NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children
Marissa Vignali, … , Christopher K. Raymond, Patrick E. Duffy
Marissa Vignali, … , Christopher K. Raymond, Patrick E. Duffy
Published February 7, 2011
Citation Information: J Clin Invest. 2011;121(3):1119-1129. https://doi.org/10.1172/JCI43457.
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Technical Advance Infectious disease

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.

Authors

Marissa Vignali, Christopher D. Armour, Jingyang Chen, Robert Morrison, John C. Castle, Matthew C. Biery, Heather Bouzek, Wonjong Moon, Tomas Babak, Michal Fried, Christopher K. Raymond, Patrick E. Duffy

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Figure 1

Read allocation for the 4 samples analyzed by NSR-seq in this study.

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Read allocation for the 4 samples analyzed by NSR-seq in this study. The...
The numbers below the sample names correspond to the total number of reads obtained for each sample. The pie charts indicate the percentage of total reads that were unaligned (dark gray) and prefiltered (black) as well as those that had a unique match to parasite (white) or human sequences (white with vertical hatching), 2–30 matches to parasite (light gray) or human sequences (light gray with vertical hatching), 2–30 matches to human and parasite sequences (medium gray), or more than 30 matches (dotted). For reads with 30 or fewer matches, the lack of shading denotes reads that match parasite sequences exclusively, vertical hatching indicates reads that match human sequences exclusively, and stippling indicates reads that match both parasite and human sequences.