| 1. |
- Chan, Sherwin, et al.
(författare)
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Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor
- 2017
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Ingår i: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.
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| 2. |
- Ch'ng, Jun-Hong, et al.
(författare)
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Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.
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| 3. |
- Quintana, Maria del Pilar, et al.
(författare)
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Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface
- 2018
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection.
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| 4. |
- Quintana, Maria del Pilar, et al.
(författare)
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SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion
- 2018
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 13:8
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Tidskriftsartikel (refereegranskat)abstract
- Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.
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| 5. |
- Quintana Varon, Maria del Pilar
(författare)
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Surface antigens in Plasmodium falciparum malaria : PfEMP1 and SURFIN4.2
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Plasmodium falciparum malaria is an infectious disease that on despite of the ongoing eradication efforts is still endemic in more than 100 countries, sometimes causing severe disease that leads to the death of around half a million people per year. Malaria pathology is tightly associated with the parasite cycle inside the human red blood cells (RBCs). Central to this cycle is the initial invasion by the merozoite and the extensive RBC modifications induced by the parasite, transporting proteins to the RBC cytoplasm and membrane. The P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) transported to the surface of the parasitized RBC (pRBC) and the surface-associated interspersed protein 4.2 (SURFIN4.2) present both at the pRBC surface as well as at the merozoite apex and surface, are the major focus of this thesis. PfEMP1 is the major surface antigen and mediates rosetting (binding of parasitized RBCs (pRBCs) to two or more RBCs), a parasite phenotype associated with the development of severe disease. The most N-terminal segment of this protein (the NTSDBL1α domain) has been identified as the ligand for rosetting and naturally acquired antibodies targeting this particular protein protect against severe disease development. In this study we wanted to address the specific regions in PfEMP1 and in other protein targets recognized by rosette-disrupting antibodies (generated upon immunization with recombinant PfEMP1 or naturally acquired during P. falciparum infection). We also wanted to explore other functional roles of these antibodies. A panel of antibodies (monoclonal and polyclonal) against rosette-mediating NTS-DBL1α domains was produced by animal immunization. The antibodies were analyzed with particular attention to their capacity to recognize the surface of the pRBC, disrupt the rosettes formed by homologous parasites and induce phagocytosis by monocytic cells. Additionally, the specific epitopes recognized by the majority of these antibodies were successfully mapped to a specific region of subdomain 3 (SD3) of the DBL1α domain, regardless of the parasite strain used. These results suggested this region as a major target of anti-rosetting antibodies. Most of these antibodies also induced opsonization for phagocytosis, a role that could be of great importance during pRBCs clearance in vivo. Interestingly, some of the antibodies with high opsonizing activity did not disrupt rosettes, indicating that other epitopes besides those involved in rosetting are exposed on the pRBC surface and are able to induce functional antibodies that could provide protection. The naturally acquired antibodies in sera from children living in a malaria endemic region were also investigated. The ability of these antibodies to recognize three parasite-derived surface proteins (PfEMP1, RIFIN-A and SURFIN4.2) was assessed. Different variables were also measured in the presence of these sera samples, including rosetting rate, surface reactivity and opsonization for phagocytosis on a rosetting model parasite grown in group O or group A RBCs. The data showed that the acquired immune response developed during natural infection could recognize the pRBC surface and more importantly could induce pRBC phagocytosis and in a few cases disrupt the rosettes formed by a heterologous parasite model. These activities however had limited access to the pRBCs inside a rosette formed with group A RBCs, where these cells act as a shield for the pRBCs, protecting it from antibodies’ recognition therefore impairing their effector function. This study also suggested that SURFIN4.2 previously identified at the pRBC surface could be involved in rosette formation, either as a direct ligand or as an accessory element for rosette strengthening. The suggestion of SURFIN4.2 as a possible mediator in rosetting prompted us to deepen the study of this protein, however, the initial results steered the approach to this protein from the rosetting phenomenon towards a more striking and understudied role of this protein during the invasion process. Using antibodies against the N-terminus, the protein was observed at the surface of the merozoite but more strikingly also in the neck of the rhoptries. The protein was shed into culture supernatant upon schizont rupture and was associated with GLURP (Glutamate Rich Protein) and RON-4 (Rhoptry Neck Protein 4) to form a complex we named SURGE (SURFIN4.2-RON-4-GLURP complEx). Importantly, SURFIN4.2 was detected at the apex of the merozoite during merozoite initial attachment and active invasion into the RBCs. The exact functional role of SURGE remains to be determined, but the presence of RON-4, a protein confined to the moving junction (MJ), strongly suggests a role in strengthening the stable contact between the merozoite apex and the RBC, possibly as and additional RBC adhesion molecule. Supporting the involvement of the protein complex during the invasion process, antibodies against the N-terminus of SURFIN4.2 partially inhibited invasion.
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