CRISPR Screens Identify
            <i>Toxoplasma</i>
            Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells

Krishnamurthy, Shruthi; Maru, Parag; Wang, Yifan; Bitew, Mebratu A; Mukhopadhyay, Debanjan; Yamaryo‐Botté, Yoshiki; Paredes-Santos, Tatiana C.; Sangaré, Lamba Omar; Swale, Christopher; Botté, Cyrille Y.

doi:10.1128/mbio.00060-23

Cited by 26 publications

(22 citation statements)

References 121 publications

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“…Despite much effort from the research community, many apicomplexan proteins still need to be annotated owing to their uniqueness and absence in other model organisms. The CRISPR screens enabled the researchers to investigate hundreds to thousands of genes in Toxoplasma at once in vitro culture or in vivo infection to identify essential genes for parasite growth, metabolism, drug resistance, differentiation, host-pathogen interaction, and virulence (10)(11)(12)(13)(14)(15)(25)(26)(27)(28)(29)(30)(31)(32)(33). Most in vivo CRISPR screens in Toxoplasma have been focused on identifying secreted virulence effectors such as rhoptry bulb proteins (ROPs) and dense granule proteins (GRAs) (11)(12)(13).…”

Section: Discussionmentioning

confidence: 99%

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

Tachibana,

Sasai,

Yamamoto

2024

Preprint

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The research field to identify and characterize virulence genes in Toxoplasma gondii has been dramatically advanced by a series of in vivo CRISPR screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1000 proteins remained unassigned and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several in vivo fitness-conferring genes that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations.

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Section: Discussionmentioning

confidence: 99%

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

Tachibana,

Sasai,

Yamamoto

2024

Preprint

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“…However, the known restriction mechanisms in humans display significant variability among different cell types, lacking a universally applicable one. This leads to a notable difference between genes critical for protecting the parasite against the IFNγ response in human and murine cells and mice (13, 36, 37, 40, 41). The phenomenon of premature egress and host cell death, observed here in various human cell types, whether primary or immortalized, appears to be unique to nonimmune human cells.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, RARRES3 is unlikely to be responsible for the observed cell death phenotype in this study. The GRA57/GRA70/GRA71 complex plays a pivotal role in blocking early egress in IFNγ primed HFFs (36, 37), and intriguingly, the components of this complex ranked among the top 200 hits in the current CRISPR screens. However, the most significant impact on parasite survival in these screens was attributed to the MYR translocon components.…”

Section: Discussionmentioning

confidence: 99%

A Combination of Four Nuclear Targeted Effectors ProtectsToxoplasmaAgainst Interferon Gamma Driven Human Host Cell Death During Acute Infection

Henry,

Sibley,

Rosenberg

2023

Preprint

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In both mice and humans, Type II interferon-gamma (IFNγ) is crucial for regulation ofToxoplasma gondii(T. gondii) infection, during acute or chronic phases. To thwart this defense,T. gondiisecretes protein effectors hindering the host’s immune response. For example,T. gondiirelies on the MYR translocon complex to deploy soluble dense granule effectors (GRAs) into the host cell cytosol or nucleus. Recent genome-wide loss-of-function screens in IFNγ-primed primary human fibroblasts identified MYR translocon components as crucial for parasite resistance against IFNγ driven vacuole clearance. However, these screens did not pinpoint specific MYR-dependent GRA proteins responsible for IFNγ signaling blockade, suggesting potential functional redundancy.Our study reveals thatT. gondiidepends on the MYR translocon complex to prevent host cell death and parasite premature egress in human cells stimulated with IFNγ post-infection, a unique phenotype observed in various human cell lines but not in murine cells. Intriguingly, inhibiting parasite egress did not prevent host cell death, indicating this mechanism is distinct from those described previously. Genome-wide loss-of-function screens uncovered TgIST, GRA16, GRA24, and GRA28 as effectors necessary for a complete block of IFNγ response. GRA24 and GRA28 directly influenced IFNγ driven transcription, GRA24’s action depended on its interaction with p38 MAPK, while GRA28 disrupted histone acetyltransferase activity of CBP/p300. Given the intricate nature of the immune response toT. gondii, it appears that the parasite has evolved equally elaborate mechanisms to subvert IFNγ signaling, extending beyond direct interference with the JAK/STAT1 pathway, to encompass other signaling pathways as well.

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“…Other CRISPR/Cas9 knockout screens in Toxoplasma identified genes ( gra45 , facilitates the secretion of Toxoplasma proteins, extending beyond the PMV (Wang et al., 2020); six genes of which five encode GRAs (Krishnamurthy et al., 2023); gra57 , gra70 and gra71 which encode proteins that form a complex that boost parasites' capacity to persist in IFNγ‐activated fibroblasts (Lockyer et al., 2023)) that regulate parasite growth in naïve and IFNγ‐activated macrophages or IFNγ‐activated fibroblasts, respectively. A CRISPR/Cas9 knockout screen of ~200 Toxoplasma genes was also used to study the transition from tachyzoites to latent bradyzoites, a critical step in chronic infections.…”

Section: Crispr/cas Knock‐out Screens: a Powerful Approach For Forwar...mentioning

confidence: 99%

Whole‐genome CRISPR screens to understand Apicomplexan–host interactions

Hesping,

Boddey

2024

Molecular Microbiology

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Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole‐genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite–host interactions.

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CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells

Abstract: 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.

Cited by 26 publications

References 121 publications

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

A Combination of Four Nuclear Targeted Effectors ProtectsToxoplasmaAgainst Interferon Gamma Driven Human Host Cell Death During Acute Infection

Whole‐genome CRISPR screens to understand Apicomplexan–host interactions

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