Paracrine rescue of MYR1-deficientToxoplasma gondiimutants reveals limitations of pooledin vivoCRISPR screens

Abstract

ABSTRACTToxoplasma gondiiis an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins inToxoplasmavirulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooledin vivoCRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at themyr1locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasitesin vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerfulin vivoCRISPR screens and point towards key functions for MYR1-dependentToxoplasma-host interactions beyond the infected cell.SIGNIFICANCE STATEMENTPooled CRISPR screens are powerful tools to interrogate gene function in a high-throughput manner. Genes conferring fitness advantages or disadvantages upon disruption can be identified by sequencing. However, inToxoplasma gondiipooled CRISPR screens in mice, fitness defects for some selected mutants drastically diverge from those observed in single-strain infections. Here, we show that a growth defect of a singleToxoplasmagene deletion mutant is rescued if co-infected with wildtype parasites. These results shine light onToxoplasma’s ability to subvert the host response beyond the infected cell, and highlight an important limitation of pooled CRISPR screens in mice. This limitation is probably encountered in CRISPR screens in general where paracrine effects occur.