Toxoplasma TgAtg8-TgAtg3 Interaction Primarily Contributes to Apicoplast Inheritance and Parasite Growth in Tachyzoite

Abstract

ABSTRACT The apicoplast, which harbors key pathways involved in biosynthesis of vital metabolites, is a unique and essential nonphotosynthetic plastid organelle in apicomplexan parasites. Intriguingly, autophagy-related protein 8 (Atg8), a highly conserved eukaryotic protein, can localize to the outermost membrane of the apicoplast and modulate its inheritance in both Toxoplasma and Plasmodium parasites. The Atg8-Atg3 interaction plays a key role in Atg8 lipidation and localization, and our previously work in Toxoplasma has suggested that the core Atg8-family interacting motif (AIM) in TgAtg3, 239FADI242, and the R27 residue of TgAtg8 contribute to TgAtg8-TgAtg3 interaction in vitro. However, little is known about the function of this interaction or its importance in tachyzoite growth in Toxoplasma gondii. Here, we generated two complemented cell lines, TgAtg3F239A/I242A and TgAtg8R27E, based on the TgAtg3 and TgAtg8 conditional knockdown cell lines, respectively. We found that both mutant complemented cell lines were severely affected in terms of tachyzoite growth and displayed delayed death upon conditional knockdown of endogenous TgAtg3 or TgAtg8. Intriguingly, both complemented lines appeared to be defective in TgAtg8 lipidation and apicoplast inheritance. Moreover, we showed that the interaction of TgAtg8 and TgAtg3 is critical for TgAtg8 apicoplast localization. In addition, we found that the TgAtg3F239A/I242A complemented line exhibits an integral mitochondrial network upon ablation of endogenous TgAtg3, which is distinct from TgAtg3-depleted parasites with a fragmented mitochondrial network. Taken together, this work solidifies the contribution of the TgAtg8-TgAtg3 interaction to apicoplast inheritance and the growth of T. gondii tachyzoites. IMPORTANCE Toxoplasma gondiiis a widespread intracellular parasite infecting a variety of warm-blooded animals, including humans. Current frontline treatment of toxoplasmosis suffers many drawbacks, including toxicity, drug resistance, and failure to eradicate tissue cysts, underscoring the need to identify novel drug targets for suppression or treatment of toxoplasmosis. TgAtg8 is thought to serve multiple functions in lipidation and is considered essential to the growth and development of both tachyzoites and bradyzoites. Here, we show that Toxoplasma gondii has adapted a conserved Atg8-Atg3 interaction, required for canonical autophagy in other eukaryotes, to function specifically in apicoplast inheritance. Our finding not only highlights the importance of TgAtg8-TgAtg3 interaction in tachyzoite growth but also suggests that this interaction is a promising drug target for the therapy of toxoplasmosis.

Keywords

• Toxoplasma
• autophagy
• Atg8
• Atg3
• protein-protein interaction
• Atg8-family interacting motif