Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. Author summary Malaria is a significant global health problem caused by a parasite that invades and multiplies within red blood cells, an invasion cycle resulting in death and disease. Plasmodium falciparum is the parasite that causes most malaria deaths. In most higher organisms, proteins can be marked by addition of a ubiquitin 'tag', which determines how quickly they will be broken down or their location in the cell. In this study we found that many parasite proteins are modified with a ubiquitin tag while the parasite matures within red blood cells and that even more proteins seem to be tagged in the extracellular invasive merozoite form. Some small molecules that inhibit the addition of this tag, and in particular one compound that inhibits the activation of the ubiquitin tag, blocked parasite growth. We show that this compound inhibits the parasite enzyme that activates ubiquitin and kills the parasite before it can finish growing and multiplying, and that the same effect is achieved by knocking out the gene for this enzyme. These results point to the importance of ubiquitin tags in the parasite life cycle and highlight the potential of this ubiquitin pathway as a target for the development of new antimalarial drugs.