The metabolic role of the ferredoxin redox system in apicomplexan parasites

Abstract

Apicomplexan parasites, including Plasmodium sp. (the causative agent of malaria) and Toxoplasma gondii (causing toxoplasmosis), are a large phylum of unicellular, obligate intracellular organisms. They harbor an essential plastid-like organelle, named the apicoplast. As the apicoplast was derived via secondary endosymbiosis of a red algae, it possesses several metabolic pathways that differ from the human host, and since perturbing its function results in parasite death, the organelle is an attractive target for drugs. Within the apicoplast, an electron transfer system is required for several enzymatic reactions; this is provided by the ferredoxin redox system. It consists of the plant-type ferredoxin-NADP+ reductase (ptFNR) and its redox partner plant-type ferredoxin (ptFd). Via protein-protein interactions, ptFd provides electrons to several enzymes of the isoprenoid pathway and to the lipoic acid synthase (LipA), which is involved in the fatty acid synthesis. Thus, the plant-type ferredoxin redox system in apicomplexan parasites is a promising drug target due to its potential involvement in several essential metabolic processes. Recently published work demonstrated that ptFd in T. gondii (TgFd) is an essential protein. A tetracycline-inducible knock-down (ikd) approach was used to replace the endogenous single copy of TgFd with a myc-tagged copy (TgFdmyc) by double cross-over homologous recombination, and severe growth inhibition of parasites was observed upon Fd depletion. Metabolomic analyses show a 30% decrease in C14:0 fatty acids and a significantly lower gliding motility (20%) in the TgFd ikd strain compared to the TgFd ikd complemented (TgFd cikd) strain. In this thesis, targeted metabolomic analysis of the isoprenoid biosynthesis metabolites demonstrates that T. gondii Fd has an essential physiological function as an electron donor for the last two enzymes of the pathway. Furthermore, results of this work show that inhibition of the host isoprenoid biosynthesis contributes to the slow onset of death in TgFd knockdown parasites, indicating that the lack of isoprenoid precursors after induced Fd knockdown can to some extent be compensated by host-derived isoprenoids. Fd has been implicated in artemisinin resistance in P. falciparum; a single point mutation in P. falciparum ptFd (PfFd; D97Y) has been found in genome-wide association studies to be associated with artemisinin (ART) resistant Kelch13 mutant strains. In this thesis an ART resistant P. falciparum line containing the PfFd mutation iv was genetically reverted to PfFd wildtype using CRISPR/Cas9 and characterised in a ring stage survival assay, in order to evaluate the role of Fd in resistance to artemisinin. While preliminary results of this study have shown a decrease in proliferation of the transgenic ART-resistant P. falciparum line, meanwhile it has been demonstrated elsewhere that the Fd D97Y mutation does not contribute directly to ART resistance. A reverse two-hybrid system (RTHS) for TgFd and its interacting proteins TgFNR and TgLipA was established previously in E. coli, with the aim to use it with the split intein circular ligation of peptides and proteins (SICLOPPS) methodology for screening a large library of genetically encoded small cyclic peptides for their ability to interrupt protein-protein interactions of Fd. In this thesis, cyclic peptide libraries of different sizes were generated, which can now be used for further research to screen for peptides that interrupt the interactions between TgFd and TgFNR and TgLipA. Together, the findings of this study contribute to a better understanding of the metabolic role of Fd in T. gondii and P. falciparum, supporting its importance for the parasite’s metabolism and underlining its potential as a drug target in apicomplexan parasites. Show more