RT Journal Article SR Electronic T1 Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.05.13.093401 DO 10.1101/2020.05.13.093401 A1 David Smith A1 Geetha Kannan A1 Isabelle Coppens A1 Fengrong Wang A1 Hoa Mai Nguyen A1 Aude Cerutti A1 Tracey L. Schultz A1 Patrick A. Rimple A1 Manlio Di Cristina A1 Sébastien Besteiro A1 Vern B. Carruthers YR 2020 UL http://biorxiv.org/content/early/2020/05/15/2020.05.13.093401.abstract AB Many of the world’s warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including up to an estimated one third of the global human population. The cellular processes that permit long-term parasite persistence within the cyst are largely unknown, not only for T. gondii but also for related coccidian parasites that impact human and animal health. A previous study revealed an accumulation of autophagic material in the lysosome-like Vacuolar Compartment (VAC) of chronic stage bradyzoites lacking functional cathepsin L protease (TgCPL) activity. Furthermore, it was shown that TgCPL knockout bradyzoites have compromised viability, indicating the turnover of autophagic material could be necessary for bradyzoite survival. However, the extent to which autophagy itself contributes to bradyzoite development and fitness remained unknown. Herein we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed structural abnormalities occurring in Δatg9 bradyzoites, including disorganization of the inner membrane complex and plasma membrane, the occurrence of multiple nuclei within a single bradyzoite cell, as well as various anomalies associated with the VAC. TgATG9-deficient bradyzoites accumulated significantly less undigested material in the VAC upon inhibition of TgCPL activity, suggesting that autophagy contributes material to the VAC for degradation. Intriguingly, abnormal mitochondria networks were observed in TgATG9-deficient bradyzoites. They were thin and elongated and often adopted a horseshoe conformation. Some abnormal mitochondrial structures were found to contain numerous different cytoplasmic components and organelles. Bradyzoite fitness was found to be drastically compromised, both in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.Competing Interest StatementThe authors have declared no competing interest.