Dissecting the distinct metabolic roles of the ferredoxin redox system
FRANK SEEBER (RKI) in partnership with Christina Spry (ANU), Kevin Saliba (ANU), and Alexander Maier (ANU)

Plasmodium sp. and Toxoplasma gondii contain a specialized organelle, termed apicoplast, which harbours several essential metabolic pathways, e.g. isoprenoid and fatty acid synthesis. The plant-type ferredoxin (Fd) redox system constitutes the only known electron transfer system in the apicoplast. We previously showed that Fd provides electrons to enzymes of the isoprenoid pathway and to lipoic acid synthetase (LipA) via protein interactions. The apicoplast is also a site for iron-sulfur cluster synthesis.  This process requires a reducing source, which likely is Fd. Our unpublished work show that Fd in T. gondii (TgFd) is an essential gene. However, its individual contributions to the distinct Fd-dependent pathways are unknown. Moreover, the reported association between polymorphisms in P. falciparum Fd and artemisinin resistance [1] warrants investigation of the underlying molecular mechanism.

We have successfully implemented a reverse two-hybrid system for TgFd and its interacting proteins ferredoxin-NADP+ reductase (FNR) and TgLipA in E. coli. Together with the ‘split intein-mediated circular ligation of peptides and proteins’ technology [2] this constitutes a powerful system that in E. coli allows screening of a large library of genetically encoded small cyclic peptides for their ability to prevent or interrupt protein interactions (“dissociators”). We propose to use dissociators derived from systematic screens with TgFd and PfFd in E. coli to disrupt the distinct Fd-dependent pathways. Hits will be validated by inducible expression of cyclic peptides in the apicoplast, a method we have recently established for T.gondii. The resulting phenotypes in the transgenic parasite lines will be determined with an emphasis on parasite replication and growth, morphology, and biochemical profiles, e.g. fatty acid or isoprenoid content.

Binding of candidate cyclic peptides to proteins will be tested at ANU (C. Spry/K. Saliba) by biophysical techniques (differential scanning fluorimetry and isothermal titration calorimetry). These assays will reveal binding affinities and localize peptide-binding sites on Fd-interacting proteins. PfFNR will also be analysed by 1H-15N- NMR studies to map the interface with PfFd.

Isoprenoid biosynthesis is the only essential apicoplast function during Plasmodium blood stage growth and exogenous supply of isopentenyl pyrophosphate during parasite selection can rescue apicoplast defects [3]. Using this approach, a Plasmodium falciparum Fd-knockout will be generated in collaboration with A. Maier (ANU). This parasite line will allow us to devise experiments to validate the potential role of PfFd in artemisinin resistance by complementation with a set of Fd mutants and quantifying artemisinin sensitivity.

Interlinkages: Melanie Rug (ANU), Christian Schmitz-Linneweber (HUB)

References

1. Miotto, O. et al. (2015) Nat. Genet. 47:226-234.

2. Horswill, A.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:15591-15596

3. Yeh, E. and Derisi, J. (2011) Plos Biol. 9:e1001138