Dataset Information

Proteomics pipeline for identifying variant proteins in Plasmodium falciparum parasites isolated from children presenting with malaria

ABSTRACT: Plasmodium falciparum variant antigens named erythrocyte membrane protein 1 (PfEMP1) are important targets for developing a protective immunity to malaria caused by P. falciparum. One of the major challenges in P. falciparum proteomics studies is identifying PfEMP1s at the protein level due to antigenic variation. To identify these PfEMP1s using shotgun proteomics, we developed a pipeline that searches high resolution mass spectrometry spectra against a custom protein sequence database. An algorithm, LAX, was developed as part of the pipeline that matches sequences to the most similar PfEMP1 and calculates a weight value used for PfEMP1 protein inference. The pipeline was first validated in the analysis of a laboratory strain with a known PfEMP1, then it was implemented on the analysis of parasite isolates from malaria-infected pregnant women and finally on the analysis of parasite isolates from malaria infected children where there was an increase of PfEMP1s identified in 28 out of 31 isolates.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Patricia Gonzales Hurtado

LAB HEAD: Michal Fried

PROVIDER: PXD012958 | Pride | 2019-09-27

REPOSITORIES: Pride

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Publications

Proteomics Pipeline for Identifying Variant Proteins in Plasmodium falciparum Parasites Isolated from Children Presenting with Malaria.

Gonzales Hurtado Patricia A PA Morrison Robert R Ribeiro Jose M C JMC Magale Hussein H Attaher Oumar O Diarra Bacary S BS Mahamar Almahamoudou A Barry Amadou A Dicko Alassane A Duffy Patrick E PE Fried Michal M

Journal of proteome research 20191008 11

Plasmodium falciparum variant antigens named erythrocyte membrane protein 1 (PfEMP1) are important targets for developing a protective immunity to malaria caused by P. falciparum. One of the major challenges in P. falciparum proteomics studies is identifying PfEMP1s at the protein level due to antigenic variation. To identify these PfEMP1s using shotgun proteomics, we developed a pipeline that searches high-resolution mass spectrometry spectra against a custom protein sequen ...[more]

PMID: 31549843

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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.

Dataset Information

Proteomics pipeline for identifying variant proteins in Plasmodium falciparum parasites isolated from children presenting with malaria

Publications

Proteomics Pipeline for Identifying Variant Proteins in <i>Plasmodium falciparum</i> Parasites Isolated from Children Presenting with Malaria.

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