In Vivo CRISPR Screen Identifies TgWIP as a Toxoplasma Modulator of Dendritic Cell Migration

Abstract

Toxoplasma can reach distant organs, especially the brain, leading to a lifelong chronic phase. However, genes involved in related in vivo processes are currently unknown. Here, we use focused CRISPR libraries to identify Toxoplasma genes that affect in vivo fitness. We focus on TgWIP, whose deletion affects Toxoplasma dissemination to distant organs. We show that TgWIP is secreted into the host cell upon invasion and interacts with the host WAVE regulatory complex and SHP2 phosphatase, both of which regulate actin dynamics. TgWIP affects the morphology of dendritic cells and mediates the dissolution of podosomes, which dendritic cells use to adhere to extracellular matrix. TgWIP enhances the motility and transmigration of parasitized dendritic cells, likely explaining its effect on in vivo fitness. Our results provide a framework for systemic identification of Toxoplasma genes with in vivo effects at the site of infection or on dissemination to distant organs, including the brain.

A genome-wide loss-of-function screen identifiesToxoplasma gondiigenes that determine fitness in interferon gamma-activated murine macrophages

“…For example, genes involved in lipid biosynthesis or transport were enriched in IFNγ-activated BMDMs: three GNS1/SUR4 family members involved in long-chain fatty acid elongation and sphingolipid/ceramide biosynthesis (TGGT1_253880, TGGT1_205350, TGGT1_242380), TGGT1_229140 (enoyl-CoA hydratase like-betaoxidation), GRA38 (an orthologue of GRA39 of which the knockout has been shown to accumulate lipid (54)), ATP-binding cassette (ABC) transporters ABCG87 and ABCG96, which have been shown to mediate cholesterol transport (55), and TGGT1_266640 (Acetyl-coenzyme A synthetase 2). A GNS1/SUR4 family member, TGGT1_205350, was recently identified as a gene important during in vivo infection of the peritoneum (49). IFNγ stimulation significantly downregulated the cholesterol biosynthesis pathway in BMDMs as has been shown by others (56).…”

“…The complete set of Toxoplasma genes involved in these processes is currently unknown. We recently published an in vivo loss-of-function screen using a focused sgRNA library enriched in GRA-targeting sgRNAs to determine the in vivo fitness contribution of Toxoplasma genes in the mouse peritoneum and in distant organs (49). It is likely that a significant fraction of the Toxoplasma genes that determined fitness at the site of infection were important for resisting the toxoplasmacidal mechanisms of IFNγ-activated cells or determined parasite growth and survival inside macrophages, the cell type preferentially infected by Toxoplasma.…”

“…(Figure 2A to 2D). We previously determined the contribution to the in vivo fitness of 217 Toxoplasma genes (49). Of the 24 genes that determined in vivo fitness, two genes (TGGT1_205350 and GRA22) were present in the top hits for fitness in IFNγ-activated BMDMs ( Table 1).…”

“…Murine BMDMs were isolated from 5 to 8 weeks old female C57BL/6J mice or A/J mice (The Jackson Laboratory) as previously described (90), and obtained by culturing murine bone marrow cells in DMEM, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1x non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin, 10 mg/mL gentamicin and 20% L929 conditioned medium for 5 to 7 days. Rat BMDMs were isolated from 6 to 8 weeks old female Brown Norway rats (Charles River Laboratories) as previously described (49), and obtained by culturing rat bone marrow cells in DMEM, 20% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1x non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin, 10 mg/mL gentamicin and 30% L929 conditioned medium for 5 to 7 days.…”

Convergent Met and voltage-gated Ca2+channel signaling on Ras-Erk MAPK drives migratory activation of dendritic cells parasitized byToxoplasma gondii

“…Early on, tachyzoites encounter DCs, which play a determinant role in mounting a robust protective immune response (Liu et al, 2006). Paradoxically, T. gondii exploits the inherent migratory ability of DCs for dissemination via a ‘Trojan horse’ mechanism (Courret et al, 2006; Lambert et al, 2006; Lambert et al, 2009; Sangare et al, 2019). Within minutes of active invasion by T. gondii , DCs adopt a hypermigratory phenotype that mediates rapid systemic dissemination in mice (Kanatani et al, 2017; Weidner and Barragan, 2014).…”