A Mevalonate Bypass System Facilitates Elucidation Of Plastid Biology In Malaria Parasites

Author summaryMalaria parasites rely on an organelle called the apicoplast for growth and survival. Antimalarial drugs such as azithromycin inhibit basic processes in the apicoplast and result in the disruption of the organelle. Surprisingly, addition of a single metabolite, isopentenyl pyrophosphate (IPP), allows the parasites to survive in culture after disruption of the apicoplast. Unfortunately, using IPP to study this phenomenon has several limitations: IPP is prohibitively expensive, has to be used at high concentrations, and has a half-life less than 5 hours. To address these problems, we engineered parasites to express four enzymes from an alternative pathway capable of producing IPP in the parasites. We validated this new system and used it to metabolically label essential metabolites, to delete an essential apicoplast protein, and to characterize the state of apicoplast-disrupted parasites. A key finding from these studies comes from transcriptomic and metabolomic analysis of parasites treated with the drug azithromycin. We found that apicoplast disruption results in few changes in parasite metabolism. In particular, the expression of hundreds of nuclear-encoded apicoplast proteins are not affected by disruption of the apicoplast organelle, making it likely that apicoplast metabolic pathways and processes are still functional in apicoplast-disrupted parasites.Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.