ABSTRACT
Plasmodium vivax is the key obstacle to malaria elimination in Asia and Latin America, largely attributed to its ability to form resilient ‘hypnozoites’ (sleeper-cells) in the host liver that cause relapsing infections. The decision to form hypnozoites is made early in the liver infection and may be set in sporozoites prior to invasion. To better understand these early stages of infection, we undertook transcriptomic and histone epigenetic characterization of P. vivax sporozoites. Through comparisons to recently published proteomic data for the P. vivax sporozoite, our study highlights the loading of the salivary-gland sporozoite with proteins required for cell traversal and invasion and transcripts for infection of and development within hepatocytes. Though highly transcribed, these transcripts are not detectable as proteins, indicating they may be regulated in the sporozoite through translational repression. We also undertook differential transcriptomic studies comparing the sporozoite with newly published mixed blood-stage and mixed versus hypnozoite-enriched liver stage transcriptomes. These comparisons indicate multiple layers of transcriptional, post-transcriptional and post-translational control active in the sporozoite stage and to a lesser extent hypnozoites, but largely absent in mixed liver or blood-stages. Common transcripts up-regulated in sporozoites and hypnozoites compared to mixed stages include several AP2 transcription factors, translational repressors and histone epigenetic regulators, as well as genes linked to dormancy/persistence in bacteria, amoebae and plants. We characterise histone epigenetic modifications in the P. vivax sporozoite and explore their role in regulating transcription. This work shows a close correlation between H3K9ac marks and transcriptional activity, with H3K4me3 and H3K9me3 appearing to act as general markers of euchromatin and heterochromatin respectively. We also show little evidence of transcriptional activity in the (sub)telomeres in sporozoites and discuss potential roles of AP2 transcription factors, including ApiAP2-L in regulating this stage. Collectively, these data indicate the sporozoite as a tightly programmed stage primed to infect the human host and identifies key targets to be further explored in liver stage models.
Author summary Our study is the first to use RNA-seq and ChIP-seq technologies to comprehensively characterize the P. vivax sporozoite transcriptome and histone epigenome, and the first to integrate these technologies with proteomic studies to explore translational repression in the sporozoite of any Plasmodium spp. Our study improves on previous work in this field and is supported by recent publication of the transcriptome/histone epigenome of P. falciparum and the liver-stage transcriptomes of a related simian parasite (P. cynomolgi) that also forms dormant liver stages. Collectively, these data characterize the infectious sporozoite as a highly programmed and tightly regulated, ‘torpedo’-like stage primed to infect the human host and suggests that hypnozoite formation is mediated both by differential signals in the sporozoite and by developmental arrest regulators that suppress further development to the schizont stage. This work reveals the primary importance of translational repression and chromatin epigenetics in this process. Our study provides a foundation for exploring the genetic differences between sporozoites derived from parasites with broadly differing hypnozoite formation and relapse phenotypes and highlights genes of major importance in liver stage development that may now be rationally investigated (including as potential drug targets) in relevant models.