Reconstituted ferredoxin-MEP pathway of Apicomplexa in E. coli as an in situ screening platform for inhibitors and essential enzyme mutations.

Abstract

The apicoplast, an essential plastid-like organelle of apicomplexan parasites, including Plasmodium spp. and Toxoplasma gondii, harbors unique metabolic pathways absent in the host. Within the apicoplast, the ferredoxin redox system consists of plant-type ferredoxin-NADP+ reductase (ptFNR) and its redox partner, plant-type ferredoxin (ptFd). It donates electrons to the last two enzymes in the essential methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. To establish an easy-to-handle platform for screening for enzyme inhibitors or functional mutations of the P. falciparum MEP pathway in situ, we established an E. coli model where bacterial growth depended on the last enzyme IspH and its redox system ptFd and ptFNR. For this, we supplemented a flavodoxin and ispH E. coli double mutant with expression constructs for ptFd, ptFNR, and IspH from P. falciparum. These proteins could functionally replace the two essential endogenous E. coli enzymes, reconstituting the last step in the isoprenoid biosynthesis pathway of the apicoplast. To validate this strain as a screening platform we used point mutations in ptFd as a surrogate for chemical pathway inhibitors. Several single mutants were evaluated by growth assays to identify amino acids that are essential for proliferation. We verified the mutants' consequences on the depletion of MEP metabolites by LC-MS analysis. Finally, some mutants were used to complement a conditional T. gondii Fd knockout strain. The results mirrored those of the respective E. coli mutant, highlighting the model's utility in identifying functional mutations or ptFd/MEP pathway inhibitors before conducting more labor-intensive and time-consuming assays in parasites.