High-throughput identification of Toxoplasma gondii effector proteins that target host cell transcription
Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonization. We combine pooled CRISPR knockout screening with dual host-microbe single-cell RNA sequencing, a method we term dual perturb-seq, to identify the molecular mediators of these transcriptional interactions. Applying dual perturb-seq to the intracellular pathogen Toxoplasma gondii, we are able to identify previously uncharacterized effector proteins and directly infer their function from the transcriptomic data. We show that TgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify an effector, TgSOS1, that is necessary for sustained host STAT6 signaling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a tool that can be broadly adapted to interrogate host-microbe transcriptional interactions and reveal mechanisms of infection and immune evasion. Overall design: Human foreskin fibroblasts (HFFs) were infected with a pool of Toxoplasma gondii CRISPR mutants for 24 h. Direct-capture perturb-seq using 10x Genomics 3' Gene Expression with Feature Barcoding was carried out on infected cells to identify perturbations in the intracellular Toxoplasma gondii parasites that alter the transcriptome of the host cell. Three experiments were carried out: (1) A pilot experiment in which the Toxoplasma gondii parasites were transfected with two different perturb-seq plasmid vectors. (2) A pilot experiment in which the parasites were transfected with a panel of 24 plasmids. (3) A screen in which the parasites were transfected with a pool of 1,312 plasmids with sgRNAs targeting 256 genes. This screen was carried out in both unstimulated and interferon-gamma-stimulated HFFs. Three replicates were carried out for unstimulated HFFs and two for interferon-gamma-stimulated HFFs, with 2-4 Chromium Controller channels per replicate.