Evidence that disruption of apicoplast protein import in malaria parasites evades delayed-death growth inhibition

Abstract

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. Hundreds of proteins are imported across 4 membranes into the apicoplast to support its function and biogenesis. The machinery that mediates this import process is distinct from proteins in the human host and may serve as ideal drug targets. However, a significant concern is whether inhibition of apicoplast protein import will result in a \"delayed-death\" phenotype that limits clinical use, as observed for inhibitors of apicoplast housekeeping pathways. To assess the growth inhibition kinetics of disrupting apicoplast protein import, we targeted a murine dihydrofolate reductase (mDHFR) domain, which is stabilized by the compound WR99210, to the apicoplast to enable inducible blocking of apicoplast-localized protein translocons. We show that stabilization of this apicoplast-targeted mDHFR disrupts parasite growth within a single lytic cycle in an apicoplast-specific manner. Consistent with inhibition of apicoplast protein import, stabilization of this fusion protein disrupted transit peptide processing of endogenous apicoplast proteins and caused defects in apicoplast biogenesis. These results indicate that disruption of apicoplast protein import avoids delayed-death growth inhibition and that target-based approaches to develop inhibitors of import machinery may yield viable next-generation antimalarials.\n\nImportanceMalaria 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, including import machinery that transports hundreds of critical proteins into the apicoplast. Unfortunately, some drugs targeting apicoplast pathways show delayed growth inhibition, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. We used a chemical biology approach to disrupt apicoplast protein import and showed that chemical disruption of this pathway avoids delayed growth inhibition. Our finding indicates that prioritization of proteins involved in apicoplast protein import for target-based drug discovery efforts may aid in the development of novel fast-acting antimalarials.