PROJECT SUMMARY/ABSTRACT Human malaria caused by Plasmodium falciparum parasites remains a devastating infectious disease marked by increasing treatment failures with frontline artemisinin combination therapies. Plasmodium parasites have evolved specialized metabolic strategies that enable them to infect and grow within red blood cells. Identifying these adaptations will give insight into Plasmodium evolution and identify new parasite-specific therapeutic targets. Parasites require nutritional iron to support critical metabolic pathways that include DNA synthesis and repair, ribosome assembly, mitochondrial electron transport chain function, and isoprenoid precursor synthesis in the apicoplast organelle. Although it has been known for decades that iron chelators effectively block parasite growth inside red blood cells, the pathways and mechanisms used by parasites to obtain iron for intracellular metabolism remain sparsely defined. Identification of iron transporters has been especially challenging given low sequence similarity between many parasite proteins and well-studied proteins from other organisms, including yeast and mammals. Our long-term goal is to understand essential functions of the apicoplast whose inhibition causes immediate rather than delayed parasite death. Our overall objective in this proposal is to elucidate an essential mechanism for iron uptake by the apicoplast, which is critical for parasite synthesis of isoprenoid precursors and depends on Fe-S cluster biogenesis within this organelle. Basic mechanisms of iron uptake into the apicoplast are currently unknown. In an exciting preliminary advance, we have used an innovative affinity screen to identify a highly divergent parasite protein (Pf3D7_1248300) that is targeted to the apicoplast and shows high structural homology to pentameric metal transporters in bacteria, to which the endosymbiotic apicoplast is evolutionarily related. In proposed studies, we will test our central hypothesis that this protein is essential for parasite viability and required for iron-dependent metabolism in the apicoplast. We will test this hypothesis in two aims. First, we will use CRISPR/Cas9 to tag the gene encoding this putative transporter to encode an epitope tag and a conditional regulation system that enables ligand- dependent protein expression. We will use these tagged parasites to test and understand the sub-organellar localization and essentiality of this protein for parasite viability and apicoplast biogenesis. In a second aim, we will test if knockdown of this protein selectively impairs iron-dependent metabolism inside the apicoplast. These studies will unravel a new molecular paradigm for iron mobilization into the apicoplast organelle and unveil a potential new therapeutic target, especially as preliminary results suggest that small molecules can selectively target this protein. These studies will also involve the rigorous training of a talented female PhD student from an underrep...