Abstract
Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol “DAFT-seq” to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.
Highlights
Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development
The first transcriptomic studies of P. vivax from field isolates and its comparison with other Plasmodium species were performed with microarrays, and showed similar cascades of stage-specific gene expression found in other Plasmodium species, where most genes show a clear peak of transcript at a specific time point in the intraerthyrocytic developmental cycle (IDC), with regulation presumed to involve ApiAP2 DNA-binding p roteins[10,11,12,13]
We generated a comprehensive list of enriched P. vivax schizont genes which provides the potential of a more in-depth understanding of P. vivax merozoite invasion, which to date is largely limited to a comparison with P. falciparum invasion homologs and a handful of known P. vivax invasion gene families such as the duffy binding like proteins (DBLs), reticulocyte binding proteins (RBPs), and merozoite surface proteins (MSPs)[19,20,21,22]
Summary
Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. A new reference genome from a Papua Indonesian isolate was released that combined short- and long-read sequencing technology, and made considerable improvements in assembly and annotation (PvP01) compared to Sal[1] This has greatly improved the understanding of P. vivax genome structure and read mapping for patient isolates[9]. We generated a comprehensive list of enriched P. vivax schizont genes which provides the potential of a more in-depth understanding of P. vivax merozoite invasion, which to date is largely limited to a comparison with P. falciparum invasion homologs and a handful of known P. vivax invasion gene families such as the duffy binding like proteins (DBLs), reticulocyte binding proteins (RBPs), and merozoite surface proteins (MSPs)[19,20,21,22]
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