Laser capture microdissection enables transcriptomic analysis of dividing and quiescent liver stages of Plasmodium relapsing species

Hypnozoites, the dormant liver stage forms of the human malaria parasites Plasmodium vivax and P. ovale, present major hurdles to control and eradicate infection, especially in South America and South and South-East Asia. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. To fill this knowledge gap, we used a combination of state-of-the-art technologies to generate the transcriptome of hypnozoites derived from the monkey malaria parasite, P. cynomolgi. We developed a robust laser capture microdissection (LCM) protocol to isolate individual P. cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi, P. vivax and P. ovale, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. We are currently characterizing the roles of these factors in P. cynomolgi and P. vivax hypnozoite formation as well as applying LCM to perform single cell parasite transcriptomics of Plasmodium spp. liver stages. In conclusion, this work not only provides a primary experimentally-derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence.