Transcriptomic analysis of Plasmodium relapsing species identifies potential regulators of hypnozoite commitment and maintenance

Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual P. cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain high quality transcriptomes of these stages. Multiple parasite metabolic pathways were up- or down- regulated in the hypnozoites when compared to liver stage schizonts, leading to the identification of potential biomarkers of this quiescent state. These included three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a list of potential molecular markers of hypnozoites but also identifies regulation of gene expression as being key to the establishment and maintenance of quiescence.