Chemical Rescue of Malaria Parasites Lacking an Apicoplast Defines Organelle Function in Blood-Stage Plasmodium falciparum

Abstract

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development. Malaria caused by Plasmodium spp parasites is a profound human health problem that has shaped our evolutionary past and continues to influence modern day with a disease burden that disproportionately affects the world's poorest and youngest. New anti-malarials are desperately needed in the face of existing or emerging drug resistance to available therapies, while an effective vaccine remains elusive. A plastid organelle, the apicoplast, has been hailed as Plasmodium's “Achilles' heel” because it contains bacteria-derived pathways that have no counterpart in the human host and therefore may be ideal drug targets. However, more than a decade after its discovery, the essential functions of the apicoplast remain a mystery, and without a specific pathway or function to target, development of drugs against the apicoplast has been stymied. In this study, we use a simple chemical method to generate parasites that have lost their apicoplast, normally a deadly event, but which survive—“rescued” by the addition of an essential metabolite to the culture. This chemical rescue demonstrates that the apicoplast serves only a single essential function, namely isoprenoid precursor biosynthesis during blood-stage growth, validating this metabolic function as a viable drug target. Moreover, the apicoplast-minus Plasmodium strains generated in this study will be a powerful tool for identifying apicoplast-targeted drugs and as a potential vaccine strain with significant advantages over current vaccine technologies.