Disruption of Apicoplast Biogenesis by Chemical Stabilization of an Imported Protein Evades the Delayed-Death Phenotype in Malaria Parasites

Malaria parasites (Plasmodium spp.) contain a nonphotosynthetic plastid organelle called the apicoplast, which houses essential metabolic pathways and is required throughout the parasite life cycle. The biogenesis pathways responsible for apicoplast growth, division, and inheritance are of key interest as potential drug targets. Unfortunately, several known apicoplast biogenesis inhibitors are of limited clinical utility because they cause a peculiar “delayed-death” phenotype in which parasites do not stop replicating until the second lytic cycle posttreatment. Identifying apicoplast biogenesis pathways that avoid the delayed-death phenomenon is a priority. Here, we generated parasites targeting a murine dihydrofolate reductase (mDHFR) domain, which can be conditionally stabilized with the compound WR99210, to the apicoplast. Surprisingly, chemical stabilization of this exogenous fusion protein disrupted parasite growth in an apicoplast-specific manner after a single lytic cycle. WR99210-treated parasites exhibited an apicoplast biogenesis defect beginning within the same lytic cycle as drug treatment, indicating that stabilized mDHFR perturbs a non-delayed-death biogenesis pathway. While the precise mechanism-of-action of the stabilized fusion is still unclear, we hypothesize that it inhibits apicoplast protein import by stalling within and blocking translocons in the apicoplast membranes. IMPORTANCE Malaria is a major cause of global childhood mortality. To sustain progress in disease control made in the last decade, new antimalarial therapies are needed to combat emerging drug resistance. Malaria parasites contain a relict chloroplast called the apicoplast, which harbors new targets for drug discovery. Unfortunately, some drugs targeting apicoplast pathways exhibit a delayed-death phenotype, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. Identification of druggable apicoplast biogenesis factors that will avoid the delayed-death phenotype is an important priority. Here, we find that chemical stabilization of an apicoplast-targeted mDHFR domain disrupts apicoplast biogenesis and inhibits parasite growth after a single lytic cycle, suggesting a non-delayed-death target. Our finding indicates that further interrogation of the mechanism-of-action of this exogenous fusion protein may reveal novel therapeutic avenues.