Global transcriptomic changes of either cell cycle arrested parasites or cell cycle re-entered asexual P. falciparum 3D7 parasites.

Parasitic protists, including Plasmodium falciparum malaria parasites, evolved sophisticated biological features to adapt and survive in e.g. mosquito vectors and mammalian hosts. The parasite’s life cycle is extraordinarily controlled, oscillating between quiescent stages (e.g. sporozoites or gametocytes) and stages of intense proliferation. The atypical mode of asexual reproduction in erythrocytes (schizogony: asynchronous nuclear division in the absence of cytokinesis), is clearly divergent from cell division in higher eukaryotes but the mechanisms that control cell cycle progression are poorly understood. Here, depletion of exogenous factors was used to induce reversible cell cycle arrest and allowed transcriptomic investigations of cell cycle control mechanisms operating in the parasite. We show that in early stages of erythrocyte infection, parasites enter a quiescent, G0-like state due to implementation of a G1 restriction point prior to G1/S transition. This quiescence is reversible, with cells making a clear decision to re-engage the proliferation machinery in response to proliferation stimuli. Cell cycle arrest is an adaptive response rather than merely prolongation of the G1 phase, as demonstrated by a distinct transcriptome. The quiescence-proliferation decision-making process in malaria parasites has hallmarks of unique regulatory principles: quiescence is associated with a deregulation of the pre-replicative complex, underlined by transcriptional control of kinases, including PfPK5 as putative functional homologue of the master mammalian cell cycle regulator, CDK1. Cell cycle re-entry is highly coordinated and mediated by a set of early responsive transcripts involving NIMA and Aurora kinases, ApiAP2 transcription factors and calcium signaling. We therefore show that Apicomplexa are able to perform quiescence-proliferation decision-making in a highly coordinated fashion, using atypical cell cycle regulation machinery. Two sets of independent oligonucleotide DNA microarrays were performed to analyze the global transcriptomic changes P. falciparum parasites using either DFMO-arrested parasites or DFMO-arrested parasites followed by putrescine-reversal. DNA microarrays were performed using custom microarray slides that contained 12 468 oligos (60-mer, Agilent Technologies) based on the complete P. falciparum genome. Synchronized ring-stage parasitized erythrocytes (5% hematocrit, 10-15% parasitemia) were incubated with DFMO (IC90) at 37˚C prior to sampling (Arrested time points: A1=21 h post treatment (hpt); A2=29 hpt) or additionally treated after 24 h DFMO pressure with 2 mM putrescine dihydrochloride to induce the re-entry and onset of the cell cycle (Re-entry time points: Re1=27 hpt; Re2=30 hpt and Re3=36 hpt). For each time point, two biological repeats were performed, each with two technical repeats. From 50 ml parasite cultures sourced at specific time points, RNA isolation, cDNA synthesis and dye coupling with Cy3 (reference pool) or Cy5 (samples) were performed as described in van Brummelen, A. C. et al. The Journal of biological chemistry 284, 4635-4646, doi:10.1074/jbc.M807085200 (2009). The slides were scanned using a GenePix™ 4000B scanner.