Reconstitution of an essential metabolic pathway of Plasmodium falciparum in Escherichia coli, facilitating functional and inhibitor screens

Introduction: New and highly selective drug targets for the malaria-causing parasite Plasmodium falciparum are still needed. The ferredoxin redox system (Fd-FNR), located in a plant-derived organelle called apicoplast, is essential for parasite survival due to its involvement in the methylerythritol phosphate pathway (MEP) for isoprenoid biosynthesis. However, in vitro screens for inhibitors are challenging due to the labile nature of the recombinant iron-sulfur proteins involved.

Goal: To develop an in situ model in E. coli which reports essential functions of PfFd and its downstream electron acceptors (i.e. the terminal enzymes of the MEP pathway, shared by both organisms) as growth/intermediate/no growth.

Materials & Methods: Deletion of the two essential genes of E. coli, flavodoxin (EcFldA) and MEP enzyme EcIspH, and inducible expression of the respective P. falciparum proteins (PfFd, PfFd reductase (PfFNR) and PfIspH), followed standard procedures.

Results: We report for the first time a double mutant of E. coli FldA and IspH. It is dependent on the mevalonate bypass system which provides the essential metabolite IPP. Removing mevalonate makes the strain entirely dependent on the simultaneous expression of plasmid-encoded PfFd, PfFNR, PfIspH, which documents that the three parasite proteins are required functionally active to replace the respective endogenous E. coli proteins. This system will allow initial functional tests to be performed. As a proof of concept, several amino acids of PfFd suspected to be involved in the interaction with PfIspH were individually mutated and the respective redox function could be evaluated by simple growth assays. Metabolomic studies are under way to characterize the mutants' consequences on the MEP.

Summary: We describe an engineered E. coli strain dependent on three P. falciparum proteins, PfFd, PfNR and the terminal MEP enzyme PfIspH, which will be very useful for functional as well as drug screening purposes of this essential parasite pathway before findings are validated in the parasite.