Unknown,Transcriptomics,Genomics,Proteomics

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Different PfEMP1-expressing Plasmodium falciparum variants induce divergent endothelial transcriptional responses during cytoadherence

ABSTRACT: The pathogenesis of severe malaria is complex and involves several pathways that influence host inflammation and endothelial function. The human malaria parasite Plasmodium falciparum is responsible for the majority of mortality and morbidity caused by malaria infection and differs from other human malaria species in the degree of accumulation of parasite-infected red blood cells in the microvasculature, known as cytoadherence or sequestration. In P. falciparum, cytoadherence is mediated by a protein called PfEMP1 which, due to its exposure to the host immune system, undergoes antigenic variation resulting in the expression of different PfEMP1 variants on the infected erythrocyte membrane. These PfEMP1s contain various combinations of adhesive domains, which allow for the differential engagement of a repertoire of endothelial receptors on the host microvasculature, with specific receptor usage associated with severe disease. Cytoadherence results in perturbation of the micro-circulation as well as direct effects on endothelial cells promoted by receptor-mediated signalling. We used a co-culture model of cytoadherence incubating human brain microvascular endothelial cells with erythrocytes infected with two parasite lines expressing different PfEMP1s; IT4var14 (long-form; ups B) that binds strongly to human brain microvascular endothelial cells mainly via ICAM-1, and IT4var 37 (short-form; ups C) that does not bind brain endothelium but shows high levels of binding to human dermal microvascular endothelial cells via CD36. We determined the transcriptional profile of the endothelial cells following different incubation periods with infected erythrocytes, identifying different transcriptional profiles of pathways involved in the pathology of severe malaria, such as inflammation, apoptosis and barrier integrity, induced by the two PfEMP1 variants.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Homo sapiens

SUBMITTER: Leo Zeef

PROVIDER: E-MTAB-12730 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In temperate and sub-tropical regions of Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. This can be attributed to many factors: one of which is the expression of clonally variant families of adhesion proteins: PfEMP1 on the iRBC surface. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variable surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison of PFC and PFU) have been observed. The exportome has been analyzed using similar parameters. GO term based functional enrichment of differentially regulated genes identified, up-regulated genes stastically enriched (p<0.5) to critical biological processes. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group A and B genes whose proteins are predicted to interact with CD36 receptor in the host as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variable surface antigens and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention. Plasmodium falciparum isolates were collected from patients (n=12) with differing clinical conditions. The patients exhibited symptoms categorized as uncomplicated (n=5) or complicated malaria (n=7). Criteria for determination of complicated disease were based on World Health Organization year 2000 guidelines. Microarray array based transcriptional profiling was carried out to find out differentailly expressed genes in complicated malaria isolates (non-cerebral malaria) compared to un-complicated malaria isolates.

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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Different PfEMP1-expressing Plasmodium falciparum variants induce divergent endothelial transcriptional responses during cytoadherence

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