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

Abstract

SummaryDormant 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 Plasmodium 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 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 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.

RNA-Seq Analysis Illuminates the Early Stages ofPlasmodiumLiver Infection

“…Hierarchical clustering of gene expression datasets from different stages of the Plasmodium life cycle (7, 8, 26, 32, 34, 52–54). Datasets generated in this study are in bold.…”

“…Yet, only a handful of transcriptome analyses have been completed in the LS relative to other parasite forms, likely owing to the technical challenges associated with studying this stage. Still, these studies have provided important insight into LS-specific biological processes (5), including hypnozoite markers (6, 7), through comparative gene expression analysis with other stages (8), even at single-cell resolution (9). These studies examined gene expression upon the establishment of a LS-trophozoite (24 hours post-infection and thereafter); however, the early stages of LS infection (0–24 hours post-infection) for any Plasmodium species remains unresolved.…”

Transcriptome and histone epigenome ofPlasmodium vivaxsalivary-gland sporozoites point to tight regulatory control and potential mechanisms for liver-stage differentiation

“…A further nine have an ortholog in P. cynomolgi (which also forms hypnozoites) but not the closely related P. knowlesi (which does not form hypnozoites) and include ‘ msp7 ’-like (PVP01_1219600, PVP01_1220300 and PVP01_1219900), ‘ msp3 ’-like (PVP01_1031300), Pv-fam-e genes (PVP01_0302100, PVP01_0524500 and PVP01_0523400), a serine-threonine protein kinase (PVP01_0207300) and a RecQ1 helicase homolog (PVP01_0717000). Notably, the P. cynomolgi ortholog of PVP01_0207300, PCYB_021650, is transcriptionally up-regulated in hypnozoites relative to replicating schizonts [23], indicating a target of significant interest when considering hypnozoite formation and/or biology and suggesting that the list here may contain other genes important in hypnozoite biology.…”

“…Although in-vitro liver stage assays and humanised mouse models are being developed [13], ‘omics analysis of P. vivax liver stage dormancy has until recently [22] been impossible and even now is in its early stages. Recent characterization [23] of liver-stage (hypnozoites and schizonts) of P. cynomolgi (a related and relapsing parasite in macaques) provides valuable insight, but investigations in P. vivax directly are clearly needed. The systems analysis of P. vivax sporozoites that reside in the mosquito salivary glands and are poised for transmission and liver infection offer a key opportunity to gain insight into P. vivax infection.…”

Inducing controlled cell cycle arrest and re-entry during asexual proliferation ofPlasmodium falciparummalaria parasites

“…Moreover, it is well established that the parasite can respond to artemisinin-induced stress by delaying their cell cycle progression and inducing a state of dormancy during early ring-stage development17 . Dormancy is also observed in P. vivax hypnozoites during liver stage development52 . However, the cell cycle arrest and quiescence observed here is clearly distinct from these examples of dormancy, as (i) it does not delay ring-stage development, (ii) it is fully reversible, (iii) it does not result in irreversible damage to the parasite (in contrast to other perturbations including treatment with the CDK inhibitor…”