Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2—the last two clustering with the eukaryotic eIF2α kinases—as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival.

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Plasmodium is auxotrophic for most AA however, it does not have either a TOR complex nor the GCN2-downstream transcription factors. Although Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernatory-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on a GCN2-like sensing pathway to respond to AA fluctuations. A kinome analysis identified eIK1 and eIK2 – both of which cluster with the eukaryotic eIF2α kinases - as critical for Plasmodium ability to sense and respond to distinct AA-limiting conditions. Such AA sensing pathway is temporally regulated by these kinases at distinct life cycle stages and allows parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data identify a previously unknown set of heterogeneous responses to AA depletion, mediated by an AA-sensing mechanism that is critical for modulating parasite cell cycle and survival.