The artemisinin-induced dormant stages of Plasmodium falciparum exhibit hallmarks of cellular quiescence/senescence and drug resilience.

Recrudescent infections with the human malaria parasite, Plasmodium falciparum, presented traditionally the major setback of artemisinin-based monotherapies. Although the introduction of artemisinin combination therapies (ACT) largely solved the problem, the ability of artemisinin to induce dormant parasites still poses an obstacle for current as well as future malaria chemotherapeutics. Here, we use a laboratory model for induction of dormant P. falciparum parasites and characterize their transcriptome, drug sensitivity profile, and cellular ultrastructure. We show that P. falciparum dormancy requires a ~ 5-day maturation process during which the genome-wide gene expression pattern gradually transitions from the ring-like state to a unique form. The transcriptome of the mature dormant stage carries hallmarks of both cellular quiescence and senescence, with downregulation of most cellular functions associated with growth and development and upregulation of selected metabolic functions and DNA repair. Moreover, the P. falciparum dormant stage is considerably more resistant to antimalaria drugs compared to the fast-growing asexual stages. Finally, the irregular cellular ultrastructure further suggests unique properties of this developmental stage of the P. falciparum life cycle that should be taken into consideration by malaria control strategies.

Stochastic expression of invasion genes in Plasmodium falciparum schizonts.

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental "checkpoint"; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines.