Surface Of Plasmodium Falciparum-infected Erythrocytes Research Articles

An Improved Method for Assessing Antigen Presentation on the Surface of Plasmodium falciparum-Infected Erythrocytes by Immuno-Electron Microscopy.

Immuno-electron microscopy can detect and localize antigens in cells or tissues at a resolution of several nanometers. In the case of P. falciparum-infected erythrocytes, immuno-EM studies are frequently hampered by the electron-dense nature of the hemoglobin and access of antibodies to antigenic sites, particularly if the targeted protein is presented on the host cell surface or lies in proximity to the host cell cytoskeleton. Here, we describe an improved immuno-EM protocol that overcomes these problems. The improved signal to noise ratio and the enhanced access to antigenic sites now allows one to obtain information regarding target density and distribution and, hence, additional insights into the architecture and function of parasite-induced, or -affected, structures.

Assessing Antigen Presentation on the Surface of Plasmodium falciparum-Infected Erythrocytes by Photoactivated Localization Microscopy (PALM).

Super-resolution microscopy in the form of photoactivated localization microscopy (PALM) offers the possibility of counting single molecules in a cell, a cellular compartment or a molecular complex. PALM can, therefore, underpin molecular and biochemical processes with a numeric and stoichiometric understanding of the interacting players. Here, we introduce the physical principles underlying PALM and provide a step-by-step protocol of how to apply PALM to questions related to the biology and pathophysiology of P. falciparum and other malaria parasites.

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones

PfEMP1 is a family of adhesive proteins expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs), where they mediate adhesion of IEs to a range of host receptors. Efficient PfEMP1-dependent IE sequestration often depends on soluble serum proteins, including IgM. Here, we report a comprehensive investigation of which of the about 60 var gene-encoded PfEMP1 variants per parasite genome can bind IgM via the Fc part of the antibody molecule, and which of the constituent domains of those PfEMP1 are involved. We erased the epigenetic memory of var gene expression in three distinct P. falciparum clones, 3D7, HB3, and IT4/FCR3 by promoter titration, and then isolated individual IEs binding IgM from malaria-unexposed individuals by fluorescence-activated single-cell sorting. The var gene transcription profiles of sub-clones measured by real-time qPCR were used to identify potential IgM-binding PfEMP1 variants. Recombinant DBL and CIDR domains corresponding to those variants were tested by ELISA and protein arrays to confirm their IgM-binding capacity. Selected DBL domains were used to raise specific rat anti-sera to select IEs with uniform expression of candidate PfEMP1 proteins. Our data document that IgM-binding PfEMP1 proteins are common in each of the three clones studied, and that the binding epitopes are mainly found in DBLε and DBLζ domains near the C-terminus.

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

We previously described two donors in whom the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 191, was inserted between the V and the DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs)2. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. We report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to IEs without conferring enhanced protection against febrile malaria. By analyzing the antibody-producing B cell clones at the protein, cDNA and gDNA level, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the gDNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientation and frame compatible with expression. Collectively, these results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against IEs and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Production, crystallization and X-ray diffraction analysis of two nanobodies against the Duffy binding-like (DBL) domain DBL6ℇ-FCR3 of the Plasmodium falciparum VAR2CSA protein.

The VAR2CSA protein has been closely associated with pregnancy-associated malaria and is recognized as the main adhesin exposed on the surface of Plasmodium falciparum-infected erythrocytes. Chondroitin sulfate A was identified as the main host receptor in the placenta. Single-domain heavy-chain camelid antibodies, more commonly called nanobodies, were selected and produced against the DBL6ℇ-FCR3 domain of VAR2CSA. Crystals of two specific nanobodies, Nb2907 and Nb2919, identified as strong binders to DBL6ℇ-FCR3 and the full-length VAR2CSA exposed on the surface of FCR3 P. falciparum-infected erythrocytes, were obtained. Crystals of Nb2907 diffract to 2.45 Å resolution and belong to space group C2 with unit-cell parameters a=136.1, b=78.5, c=103.4 Å, β=118.8°, whereas Nb2919 crystals diffract to 2.15 Å resolution and belong to space group P4₃2₁2 with unit-cell parameters a=b=62.7, c=167.2 Å.

A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three‐dimensional tomography in the caveola–vesicle complexes (Schüffner's dots) of infected erythrocytes is a member of the PHIST family

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81(95) and PcyPHIST/CVC-81(95) , analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81(95) is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81(95) localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81(95) gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81(95) is essential for survival of these malaria parasites.

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.

Acquisition of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes during pregnancy

Pregnancy-associated malaria is characterized by Plasmodium falciparum adherence to chondroitin sulfate A (CSA) in placenta, through a particular variant surface antigen (VSA). VSA CSA-specific IgG are involved in protection against placental malaria. In order to assess the relationship between VSA CSA-specific antibody responses and parity as well as protection against placental malaria, the occurrence of P. falciparum infection was assessed in 306 pregnant women from a low malaria transmission area of Senegal. Anti-VSA CSA antibodies against three placental parasite isolates were measured by flow cytometry, at enrollment and delivery. Placental infection prevalence rates were highest in primigravidae, but no clear decreasing trend was observed from the second pregnancy onwards. Anti-VSA CSA antibody prevalence rates increased with parity. Both anti-VSA CSA antibody prevalence rates and levels increased during pregnancy only in women infected with P. falciparum. Although a single or a very limited number of P. falciparum infections were able to induce an anti-VSA CSA antibody response, the level or the quality of this response did not appear to confer protection against placental malaria infection.

Antibodies from malaria-exposed pregnant women recognize trypsin resistant epitopes on the surface of Plasmodium falciparum-infected erythrocytes selected for adhesion to chondroitin sulphate A

BackgroundThe ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM).MethodsFluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for adhesion to CSA. The effect of proteolytic digestion of parasite erythrocyte surface antigens on surface IgG binding and adhesion to CSA and hyaluronic acid (HA) was also studied.ResultsP. falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved labelling technique for the detection of VSA expressed by CSA binding isolates has also been described.ConclusionThe VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P. falciparum erythrocyte surface protein that can be used as a malaria in pregnancy vaccine has not yet been achieved.

An alternative agglutination assay to measure antibodies to variant surfaceantigens of Plasmodium falciparum-infected erythrocytes

Agglutination assays detect antibodies to variant parasite antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes. Standard techniques require immediate analysis limiting the number of samples that can be processed simultaneously and preclude re-examination of slides. Fixed Giemsa-stained smears allow long-term storage and re-examination, without fluorescence microscopy.

Effect of var gene disruption on switching in Plasmodium falciparum.

The molecular mechanisms underpinning switching of variant antigens on the surface of Plasmodium falciparum-infected erythrocytes are poorly understood. We tested the hypothesis that insertional disruption of the A4var gene, one of two var genes located within the subtelomeric region of one end of chromosome 13, would result in a preferential switch in transcription to the adjacent R29var gene upon rosette selection. In this way, we aimed to mimic the preferential transcription of R29var in rosetting R29 parasites, a parasite line in which the A4var gene is deleted through a chromosome end truncation. Initial analysis of the knock-out parasite lines shows that the insertional disruption of the A4var gene prevents A4 PfEMP1 expression, but that switching transcription to other var gene variants is unaffected. Furthermore, analysis of var transcription in the knock-out parasite line during rosette selection shows that, rather than facilitating a switch to R29var gene transcription, this event was suppressed in the transfectants. These data, and the implications for epigenetic transcriptional control of var genes, are discussed.

Nonimmune IgM, but not IgG binds to the surface of Plasmodium falciparum-infected erythrocytes and correlates with rosetting and severe malaria.

Recent work suggests that IgG and IgM from nonimmune human serum (natural antibodies) bind to the surface of Plasmodium falciparum-infected erythrocytes and contribute to rosette formation by stabilizing the interaction between infected and uninfected erythrocytes. Here we show, in both laboratory clones and field isolates, that only IgM but not IgG is detected on the surface of infected cells. In field isolates, there was a strong positive correlation between IgM binding and rosette formation (Spearman's rank correlation coefficient p = 0.804, P < 0.001). Both rosette formation and IgM binding were associated with severe malaria, although statistical analysis indicates that rosette formation is the more strongly associated variable. Rosette formation, but not IgM binding, was also associated with malarial anemia. We conclude that IgM is the predominant class of natural antibodies binding to the surface of infected erythrocytes. However, we could not confirm previous suggestions that infected erythrocytes are coated with nonimmune IgG, which could lead to their interaction with host Fcgamma receptors.

Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum.

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

Multiple ligands for cytoadherence can be present simultaneously on the surface of Plasmodium falciparum-infected erythrocytes.

A major virulence factor of Plasmodium falciparum is the adherence of parasitized erythrocytes to the wall of postcapillary venules via a specific interaction between parasite-derived erythrocyte surface ligands and receptors on endothelial cells. To study this phenomenon in vitro, we selected a parasite population that expressed at least two different ligands and demonstrated that parasitized cells may coexpress ligands with specificity for multiple receptors. This selected parasite line had several antigenic and cytoadherence characteristics that were different from those of the parent line. Single parasitized erythrocytes were able to adhere to three distinct receptors via at least two separate ligands; a trypsin-sensitive molecule mediated cytoadherence to CD36 and intercellular adhesion molecule 1 and a trypsin-insensitive molecule(s) was responsible for adherence to a third receptor on the surface of melanoma cells. We present evidence that this newly discovered receptor for cytoadherence is an N-linked glycosaminoglycan, as treatment of melanoma cells with endoglycosidase H abolished cytoadherence. These observations emphasize the adaptability of P. falciparum and the complexity of the cytoadherence phenomenon.

Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum-infected erythrocytes.

We have identified strain-specific antigens with Camp and St. Lucia strains of P. falciparum of Mr approximately 285,000 and approximately 260,000, respectively. These strain-specific antigens were metabolically labeled with radioactive amino acids, indicating that they were of parasite origin rather than altered host components. These proteins had the properties of a molecule exposed on the surface of infected erythrocytes (IE). First, the proteins are accessible to lactoperoxidase-catalyzed radioiodination of IE. Second, the radioiodinated proteins were cleaved by low concentrations of trypsin (0.1 microgram/ml). Third, these antigens were immunoprecipitated after addition of immune sera to intact IE. Fourth, the strain-specific immuno-precipitation of these proteins correlated with the capacity of immune sera to block cytoadherence of IE in a strain-specific fashion. Fifth, the strain-specific antigen had detergent solubility properties (i.e., insolubility in 1% Triton X-100, solubility in 5% sodium dodecyl sulfate) similar to the variant antigen of P. knowlesi, which has been proven to be a malarial protein exposed on the erythrocyte surface.

Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes.

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.