ToxoplasmaTgATG9 is critical for autophagy and long-term persistence in tissue cysts

Abstract

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 ∆atg9 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 TgATG9deficient 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 postinfection. 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.

In vitromaturation ofToxoplasma gondiibradyzoites in human myotubes and their metabolomic characterization

“…Hence, these changes may reflect a lowered demand for protein biosynthesis and may also be associated with an additional source. Autophagy and proteolysis through autophagy-related protein (ATG9) (Smith et al, 2020) and cathepsin L (Dou et al, 2014) has been shown to be essential in bradyzoites and may constitute an endogenous source for amino acids. Interestingly, restriction of some exogenous amino acids can be used to facilitate stage conversion (Cerutti et al, 2020; Jeffers et al, 2018).…”

“…Interestingly, restriction of some exogenous amino acids has been used to facilitate stage conversion 63, 73 suggesting that alternative sources may represent adaptions to limiting supply of exogenous amino acids. These include obligate autophagy and proteolysis through autophagy-related protein (ATG9) 19 and cathepsin L 74 and lower amino acid demand by translational repression through the eukaryotic initiation factor 2α 75 . The diversification of nutrient sources would resemble the stringent metabolic response of Leishmania mexicana amastigotes that also adopt a slow growing phenotype within the phagolysosome of macrophages 76 .…”

“…Moreover, T. gondii cysts survive extended atovaquone exposure in vivo (Araujo et al, 1991; Ferguson et al, 1994b), indicating that their mitochondrial electron transport chain is not strictly essential. Similarly, bradyzoites can also be induced by limiting supply of exogenous amino acids (Ferreira da Silva Mda et al, 2008) and their viability depends on proteolysis in their plant-like vacuolar compartment (Di Cristina et al, 2017) and on autophagy (Smith et al, 2020). Collectively, these data indicate a substantial remodeling of metabolic homeostasis in bradyzoites as a strategy to cope with nutrient limitations (Lüder and Rahman, 2017).…”