Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.
Summary
The virulence of eukaryotic parasites like
Toxoplasma gondii
depends on their capacity to escape from the host immune response and disseminate throughout the host organism. However,
Toxoplasma
gene products essential for its
in vivo
pathogenesis remain uncharacterized. Here, we present the complete workflow of a CRISPR-Cas9
in vivo
loss-of-function screen to identify
Toxoplasma
fitness-conferring genes. This protocol can be used to uncover gene products that play a role in
Toxoplasma
immune evasion, nutrient acquisition, dissemination, and tissue colonization.
For complete details on the use and execution of this protocol, please refer to
Sangaré et al. (2019)
.
Yifan Wang works in the field of molecular parasitology with a focus on host-pathogen interactions. In this mSphere of Influence article, he reflects on how papers entitled “A genome-wide CRISPR screen in
Toxoplasma
identifies essential apicomplexan genes” by S. M. Sidik, D. Huet, S. M. Ganesan, M.-H. Huynh, et al. (Cell 166:1423.e12–1435.e12, 2016,
https://doi.org/10.1016/j.cell.2016.08.019
) and “Mapping host-microbe transcriptional interactions by dual Perturb-seq” by S. Butterworth, K. Kordova, S. Chandrasekaran, K. K. Thomas, et al. (bioRxiv,
https://doi.org/10.1101/2023.04.21.537779
) made an impact on his research and changed the way he thinks how functional genomics and high-throughput screens provide novel insights into pathogen pathogenesis.
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