Abstract
BackgroundEncoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40–60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates.MethodsWe developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data.ResultsUsing ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria.ConclusionETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1.ETHA is available at: https://sourceforge.net/projects/etha/.
Highlights
Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease
We show that all P. falciparum genomes from uncomplicated malaria infections harbor var2csa and var gene subclasses previously associated with severe malaria
The varying amount of data per sample depended on the percent host contamination, the variation associated with multiplexing samples in an Illumina run, the number of Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) cells sequenced, and the variation inherent to each SMRT cell (Additional file 1: Tables S1 and S2)
Summary
Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Acquired immunity to malaria appears to occur, at least in part, through the acquisition of antibodies to parasite antigens expressed on the surface of infected erythrocytes. Compared to other P. falciparum genes, most var genes exhibit extreme diversity, with less than 50% shared amino acid sequence identity [12]. Such diversity has proven a major obstacle to the development of strategies to amplify and sequence new var genes from clinical samples, hampering the understanding of var gene biology and the role of PfEMP1 in clinical disease. It has not been possible to sequence vars with adequate completeness and from a number of samples sufficient to associate specific var elements with clinical outcomes such as pregnancy-associated or severe malaria [14]
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