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Plasmodium falciparum PfEMP1 DiQ-BioID data

ABSTRACT: PfEMP1 is the major virulence factor of Plasmodium falciparum parasites. PfEMP1 is expressed as an integral membrane protein on the surface of infected red blood cells. As a part of this project, the BioID system was utilized to biotinylate proximal proteins of a functional PfEMP1. In order to do this, selection linked integration (SLI) was used to modify a specific PfEMP1 (PfIT_060021400; IT4var01) by fusing it with the promiscuous biotin ligase BirA* and obtain a parasite population predominantly expressing this modified variant. Three separate IT4 cell lines were generated where BirA* was fused to different positions of PfEMP1. In the first cell line (Position 1), BirA* is C-terminal to the ATS domain of PfEMP1, in the second cell line (position 2), BirA* is between the ATS and the transmembrane domain and in the third cell line (position 3), BirA* is between the transmembrane and the extracellular binding domains. Biotinylated proteins extracted with either triton or SDS containing buffers (using sequential extraction of the same parasite pellet) were isolated in large scales and subsequently analyzed via mass spectrometry in three separate experiments for the three BioID cell line as well as for IT4 control parasites (parent). The experiments resulted in a set of enriched proteins for each of the 3 cell lines (position 1-3) over IT4 parasites.

INSTRUMENT(S):

ORGANISM(S): Plasmodium Falciparum

TISSUE(S): Cell Culture

SUBMITTER: Pascal Jansen

LAB HEAD: Tobias Spielmann

PROVIDER: PXD052297 | Pride | 2025-11-13

REPOSITORIES: Pride

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			Action	DRS
	20210902_Exploris1_SA_PJ_TSext_PfEMP1_X_condition1_1.raw	Raw
	20210902_Exploris1_SA_PJ_TSext_PfEMP1_X_condition2_1.raw	Raw
	20210902_Exploris1_SA_PJ_TSext_PfEMP1_X_condition3_1.raw	Raw
	20210902_Exploris1_SA_PJ_TSext_PfEMP1_X_control4_1.raw	Raw
	20210902_Exploris1_SA_PJ_TSext_PfEMP1_Y_condition1_2.raw	Raw

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Project description:Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterised by the accumulation of infected erythrocytes in the microvasculature of the brain, due to parasite adhesins on the surface of infected erythrocytes binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We used the Human Brain Endothelial Cell line HBEC-5i to identify the malaria parasite ligands responsible for binding to human brain endothelial cells. Three P. falciparum strains (HB3, 3D7 and IT/FCR3) were selected for binding to HBEC5i and the whole transcriptome of selected and unselected parasites was analysed using a variant surface antigen-supplemented microarray chip. After selection, the only highly upregulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7 and ITvar19), that showed 11 to >100-fold higher transcription levels in selected parasites. These genes are highly diverse in sequence, but do however show strong similarities in PfEMP1 architecture. Antibodies raised to the HB3var3 variant recognized the surface of infected erythrocytes and abolished the binding of infected erythrocytes to brain endothelial cells. The subset of Group A PfEMP1 variants identified here provides a new target for interventions to treat or prevent cerebral malaria. Unselected Plasmodium falciparum parasites (Uns) only poorly cytoadhere to human brain endothelial cells (HBEC-5i). Plasmodium falciparum HB3, 3D7 and IT/FCR3 strains were selected for binding to HBEC-5i (HB3-HBEC1, HB3-HBEC2, HB3-HBEC-TNF, 3D7-HBEC and IT-HBEC). HBEC-selected and unselected cultures were tightly synchronised before a timecourse experiment was performed. 6 samples, named time points 1 to 6, were taken every 8 hours. 12μg of RNA from the unselected parasites (HB3-Uns, 3D7-Uns or IT-Uns) at each of the 6 time points was combined together to form the reference pool. The pool and 12μg of each individual time point sample from both selected and unselectedparasites were then used for cDNA synthesis. For HB3-HBEC1 (pilot experiment), each HB3-HBEC1 time point (pilot experiment) was hybridised directly with HB3-Uns1 time points. For microarray hybridizations, each cDNA sample was coupled to Cy5 (red dye) while Cy3 (green dye) was added to the pool. Cy5-labelled time point samples were mixed with the same amount of Cy3-labelled pool sample. The solution was loaded on a microarray slide and hybridized for 14–16 h.

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Plasmodium falciparum PfEMP1 DiQ-BioID data

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