A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes

Abstract

SUMMARY Apicomplexan parasites are leading causes of human and livestock diseases—like malaria and toxoplasmosis—yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the human parasite Toxoplasma gondii during infection of fibroblasts. Our analysis defines ~200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes, and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

Genetic impairment of parasite myosin motors uncovers the contribution of host cell membrane dynamics to Toxoplasma invasion forces

“…Future structural and functional studies are necessary to understand how the RON complex insertion dictates the fast remodeling of the PM and how its tension/stretching properties are changed and appropriate curvatures promoted to allow (1) PV folding and (2) ruffles/tube assembly and dynamics. An interesting candidate is the CLAMP, appropriately localized to the cell zoite TJ throughout invasion, as published in the course of the reviewing process by the team of Lourido [9]. Finally, given the harmfulness of the host cell PM remodeling on the MyoA -deficient zoite, we propose to consider host cell invasion as the competitive contribution of both a Toxoplasma motor function and a complex host cell membrane dynamics.…”

“…Early studies on major members of this phylum, specifically Toxoplasma gondii and Plasmodium spp., have highlighted the lack of host cell contribution when the parasite invasive stages, also called zoites, actively invade their respective host cells in a process completed within a few seconds [5–8]. Invasion starts with the insertion in the host cell plasma membrane (PM), by the zoite, of a multi-subunit complex (identified as the apical major antigen 1 (AMA1)-rhoptry neck (RON) complex and possibly enlarged with the recently discovered claudin-like apicomplexa microneme protein (CLAMP) [9]. This macromolecular complex connects the two cells by forming a circular tight junction (TJ) [10–13] that will act as a door of entry.…”

Gliding motility powers invasion and egress in Apicomplexa

“…The magnified view (inset) highlights the interaction between the glideosome and the moving junction, and the interactions between the moving junction and host proteins (ALG2-interacting protein X (ALIX), CBL-interacting protein of 85 kDa (CIN85), CD2-associated protein (CD2AP) and tumour susceptibility gene 101 protein (TSG101)). Claudin-like apicomplexan microneme protein (CLAMP) also follows the moving junction during invasion, but its role is unknown 169 . After invasion, the parasite is enclosed in the parasitophorous vacuole membrane (PVM).…”

EuPathDB: The Eukaryotic Pathogen Genomics Database Resource

“…A genome-wide loss of function screen using CRISPR technology is available in ToxoDB and provides a measure of a gene's contribution to parasite fitness (24 Quantitative proteomics. This new data type provides evidence for differential protein expression from experimental methods such as SILAC (25,26).…”