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A pooled image-based CRISPR screen identifies EAF1 as a T. gondii modulator of ESCRT subversion
Einar B Olafsson1,2, Christophe-Sebastien Arnold2, Jacob A Kellermeier2
1Department of Cell and Molecular Biology, Microbiology and Immunology, Uppsala University, Uppsala, Sweden.
Biorxiv : the Preprint Server for Biology
|July 17, 2026
Summary
Toxoplasma gondii parasites hijack host cell machinery using effectors like EAF1 to recruit ESCRT proteins to the PVM. This study introduces spaCR, a novel screening framework for identifying parasite effectors and their roles in host-pathogen interactions.
Area of Science:
- Cell Biology
- Parasitology
- Molecular Biology
Background:
- Intracellular pathogens manipulate host cells for replication.
- Toxoplasma gondii utilizes host ESCRT proteins at the PVM to form host-derived vesicles.
- The specific parasite effectors involved in this process are largely unknown.
Purpose of the Study:
- To identify novel Toxoplasma gondii effectors that recruit host ESCRT proteins.
- To characterize the function of identified effectors in host-pathogen interactions.
- To establish a new screening platform for linking genetic perturbations to spatial phenotypes.
Main Methods:
- Development of spaCR (spatial phenotype analysis of CRISPR-Cas9 screens), a pooled image-based screening framework.
- Integration of deep-learning for single-cell spatial phenotype classification.
- Utilized well-level barcode genotyping and regression-based deconvolution to identify gene effects.
Main Results:
- spaCR screening identified the uncharacterized effector ESCRT-association factor 1 (EAF1) as a promoter of TSG101 recruitment to the PVM.
- The known effector GRA14, a TSG101 recruiter, was also recovered.
- Targeted deletion confirmed EAF1's role in different T. gondii lineages, and IP-MS confirmed ESCRT-I and ESCRT-III component interactions.
Conclusions:
- EAF1 is identified as a novel ESCRT-associated parasite effector crucial for T. gondii's manipulation of host cells.
- The spaCR framework provides a powerful and generalizable method for dissecting host-pathogen interactions by linking CRISPR perturbations to spatial phenotypes.
