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NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

ABSTRACT: Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

SUBMITTER: Vignali M

PROVIDER: S-EPMC3046638 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

Vignali Marissa M Armour Christopher D CD Chen Jingyang J Morrison Robert R Castle John C JC Biery Matthew C MC Bouzek Heather H Moon Wonjong W Babak Tomas T Fried Michal M Raymond Christopher K CK Duffy Patrick E PE

The Journal of clinical investigation 20110207 3

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. ...[more]

PMID: 21317536

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Project description:BACKGROUND:The transcriptome of Plasmodium falciparum clinical isolates varies according to strain, mosquito bites, disease severity and clinical history. Therefore, it remains a challenge to directly interpret the parasite's transcriptomic information into a more general biological signature in a natural human malaria infection. These confounding variations can be potentially overcome with parasites derived from controlled-human malaria infection (CHMI) studies. METHODS:We performed CHMI studies in healthy and immunologically naïve volunteers receiving the same P. falciparum strain ((Sanaria® PfSPZ Challenge (NF54)), but with different sporozoite dosage and route of infection. Parasites isolated from these volunteers at the day of patency were subjected to in vitro culture for several generations and synchronized ring-stage parasites were subjected to transcriptome profiling. FINDINGS:We observed clear deviations between CHMI-derived parasites from volunteer groups receiving different PfSPZ dose and route. CHMI-derived parasites and the pre-mosquito strain used for PfSPZ generation showed significant transcriptional variability for gene clusters associated with malaria pathogenesis, immune evasion and transmission. These transcriptional variation signature clusters were also observed in the transcriptome of P. falciparum isolates from acute clinical infections. INTERPRETATION:Our work identifies a previously unrecognized transcriptional pattern in malaria infections in a non-immune background. Significant transcriptome heterogeneity exits between parasites derived from human infections and the pre-mosquito strain, implying that the malaria parasites undergo a change in functional state to adapt to its host environment. Our work also highlights the potential use of transcriptomics data from CHMI study advance our understanding of malaria parasite adaptation and transmission in humans. FUND: This work is supported by German Israeli Foundation, German ministry for education and research, MOE Tier 1 from the Singapore Ministry of Education Academic Research Fund, Singapore Ministry of Health's National Medical Research Council, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA and the German Centre for Infection Research (Deutsches Zentrum für Infektionsforschung-DZIF).

Project description:Background: The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes to each VSA gene. Methods: All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray. Results: From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly upregulated gene in rosetting parasites was ITvar9 (19 to 429-fold upregulated over six time points). Only one rif gene (IT4rifA_042) was upregulated by more than 4-fold (5-fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were upregulated by more than 2-fold. 49 non-VSA genes were upregulated in rosetting parasites by more than 3-fold in at least two time-points, although none as markedly as ITvar9. Conclusions: We demonstrate that the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation.

Dataset Information

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

Publications

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

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