Abstract
BackgroundDuring the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.MethodsWe developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy.ResultsWe demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.ConclusionsOur study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0110-6) contains supplementary material, which is available to authorized users.
Highlights
During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood
Genes in the nCounter® custom code set included genes that can distinguish between intraerythrocytic life cycle stages, highly differentially expressed genes from previous studies [8,9], and probes representing genes of interest expressed in gametocytes, heat shock, citric acid cycle, glycolysis, ATP binding, ubiquitin pathways, acyl CoA pathways, and others
Genes selected for the probe set included genes that can distinguish between intraerythrocytic life cycle stages, highly differentially expressed genes from previous in vivo studies [8,9], landmark genes for imputation, and other genes of interest
Summary
During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge. Low parasite RNA abundance, poor quality RNA, and the presence of human RNA in tissue with sequestered parasites have made approaches to measure malaria gene expression in the genome-wide scale inaccessible to date
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