Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens DOI Creative Commons
Francesca Torelli, Diogo M da Fonseca, Simon Butterworth

et al.

eLife, Journal Year: 2024, Volume and Issue: 13

Published: Dec. 10, 2024

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the transcriptome rely on MYR1 protein, which required for translocation proteins into cell. Mice infected with knock-out (KO) strains survive infection, supporting a paramount function MYR1-dependent and proliferation. However, we have previously shown mutants no growth defect pooled vivo CRISPR-Cas9 screens mice, suggesting presence parasites are wild-type at myr1 locus can rescue phenotype. Here, demonstrate not survival IFN-γ-activated murine macrophages, lacking able expand during onset infection. While ΔMYR1 restricted single-strain infections, show phenotype rescued by co-infection (WT) , independent functional adaptive immunity or key pro-inflammatory cytokines. These data major protect from clearance within cells. Instead, MYR-dependent generate permissive niche paracrine manner, rescues pool CRISPR mice. Our results highlight important limitation otherwise powerful point towards -host interactions beyond

Language: Английский

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei DOI Creative Commons
Thorey K. Jonsdottir, Martina Soledad Paoletta, Takahiro Ishizaki

et al.

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(2)

Published: Jan. 11, 2025

Abstract Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able target about half of the genome in more genetically tractable rodent malaria parasite berghei. To overcome this limitation, we developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses single cloning step generate targeting vectors with 100-bp homology arms physically linked guide RNA (gRNA) that effectively integrate into locus. We show PbHiT coupled gRNA sequencing robustly recapitulates known mutant phenotypes pooled transfections. Furthermore, provide an online resource and tagging designs entire scale-up vector production using ligation approach. This work presents for first time tool studying parasite’s biology at scale.

Language: Английский

Citations

2
A CRISPR view on genetic screens in Toxoplasma gondii DOI Creative Commons
Franziska Hildebrandt, Ana Catarina Matias, Moritz Treeck

et al.

Current Opinion in Microbiology, Journal Year: 2025, Volume and Issue: 83, P. 102577 - 102577

Published: Jan. 8, 2025

Genome editing technologies, such as CRISPR-Cas9, have revolutionised the study of genes in a variety organisms, including unicellular parasites. Today, CRISPR-Cas9 technology is vastly applied high-throughput screens to investigate interactions between Apicomplexan parasite Toxoplasma gondii and its hosts. In vitro vivo T. performed naive restrictive conditions led discovery essential fitness-conferring genes, well factors important for virulence dissemination. Recent studies adapted screening based on phenotypes unrelated survival. These advances were achieved by using conditional systems coupled with imaging, single-cell RNA sequencing phenotypic selection. Here, we review state-of-the-art technologies focus gondii, highlighting strengths, current limitations future avenues development, application other species.

Language: Английский

Citations

1
High throughput CRISPR approaches: New solutions for challenging problems DOI

Gargi Bindal,

Chitra Seetharam Misra,

Mandeep Singh

et al.

TrAC Trends in Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 118245 - 118245

Published: March 1, 2025

Language: Английский

Citations

1
Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma DOI Creative Commons
Tatiana C. Paredes-Santos, Mebratu A. Bitew, Christopher Swale

et al.

PLoS Pathogens, Journal Year: 2023, Volume and Issue: 19(7), P. e1011543 - e1011543

Published: July 27, 2023

Toxoplasma gondii is a parasite that replicates within specialized compartment called the parasitophorous vacuole (PV), which surrounded by PV membrane (PVM). To obtain essential nutrients, must transport molecules across PVM, process mediated secreted proteins GRA17 and GRA23. These form pores in PVM through small can diffuse out of PV. GRA23 are synthetically lethal, suggesting at least one pore type for survival. In ‘nutrient sensitized’ Δ gra17 strain it likely other genes become essential, because they mediate nutrient acquisition from host or involved trafficking to PVM. identify these genes, genome-wide loss-of-function screen was performed wild-type parasites, identified multiple were sick/lethal with . Several correct localization GRAs, including GRA17/GRA23, One top hits, GRA72, predicted on its deletion led formation enlarged “bubble vacuoles” reduced molecule permeability, similar what previously observed parasites. Furthermore, gra72 parasites had vitro growth virulence mice. findings suggest absence GRA17, play role proper (and GRAs) determine host-derived

Language: Английский

Citations

14
A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma DOI Creative Commons
Dylan Valleau, Saima Sidik, Luiz C. Godoy

et al.

The EMBO Journal, Journal Year: 2023, Volume and Issue: 42(23)

Published: Oct. 27, 2023

Abstract Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry are poorly understood, yet essential sustain the apicomplexan parasitic life cycle. Rhoptry appears depend on proteins secreted from another set of micronemes, which vary in function allowing binding facilitation gliding motility. Here we examine microneme protein CLAMP, previously found be necessary for Toxoplasma gondii invasion, and demonstrate its role discharge. CLAMP forms a distinct complex with two other proteins, invasion‐associated SPATR, uncharacterized name CLAMP‐linked invasion (CLIP). deficiency does not impact parasite adhesion or secretion; however, knockdown any member affects Phylogenetic analysis suggests orthologs components, CLIP, ubiquitous across apicomplexans. SPATR act as an accessory factor , but despite incomplete conservation is also during Plasmodium falciparum blood stages. Together, our results reveal new mediates following host‐cell contact.

Language: Английский

Citations

13
Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma DOI Creative Commons
Sara Chelaghma, Huiling Ke, Konstantin Barylyuk

et al.

eLife, Journal Year: 2024, Volume and Issue: 13

Published: Jan. 25, 2024

Apicomplexans are ubiquitous intracellular parasites of animals. These use a programmed sequence secretory events to find, invade, and then re-engineer their host cells enable parasite growth proliferation. The organelles micronemes rhoptries mediate the first steps invasion. Both secrete contents through apical complex which provides an opening in parasite's elaborate inner membrane (IMC) - extensive subpellicular system flattened cisternae proteinaceous meshwork that otherwise limits access cytoplasm plasma for material exchange with cell exterior. After invasion, second secretion programme drives remodelling occurs from dense granules. site(s) granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures embedded IMC have observed, but role or significance these annuli function also Here, we determined integral proteins occur specifically at sites, include SNARE proteins, sites vesicle fusion exocytosis. Specifically, show granules require cargo proteins. When is perturbed annuli, strongly impaired. therefore, represent type IMC-embedded structure specialised protein secretion, reveal there physical separation processes pre- post-invasion host-parasite interactions.

Language: Английский

Citations

4
Integrated network pharmacology, molecular docking and in vivo experiments to elucidate the extenuative mechanisms of ginseng polysaccharide against Toxoplasma gondii-induced testicular toxicity DOI
Chengquan Han, Xin Qi, Xinyu Liu

et al.

International Immunopharmacology, Journal Year: 2025, Volume and Issue: 148, P. 114147 - 114147

Published: Jan. 24, 2025

Language: Английский

Citations

0
Anin vivofitness gene ofToxoplasma, MIC11, is essential for PLP1-mediated egress from host cells DOI Creative Commons
Yuta Tachibana, Miwa Sasai, Hidetaka Kosako

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 24, 2025

Summary After invasion and replication, intracellular pathogens must egress from infected host cells . Toxoplasma gondii facilitates this process by permeabilizing releasing perforin-like protein 1 (PLP1) through induced microneme secretion. However, the precise mechanism of cell permeabilization remains enigmatic. Here, we identified secretory MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen revealed several genes including an essential gene virulence. Deletion resulted severe defects both rupture egress. Scanning mutagenesis functional motifs MIC11, mechanistic analyses demonstrated that directly associates with PLP1, regulating its activity The paralogue MIC22 compensated deletion, suggesting conserved feline-restricted stages T. discovery advances understanding how parasites disrupt to facilitate rapid successful dissemination.

Language: Английский

Citations

0
GRA12 is a common virulence factor across Toxoplasma gondii strains and mouse subspecies DOI Creative Commons
Francesca Torelli, Simon Butterworth, Eloise J. Lockyer

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: April 16, 2025

Language: Английский

Citations

0
CRISPR screens identify genes essential for in vivo virulence among proteins of hyperLOPIT-unassigned subcellular localization in Toxoplasma DOI Creative Commons
Yuta Tachibana, Miwa Sasai, Masahiro Yamamoto

et al.

mBio, Journal Year: 2024, Volume and Issue: 15(9)

Published: July 31, 2024

ABSTRACT The research field to identify and characterize genes essential for in vivo virulence Toxoplasma gondii has been dramatically advanced by a series of clustered regularly interspaced short palindromic repeats (CRISPR) screens. Although subcellular localizations thousands proteins were predicted the spatial proteomic method called hyperLOPIT, those more than 1,000 remained unassigned, their essentiality was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned performed CRISPR As result, identified several that previously unreported. We further characterized candidates, TgGTPase TgRimM, which are localized cytoplasm apicoplast, respectively. Both parasite widely conserved phylum Apicomplexa. Collectively, our current study provides resource estimating with unknown localizations. IMPORTANCE is protozoan causes severe infection immunocompromised patients or newborns. possesses 8,000 genes; however, not fully identified. apicomplexan parasites, including , developed unique organelles do exist other model organisms; thus, determining location important understanding functions. Here, used genetic screens enabled us investigate hundreds during mouse infection. screened many novel confer mice. Among top hits, virulence, Our findings will contribute how apicomplexans adapt host environment cause disease.

Language: Английский

Citations

3