Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
Autophagy is a catabolic process involved in maintaining cellular homeostasis in eukaryotic cells, while coping with their changing environmental conditions. Mechanistically, it is also a process of considerable complexity involving multiple protein factors and implying numerous protein-protein and protein-membrane interactions. The cellular material to be degraded by autophagy is contained in a membrane-bound compartment called the autophagosome. We have characterised the formation of autophagosomes in the protozoan parasite Toxoplasma gondii by following the relocalisation of autophagosome-bound TgAtg8. Thus, exploiting GFP-TgAtg8 as a marker, we showed that it is a process that is regulated and can be induced artificially by amino acid starvation. Autophagic vesicles were also observed in normally dividing intracellular parasites. Depleting Toxoplasma of the TgAtg3 autophagy protein led to an impairment of TgAtg8 conjugation to the autophagosomal membrane and, at the cellular level, to a fragmentation of the single mitochondrion of the parasite and to a severe growth arrest. We have thus found that TgAtg3-dependent autophagy is essential for normal intracellular development of T. gondii tachyzoites.