Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens

• Published in: eLife Vol. 13
• Main Authors: Torelli, Francesca, da Fonseca, Diogo M, Butterworth, Simon W, Young, Joanna C, Treeck, Moritz
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 10.12.2024; eLife Sciences Publications, Ltd; eLife Sciences Publications Ltd
• Subjects: Animals; B cells; Clustered Regularly Interspaced Short Palindromic Repeats; crispr screening; CRISPR-Cas Systems; Female; Health aspects; Host-Parasite Interactions - genetics; immunology; Infection; Macrophages; Macrophages - parasitology; Mice; Mice, Inbred C57BL; microbiology; Microbiology and Infectious Disease; Mutation; Paracrine Communication; parasitology; Proteins; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Toxoplasma; Toxoplasma - genetics; Toxoplasmosis - genetics; Toxoplasmosis - parasitology; United States; Massachusetts; Toxoplasma; infection; crispr screening; infectious disease; microbiology; immunology; parasitology
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.102592

Toxoplasma gondii is 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 in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus 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) parasites in 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 powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma -host interactions beyond the infected cell.