Rimple, P. | bioRxiv Science

bioRxiv2026

A pooled image-based CRISPR screen identifies EAF1 as a T. gondii modulator of ESCRT subversion.

Intracellular pathogens remodel host cells by redirecting cellular machinery to the host-pathogen interface. The protozoan parasite Toxoplasma gondii co-opts host ESCRT proteins at the parasitophorous vacuole membrane (PVM), where they support budding of host-derived vesicles into the vacuole. Yet the parasite effectors that direct this process remain largely unknown. Here we developed spaCR (spatial phenotype analysis of CRISPR-Cas9 screens), a pooled image-based screening framework that combines deep-learning classification of single-cell spatial phenotypes with well-level barcode genotyping and regression-based deconvolution of gene effects. Screening T. gondii secretory proteins recovered the known TSG101 recruiter GRA14 and identified an uncharacterized effector, ESCRT-association factor 1 (EAF1), that promoted TSG101 recruitment to the PVM. Targeted deletion confirmed its role across Type I and II lineages, and IP-MS recovered ESCRT-I and ESCRT-III components, including TSG101. Together, these findings identify EAF1 as an ESCRT-associated parasite effector and establish spaCR as a general framework for linking CRISPR perturbations to spatial phenotypes.

microbiology↗

bioRxiv2020

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts

Many of the worlds 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 {Delta}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 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.

microbiology↗