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Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition.

ABSTRACT: The human malaria parasite Plasmodium falciparum can cause infected red blood cells (iRBC) to form rosettes with uninfected RBC, a phenotype associated with severe malaria. Rosetting is mediated by a subset of the Plasmodium falciparum membrane protein 1 (PfEMP1) variant adhesins expressed on the infected host-cell surface. Heparin and other sulfated oligosaccharides, however, can disrupt rosettes, suggesting that therapeutic approaches to this form of severe malaria are feasible. We present a structural and functional study of the N-terminal domain of PfEMP1 from the VarO variant comprising the N-terminal segment (NTS) and the first DBL domain (DBL1?(1)), which is directly implicated in rosetting. We demonstrate that NTS-DBL1?(1)-VarO binds to RBC and that heparin inhibits this interaction in a dose-dependent manner, thus mimicking heparin-mediated rosette disruption. We have determined the crystal structure of NTS-DBL1?(1), showing that NTS, previously thought to be a structurally independent component of PfEMP1, forms an integral part of the DBL1? domain. Using mutagenesis and docking studies, we have located the heparin-binding site, which includes NTS. NTS, unique to the DBL ?-class domain, is thus an intrinsic structural and functional component of the N-terminal VarO domain. The specific interaction observed with heparin opens the way for developing antirosetting therapeutic strategies.

SUBMITTER: Juillerat A

PROVIDER: S-EPMC3069207 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition.

Juillerat Alexandre A Lewit-Bentley Anita A Guillotte Micheline M Gangnard Stéphane S Hessel Audrey A Baron Bruno B Vigan-Womas Inès I England Patrick P Mercereau-Puijalon Odile O Bentley Graham A GA

Proceedings of the National Academy of Sciences of the United States of America 20110314 13

The human malaria parasite Plasmodium falciparum can cause infected red blood cells (iRBC) to form rosettes with uninfected RBC, a phenotype associated with severe malaria. Rosetting is mediated by a subset of the Plasmodium falciparum membrane protein 1 (PfEMP1) variant adhesins expressed on the infected host-cell surface. Heparin and other sulfated oligosaccharides, however, can disrupt rosettes, suggesting that therapeutic approaches to this form of severe malaria are feasible. We present a s ...[more]

PMID: 21402930

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Project description:BackgroundRosette-formation of Plasmodium falciparum parasitized erythrocytes is of importance in the development of severe malaria. The parasite-derived molecule PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), central to rosetting, is suggested to be included in a multimeric vaccine targeting severe disease.MethodsThree recombinant NTS-DBL1?-domains of PfEMP1 were generated in Escherichia coli, purified and used for immunization of rats and goats. Antibody titres were determined in ELISA assays and responses were compared in-between different individual animals and species. Reactivity with the parasites was tested in live pRBC using FACS. B-cell epitopes prediction was carried out in silico and compared to the results obtained by peptide microarray. Screening for serological cross-reactivity with heterologous NTS-DBL1? variants was carried out by ELISA, peptide array and FACS on pRBC of different laboratory strains and patient isolates.ResultsAll three NTS-DBL1?-domains induced high titres of antibodies that were biologically active with no apparent difference between constructs covering slightly different parts of the DBL1?-sequence. The different animal species showed comparable titres of antibodies, while variations within individuals of the species could be observed.Mapping of the recognized epitopes revealed that most parts of the molecule were able to induce an antibody response with a tendency for the N and C terminal parts of the molecule for slightly higher recognition. Important differences to the epitopes predicted were found as some of the most conserved parts of the DBL1?-domain contained the main epitopes for antibody reactivity. ELISA assays and peptide microarray demonstrated substantial cross-reactivity to heterologous variants, while binding to native PfEMP1 was observed only in few combinations on the pRBC surface, underlining that mainly internal, conserved and not surface exposed parts of the DBL1?-domain are responsible for this observation.ConclusionBiologically active antibodies can be induced consistently, with high titres, in different animal species and the antibodies elicited by different constructs react with similar epitopes. Induced antibodies recognize epitopes localized in all subdomains of the DBL1?-sequence. Cross-reactivity between NTS-DBL1?-variants is common in ELISA, but rare with live pRBC emphasizing that also internal, conserved areas of PfEMP1 carry important highly immunogenic epitopes of the molecule.

Project description:BackgroundRosetting and cytoadherence of Plasmodium falciparum-infected red blood cells have been associated with severity of malaria. ICAM-1 and CD36 are the main host cell receptors, while PfEMP1-DBLalpha is a major parasite ligand, which can contribute to rosette formation. This study is aimed at demonstrating whether the highly polymorphic PfEMP1-DBLalpha sequences occurring among Thai isolates causing severe and uncomplicated malaria are associated with their ability to form rosettes and reflected the clinical outcome of the patients.MethodsTwo hundred and ninety five PfEMP1-DBLalpha sequences from Thai clinical isolates causing severe and uncomplicated malaria were evaluated by sequencing and direct comparison using the specific text string analysis functions in Microsoft Excel and Perl. The relationships between the PfEMP1-DBLalpha sequences were also analysed by network analysis. The binding abilities of parasitized red blood cells (PRBCs) to CD36, wild type ICAM-1, ICAM-1Kilifi and ICAM-1S22/A under static condition were included.ResultsTwo hundred and eighty one non-identical amino acid sequences were identified (< 95% sequence identity). When the distributions of semi-conserved features (PoLV1-4 and sequence group) within the rosetting domain PfEMP1-DBLalpha were observed, close similarity was found between isolates from the two disease groups. The sequence group 1 representing uncomplicated malaria was significantly different from the sequence group 3 representing the majority of severe malaria (p = 0.027). By using a simple non-phylogenetic approach to visualize the sharing of polymorphic blocks (position specific polymorphic block, PSPB) and cys/PoLV among DBLalpha sequences, the sequence group 1 was split from the other five sequence groups. The isolates belonging to sequence group 5 gave the highest mean rosetting rate (21.31%). However, within sequence group 2 and group 6, the isolates causing severe malaria had significantly higher rosetting rate than those causing uncomplicated malaria (p = 0.014, p = 0.007, respectively).ConclusionThis is the first report of PfEMP1-DBLalpha analysis in clinical Thai isolates using semi-conserved features (cys/PoLV and PSPBs). The cys/PoLV group 5 gave the highest rosetting rate. PfEMP1-DBLalpha domains in Thai isolates are highly diverse, however, clinical isolates from severe and uncomplicated malaria shared common sequences.

Dataset Information

Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition.

Publications

Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition.

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