Chemical genetics and a global comparative analysis of phosphorylation and phospholipids in vivo shows that PKG is the upstream regulator that induces calcium signals that enables Plasmodium to progress through its complex life cycle.

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre-and post-exocytosis. Here, we report the multi-stage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound, 49c. This scaffold inhibits the pre-exocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PfPMIX) and X (PfPMX), respectively. Conditional excision of PfPMIX revealed its crucial role in invasion, and recombinantly active PfPMIX and PfPMX cleave egress and invasion factors in a 49c sensitive manner. KeywordsMalaria; Plasmodium falciparum; Plasmodium berghei; aspartic protease; invasion; egress; exflagellation; transmission; hydroxyl-ethyl-amine scaffold; peptidomimetic inhibitor; protein maturase Malaria remains a major cause of mortality worldwide, and resistance to existing antimalarials is a growing problem, that requires the development of new drugs urgently. Aspartic proteases are potential targets for chemotherapy (1), and key contributors to Plasmodium falciparum pathogenicity (2, 3). P. falciparum possesses a repertoire of 10 aspartic proteases, named plasmepsins (PMI to X). PMIX and PMX are expressed in mature blood-stage schizonts and invasive merozoites and fulfill indispensable but unknown functions. The activity of several serine and cysteine proteases promotes the destabilization * Corresponding authors: Paco.Pino@unige.ch, Dominique.Soldati-favre@unige.ch.

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SummaryCalcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3′ and 5′UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression.

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