Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites.