1. CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells
- Author
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Krishnamurthy, Shruthi, Maru, Parag, Wang, Yifan, Bitew, Mebratu A, Mukhopadhyay, Debanjan, Yamaryo-Botté, Yoshiki, Paredes-Santos, Tatiana C, Sangaré, Lamba O, Swale, Christopher, Botté, Cyrille Y, and Saeij, Jeroen PJ
- Subjects
Medical Microbiology ,Biomedical and Clinical Sciences ,Emerging Infectious Diseases ,Infectious Diseases ,Foodborne Illness ,Genetics ,Vaccine Related ,Biodefense ,Human Genome ,Prevention ,2.1 Biological and endogenous factors ,2.2 Factors relating to the physical environment ,Aetiology ,Infection ,Inflammatory and immune system ,Humans ,Animals ,Mice ,Toxoplasma ,Parasites ,Interferon-gamma ,Clustered Regularly Interspaced Short Palindromic Repeats ,Virulence ,Protozoan Proteins ,CRISPR screen ,Toxoplasma gondii ,effector functions ,host-pathogen interactions ,interferons ,Microbiology ,Biochemistry and cell biology ,Medical microbiology - Abstract
Toxoplasma virulence depends on its ability to evade or survive the toxoplasmacidal mechanisms induced by interferon gamma (IFNγ). While many Toxoplasma genes involved in the evasion of the murine IFNγ response have been identified, genes required to survive the human IFNγ response are largely unknown. In this study, we used a genome-wide loss-of-function screen to identify Toxoplasma genes important for parasite fitness in IFNγ-stimulated primary human fibroblasts. We generated gene knockouts for the top six hits from the screen and confirmed their importance for parasite growth in IFNγ-stimulated human fibroblasts. Of these six genes, three have homology to GRA32, localize to dense granules, and coimmunoprecipitate with each other and GRA32, suggesting they might form a complex. Deletion of individual members of this complex leads to early parasite egress in IFNγ-stimulated cells. Thus, prevention of early egress is an important Toxoplasma fitness determinant in IFNγ-stimulated human cells. IMPORTANCE Toxoplasma infection causes serious complications in immunocompromised individuals and in the developing fetus. During infection, certain immune cells release a protein called interferon gamma that activates cells to destroy the parasite or inhibit its growth. While most Toxoplasma parasites are cleared by this immune response, some can survive by blocking or evading the IFNγ-induced restrictive environment. Many Toxoplasma genes that determine parasite survival in IFNγ-activated murine cells are known but parasite genes conferring fitness in IFNγ-activated human cells are largely unknown. Using a Toxoplasma adapted genome-wide loss-of-function screen, we identified many Toxoplasma genes that determine parasite fitness in IFNγ-activated human cells. The gene products of four top hits play a role in preventing early parasite egress in IFNγ-stimulated human cells. Understanding how IFNγ-stimulated human cells inhibit Toxoplasma growth and how Toxoplasma counteracts this, could lead to the development of novel therapeutics.
- Published
- 2023