Molecular mechanisms of Plasmodium falciparum placental adhesion

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA-binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood-stage cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients.     

Comparative proteomic analysis of metabolically labelled proteins from Plasmodium falciparum isolates with different adhesion properties

Background

Malaria is a huge public health problem in Africa that is responsible for more than one million deaths annually. In line with the Roll Back Malaria initiative and the Abuja Declaration, Eritrea and other African countries have intensified their fight against malaria. This study examines the impact of Eritrea's Roll Back Malaria Programme: 2000–2004 and the effects and possible interactions between the public health interventions in use.

Methods

This study employed cross-sectional survey to collect data from households, community and health facilities on coverage and usage of Insecticide-Treated Nets (ITNs), Indoor Residual Spraying (IRS), larvicidal activities and malaria case management. Comparative data was obtained from a similar survey carried out in 2001. Data from the Health Management Information System (HMIS) and reports of the annual assessments by the National Malaria Control Programme was used to assess impact. Time series model (ARIMA) was used to assess association.

Results

In the period 2000–2004, approximately 874,000 ITNs were distributed and 13,109 health workers and community health agents were trained on malaria case management. In 2004, approximately 81% households owned at least one net, of which 73% were ITNs and 58.6% of children 0–5 years slept under a net. The proportion of malaria cases managed by community health agents rose from 50% in 1999 to 78% in 2004. IRS coverage increased with the combined amount of DDT and Malathion used rising from 6,444 kg, in 2000 to 43,491 kg, in 2004, increasing the population protected from 117,017 to 259,420. Drug resistance necessitated regimen change to chloroquine plus sulfadoxine-pyrimethamine. During the period, there was a steep decline in malaria morbidity and case fatality by 84% and 40% respectively. Malaria morbidity was strongly correlated to the numbers of ITNs distributed (β = -0.125, p < 0.005) and the amount (kg) of DDT and Malathion used for IRS (β = -2.352, p < 0.05). The correlation between malaria case fatality and ITNs, IRS, population protected and annual rainfall was not statistically significant.

Conclusion

Eritrea has within 5 years attained key Roll Back Malaria targets. ITNs and IRS contributed most to reducing malaria morbidity.     

Drug susceptibility of Plasmodium falciparum from polyclonal isolates

Msp-1 and Msp-2 genes, each present as a unique copy in the genome of Plasmodium, contain polymorphic repeats in bloc 2. We studied allelic polymorphism of Msp-1 and Msp-2 by amplifying bloc 2 with a fluorescent primer, and analysing the fragment generated. We validated this method by mixing two cloned strains: chloroquine-susceptible HB3-Honduras and chloroquine-resistant FCM29-Cameroon. This method was then used to quantify the clones in natural isolates of 19 infected persons during quinine treatment. The fragment analysis method detects efficiently clone numbers and the proportions of each in isolates.     

Plasmodium falciparum: chondroitin sulfate A is the major receptor for adhesion of parasitized erythrocytes in the placenta

Plasmodium falciparum parasites that sequester in the placenta bind to the molecule chondroitin sulfate A (CSA). Women become resistant to malaria during pregnancy as they acquire antibodies that inhibit parasite adhesion to CSA, suggesting that a vaccine against placental malaria is feasible. Hyaluronic acid (HA) and non-immune IgG have also been proposed as receptors for P. falciparum adhesion in the placenta, but evidence for their roles is inconclusive. In this study, CSA, HA, and IgG were simultaneously assessed for their relative contributions to placental adhesion. Placental parasites collected in Tanzania uniformly adhered to the molecule CSA, and soluble CSA completely inhibited adhesion of most samples to placental cryosections. Three of 46 placental parasite samples also adhered to immobilized HA, but HA failed to inhibit adhesion of any placental parasites to placental cryosections. Similarly, non-immune IgG and protein A failed to inhibit adhesion of parasite samples to placental cryosection. P. falciparum adhesion in the placenta appears to be a non-redundant process that requires CSA as a receptor. Vaccines that elicit functional antibodies against CSA-binding parasites may confer resistance to pregnancy malaria.     

Plasmodium falciparum: carbohydrates as receptor sites of invasion

Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.     

Divergent binding sites on intercellular adhesion molecule-1 (ICAM-1) for variant Plasmodium falciparum isolates

Adhesion of human erythrocytes infected with the malaria parasite Plasmodium falciparum to host endothelium has been associated with severe forms of this disease. A number of endothelial receptors have been identified, and there is evidence that one of these, intercellular adhesion molecule-1 (ICAM-1), may play an important role in the pathology of cerebral malaria. Mutagenesis of domain 1 of ICAM-1, which is involved in parasite adhesion, shows that the binding sites for different parasite variants overlap to a large extent, but that there are subtle differences between them that correlate with their adhesive phenotypes. This suggests that the ability to bind to ICAM-1 has arisen from a common variant, but that subsequent changes have led to differences in binding avidity, which may affect pathogenesis. The definition of common binding determinants and the elucidation of links between ICAM-1 binding phenotype and disease will provide new leads in the design of therapeutic interventions.     

Intranuclear structures in pyrimethamine-resistant isolates of the malaria parasite Plasmodium falciparum

The ultrastructure of the malaria parasite Plasmodium falciparum is well known, both from natural infections and from culture material ( Aikawa , 1977, Langreth et al., 1978). It is noteworthy that all of these studies were done with pyrimethamine-sensitive strains, e.g. FCR-3/Gambia. Except for spindle microtubules during schizogony, no intranuclear structures have been described in any of the asexual erythrocytic stages. In the course of isolating clones from the pyrimethamine-resistant strain Honduras I/CDC (V.K. Bhasin and W. Trager , in print) and checking by electron microscopy for the presence or absence of knobs, we noticed intranuclear structures that might be correlated with pyrimethamine resistance. For comparison, we then examined the multi-drug-resistant strain Indochina 1. We present here a first report on these structures as a basis for further studies.     

A single member of the Plasmodium falciparum var multigene family determines cytoadhesion to the placental receptor chondroitin sulphate A

In high-transmission regions, protective clinical immunity to Plasmodium falciparum develops during the early years of life, limiting serious complications of malaria in young children. Pregnant women are an exception and are especially susceptible to severe P. falciparum infections resulting from the massive adhesion of parasitized erythrocytes to chondroitin sulphate A (CSA) present on placental syncytiotrophoblasts. Epidemiological studies strongly support the feasibility of an intervention strategy to protect pregnant women from disease. However, different parasite molecules have been associated with adhesion to CSA. In this work, we show that disruption of the var2csa gene of P. falciparum results in the inability of parasites to recover the CSA-binding phenotype. This gene is a member of the var multigene family and was previously shown to be composed of domains that mediate binding to CSA. Our results show the central role of var2CSA in CSA adhesion and support var2CSA as a leading vaccine candidate aimed at protecting pregnant women and their fetuses.     

Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor

Cytoadhesion of Plasmodium falciparum‐infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C–EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine‐rich interdomain region (CIDRα1) domain from a variety of domain cassette (DC) 8 and DC13 P. falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)–EPCR interaction. Overall, there was a positive correlation between CIDRα1–EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti‐EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.     

Plasmodium falciparum: differentiation of isolates with DNA hybridization using antigen gene probes

Chromosomal DNA was prepared from seven Plasmodium falciparum isolates that had been cultured in vitro and from a cloned P. falciparum line. The DNA was cleaved with restriction endonucleases, fractionated by agarose gel electrophoresis, blotted to nitrocellulose, and hybridized with a series of radioactively labeled DNA probes. The probes had been derived from cDNA clones encoding portions of P. falciparum antigens. Simple, reproducible band patterns that differed for many of the isolates were obtained. Parasite isolates collected from different continents could be readily distinguished, as could some but not all isolates collected from one restricted region of Papua New Guinea. Application of this technique for the identification and differentiation of parasite strains was explored. The patterns of hybridization observed were consistent with the proposition that blood stages of P. falciparum have a haploid genome.     

Impact of placental Plasmodium falciparum malaria on pregnancy and perinatal outcome in sub-Saharan Africa: I: introduction to placental malaria

Placental malaria is one of the major features of malaria during pregnancy and has been widely used as a standard indicator to characterize malaria infection in epidemiologic investigations. Although pathogenesis of placental malaria is only partially understood, placental sequestration of Plasmodium falciparum results in the accumulation of parasitized erythrocytes in the intervillous space, infiltration by inflammatory cells, and release of pro-inflammatory mediators, which cause pathologic alterations that could impair materno-fetal exchanges, often resulting in adverse pregnancy outcome. In this report, the impact of placental malaria on pregnancy and perinatal outcome is reviewed using data from studies conducted in sub-Saharan Africa. Generally, placental malaria was associated with increased risk of maternal anemia, HIV infection, and maternal mortality, with younger women and primigravidae more likely to be affected. A variety of adverse perinatal outcomes, including low birth weight, preterm delivery, intrauterine growth retardation, reduced fetal anthropometric parameters, fetal anemia, congenital malaria, increased mother-to-child HIV transmission, and perinatal mortality, were associated with placental malaria. There were, however, conflicting reports on whether the risk of these adverse perinatal outcomes associated with placental malaria were statistically significant. There is a clear need to strengthen the malaria prevention and intervention measures for pregnant women in sub-Saharan Africa.     

Plasmodium falciparum: diversity studies of isolates from two Colombian regions with different endemicity

The population structure of Plasmodium falciparum has been widely studied in diverse epidemiological contexts, but emphasis has been made in regions with high and stable transmission. In order to establish the genetic structure of P. falciparum in areas of Colombia with different degree of endemicity, we studied 100 samples from malaria patients of two different municipalities. The frequency of multiclonal infection in these areas and the correlation with the endemicity were carried out by comparison of the amplified products from polymorphic segments of MSP-1, MSP-2, and GLURP genes. We found low size polymorphism of the studied genes: 1 MSP-1 allele, 3 MSP-2 alleles, and 4 GLURP alleles. We conclude that the P. falciparum population in the regions studied is genetically homogeneous.     

Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites

Surface proteins from Plasmodium falciparum are important malaria vaccine targets. However, the surface proteins previously identified are highly variant and difficult to study. We used tandem mass spectrometry to characterize the variant antigens (Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)) expressed on the surface of malaria-infected erythrocytes that bind to chondroitin sulfate A (CSA) in the placenta. Whereas PfEMP1 variants previously implicated as CSA ligands were detected, in unselected parasites four novel variants were detected in CSA-binding or placental parasites but not in unselected parasites. These novel PfEMP1 variants require further study to confirm whether they play a role in placental malaria.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Plasmodium falciparum: genetic polymorphism in apical membrane antigen-1 gene from Indian isolates

A number of stage-specific antigens have been characterized for vaccine development against Plasmodium falciparum malaria. This study presents a comprehensive analysis of the sequence polymorphism in Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) in population samples from the eastern and western parts of India. This is the first study of its kind for the nearly full length PfAMA-1 gene from these regions in India. Our observations confirmed that sequence diversity of PfAMA-1 confines only to point mutations and shows 4-8% variation as compared to the prototypes. As opposed to the previous studies on PfAMA-1, our study revealed a greater degree of polymorphism in the Domain II region of PfAMA-1 protein, though signature for diversifying selection is seen throughout the gene. Our present investigation also indicates a very high degree of variation in the reported T- and B-cell epitopes of PfAMA-1. Few noteworthy and unique observations made in this study are the substitution of Cysteine residues responsible for the disulfide bond structure of the protein and the presence of premature termination after 595 amino acids in 3 of the 13 isolates under consideration. These crucial findings add new perspectives to the future of AMA-1 research and could have major implications in establishing AMA-1 as a vaccine candidate.