Identification of Toxoplasma gondii effector proteins by pooled CRISPR-Cas9 knockout screening

Abstract

Toxoplasma gondii is an intracellular eukaryotic pathogen of global clinical significance. Secretion of effector proteins by T. gondii into a host cell is critical for infection. At least two hundred putative effector proteins are secreted into the host cell, of which the vast majority are uncharacterised. In this work, I used CRISPR-Cas9 knockout screening in T. gondii to identify effector proteins involved in parasite evasion of host immune responses. From a screen of T. gondii mutants in a mouse model of infection, I identified genes that confer parasite fitness in vivo. I found that the first-identified rhoptry protein of T. gondii, ROP1, is necessary for parasite virulence and is important for evasion of the cell-autonomous, interferon-gamma-stimulated immune response in both murine and human macrophages. Then, I adapted CRISPR-Cas9 tools in T. gondii to enable pooled knockout screening with a single-cell, dual host-parasite transcriptome readout. This method, which I term dual perturb-seq, enables identification of parasite effectors that modulate host cell transcription in a high-throughput and unbiased manner. Using dual perturb-seq and additional cellular assays, I show that the dense granule protein GRA59 contributes to export of other dense granule effectors from the parasitophorous vacuole to the host cell. I also identify a rhoptry protein, newly named TgSOS1, that is required for sustained parasite-induced STAT6 signalling in the host cell and M2 polarisation of infected macrophages. Together, these genetic screens and the characterisation of newly identified effector proteins advance the understanding of how T. gondii manipulates the host cell and evades host immune responses.