transfected with CD36 or ICAM-1 receptors as target cells. ICAM-1-mediated adherence was found to be relatively pH insensitive, whereas CD36-mediated adherence was pH sensitive and inhibited by monoclonal antibodies and peptides based on a region found in human band 3 protein and named pfalhesin. Immobilized pfalhesin was used as an affinity matrix to purify CD36 from extracts of C32 amelanotic melanoma cells, which have ICAM-1 as well as CD36 receptors, and bind both parasite cell lines. We conclude that pfalhesin and CD36 constitute an adhesin/receptor pair.
The human malaria parasite Plasmodium falciparum evades host immunity by varying the antigenic and adhesive character of infected erythrocytes. We describe a large and extremely diverse family of P. falciparum genes (var) that encode 200-350 kDa proteins having the expected properties of antigenically variant adhesion molecules. Predicted amino acid sequences of var genes show a variable extracellular segment with domains having receptor-binding features, a transmembrane sequence, and a terminal segment that is a probable submembrane anchor. There are 50-150 var genes on multiple parasite chromosomes, and some are in clustered arrangements. var probes detect two classes of transcripts in steady-state RNA: 7-9 kb var transcripts, and an unusual family of 1.8-2.4 kb transcripts that may be involved in expression or rearrangements of var genes.
We have studied the surface-antigen phenotypes of infected erythrocytes from children with acute falciparum malaria in the Punjab, Pakistan. Infected erythrocytes from 15 children and their homologous acute and convalescent sera, as well as hyperimmune sera from adults living in the same area, were used to analyse the serological diversity of parasite-associated antigens expressed on the surface of erythrocytes infected with Plasmodium falciparum. Analysis included agglutination, cytoadherence/inhibition of cytoadherence to melanoma cells, and rosette formation/disruption of rosettes formed between infected and non-infected erythrocytes. We found that isolate-specific antibodies were acquired by all children during convalescence and that there was a high degree of diversity of surface-antigen phenotypes. A majority of the sera either acquired or increased by 2- to 3-fold the capacity to inhibit cytoadherence and/or agglutinate the patient's own infected erythrocytes during convalescence and a strong correlation was found between these 2 activities. Hyperimmune sera from adults agglutinated and/or inhibited cytoadherence of many, but not all, of the isolates. Rosette formation was observed in 11 of the 15 isolates; 38% of the acute and 75% of the convalescent sera had anti-rosette activity on rosettes formed by the patient's own parasites. Among the isolates there was no correlation between rosette formation and binding of infected erythrocytes to melanoma cells. Our data provide good evidence that the children developed isolate-specific antibodies during convalescence from an acute attack of falciparum malaria and suggest that there was a high degree of heterogeneity of antigens expressed on the surface of infected erythrocytes.
Women are particularly susceptible to malaria during first and second pregnancies, even though they may have developed immunity over years of residence in endemic areas. Plasmodium falciparum-infected red blood cells (IRBCs) were obtained from human placentas. These IRBCs bound to purified chondroitin sulfate A (CSA) but not to other extracellular matrix proteins or to other known IRBC receptors. IRBCs from nonpregnant donors did not bind to CSA. Placental IRBCs adhered to sections of fresh-frozen human placenta with an anatomic distribution similar to that of naturally infected placentas, and this adhesion was competitively inhibited by purified CSA. Thus, adhesion to CSA appears to select for a subpopulation of parasites that causes maternal malaria.
Plasmodium falciparum infection in pregnant women frequently leads to placental infection and low birth weight (< 2,500 grams) of the infant, particularly in the areas of high malaria transmission found in sub-Saharan Africa. Low birth weight is widely known to be an important risk factor for early infant mortality. To reduce the risk that maternal infection poses to child survival, many antenatal clinic programs recommend and provide antimalarial chemoprophylaxis, often with chloroquine (CQ) as a recommended element for antenatal care. Prior to the 1980s, despite widespread advocacy for this intervention, little was known about the effect of this intervention strategy. As an introduction to the Mangochi Malaria Research Project, which examined the efficacy of several antimalarial regimens using CQ or mefloquine in pregnant women in Malawi, we describe the background of knowledge regarding malaria infection in pregnant African women and the important elements of an intervention and prevention strategy.
The protozoan Plasmodium falciparum causes lethal malaria. Adhesion of erythrocytes infected with P. falciparum to vascular endothelium and to uninfected red blood cells (rosetting) may be involved in the pathogenesis of severe malaria. The binding is mediated by the antigenically variant erythrocyte-membrane-protein-1 (PfEMP-1), which is encoded by members of the P. falciparum var gene family. The control of expression and switching of var genes seems to lack resemblance to mechanisms operating in variant gene families of other microbial pathogens. Here we show that multiple, distinct var gene transcripts (about 24 or more) can be detected by reverse transcription and polymerase chain reaction in bulk cultures of the rosetting parasite FCR3S1.2, despite the adhesive homogeneity of the cultures. We also detected several var transcripts in single erythrocytes infected with a ring-stage parasite of FCR3S1.2, and found that different var genes are transcribed simultaneously from several chromosomes in the same cell. In contrast, we detected only one var transcript, FCR3S1.2 var-1, which encodes the rosetting PfEMP-1 protein, in individual rosette-adhesive trophozoite-infected cells, and we found only one PfEMP-1 type at the erythrocyte surface by labelling with 125iodine and immunoprecipitation. We conclude that a single P. falciparum parasite simultaneously transcribes multiple var genes but, through a developmentally regulated process, selects only one PfEMP-1 to reach the surface of the host cell.
Women are at increased risk from malaria during pregnancy, and, for unknown reasons, this risk is greatest during the first pregnancy. Plasmodium falciparum, the most virulent of the four malaria parasites of humans, adheres to a molecule called chondroitin sulphate A (CSA) on the surface of syncytiotrophoblasts (cells lining the intervillous space) and sequesters in the human placenta. Here we show that anti-adhesion antibodies, which limit the accumulation of parasites in the placenta, appear in pregnant women from Africa and Asia who have been pregnant on previous occasions (multigravidas), but not in those who are pregnant for the first time (primigravidas). Anti-adhesion antibodies against CSA-binding parasites are strain-independent and are associated with greatly reduced prevalence and density of infection. We conclude that malaria susceptibility in primigravidas is related to the lack of these anti-adhesion antibodies, and that an anti-adhesion vaccine for maternal malaria may be globally effective.
Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.
The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (alpha, beta, and gamma) and DBL domains as five sequence classes (alpha, beta, gamma, delta, and epsilon). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations--such as DBLalphaCIDRalpha, DBLdeltaCIDRbeta, and DBLbetaC2--are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.
Plasmodium falciparum–infected erythrocytes adhere to syncytiotrophoblast cells lining the placenta via glycosaminoglycans, such as chondroitin
sulfate A (CSA) and hyaluronic acid. Adherence of infected erythrocytes to host receptors is mediated by P. falciparum erythrocyte membrane protein–1 (PfEMP-1). A single PfEMP-1 domain (duffy binding-like [DBL]–3, of the γ sequence class) from
laboratory-adapted strains is thought to be responsible for binding to CSA. In this study, DBL-γ domains expressed by placental
P. falciparum isolates were shown to have an affinity to CSA. All parasite populations accumulating in infected placentas express only
1 variant of PfEMP-1, each of which contains a DBL-γ domain with CSA binding capacities. Furthermore, sequence analysis data
provide evidence for antigenic conservation among the DBL-γ sequences expressed by different placental parasites. This study
offers a close reflection of the process of parasite adhesion in the placenta and is crucial to the understanding of the pathogenesis
of malaria during pregnancy
We sought genetic evidence for the importance of host-parasite interactions involving CD36 in severe malaria. We identified a non-sense mutation in Cd36 gene and looked at the influence of this mutation on the outcome of malaria infection in 693 African children with severe malaria and a similar number of ethnically matched controls. We showed that heterozygosity for this mutation is associated with protection from severe disease (OR 0.74, 95% CI 0.55-0.99; p=0.036). These findings suggest that this Cd36 mutation might have a complex effect on malaria infection by decreasing parasite sequestration, and also by decreasing host immune responses.
Otherwise clinically immune women in areas endemic for malaria are highly susceptible to Plasmodium falciparum malaria during their first pregnancy. Pregnancy-associated malaria (PAM) is characterized by placental accumulation of infected
erythrocytes that adhere to chondroitin sulfate A (CSA). Susceptibility to PAM decreases with increasing parity, apparently
due to acquisition of antibodies directed against the variant surface antigens (VSAs) that mediate the adhesion to CSA (VSACSA). This study found that levels of VSACSA-specific antibodies depend on endemicity, that anti-VSACSA IgG is acquired during gestation week 20, and that plasma levels of the antibodies decline during the postpartum period.
There is evidence that VSACSA-specific antibodies are linked to placental infection and that high antibody levels contribute to the control of placental
infection by inhibiting parasite adhesion to CSA. Data suggest that VSACSA is a target for vaccination against PAM
Many pathogens that either rely on an insect vector to complete their life cycle (e.g., Trypanosoma spp. and Borrelia spp.) or exist in a unique ecological niche where transmission from host to host is sporadic (e.g., Neisseria spp.) have evolved strategies to maintain infection of their mammalian hosts for long periods of time in order to ensure their survival. Because they have to survive in the face of a fully functional immune system, a common feature of many of these organisms is their development of sophisticated strategies for immune evasion. For the above organisms and for malaria parasites of the genus Plasmodium, a common theme is the ability to undergo clonal antigenic variation. In all cases, surface molecules that are important targets of the humoral immune response are encoded in the genome as multicopy, nonallelic gene families. Antigenic variation is accomplished by the successive expression of members of these gene families that show little or no immunological cross-reactivity. In the case of malaria parasites, however, some of the molecules that undergo antigenic variation are also major virulence factors, adding an additional level of complication to the host-parasite interaction. In this review, we cover the history of antigenic variation in malaria and then summarize the more recent data with particular emphasis on Plasmodium falciparum, the etiological agent of the most severe form of human malaria.
To approve a theoretical basis for the molecular pathogenesis of human cerebral malaria and treatment with prevention.
The blood samples were collected from 24 patients with cerebral malaria, 143 with falciparum malaria, 34 with vivax malaria and 20 healthy controls from the endemic areas of Yunnan Province, China. Using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique, we determined the molecular mass (Mr) of these Plasmodium falciparum (P. falciparum) erythrocyte membrane protein 1 (PfEMP1) molecules.
Our findings indicate that higher molecular mass (260 kDa-320 kDa) forms of PfEMP1 were expressed on parasitized erythrocyte (PE) from human cerebral malaria patients. Compared with PfEMP1 expressed on PE from human cerebral malaria patients, the expression of PfEMP1 and Plasmodium vivax (P. vivax) erythrocyte membrane protein 1 (PvEMP1) on PE from falciparum malaria patients and vivax malaria patients did not have multiple bands of PfEMP1 of > or = 260 kDa, but had a PfEMP1 with molecular mass of 240 kDa and a PvEMP1 with molecular mass of 180 kDa band separately. Healthy controls expressed an EMP of molecular mass of 140 kDa.
Results confirm the antigenic variation of higher molecular mass of PfEMP1 whose molecular mass is equal to or exceeds 260 kDa-320 kDa on PE of patients with cerebral malaria. Our results show that the binding of large antigenic variability PfEMP1 molecular mass of 260 kDa-320 kDa on PE from human cerebral malaria patients with diverse receptor molecules on the endothelial cell (EC) of the cerebral microvessels may be involved in the molecular pathogenesis of cerebral malaria.