GRA47 and GRA72 areToxoplasma gondiipore-forming proteins that influence small molecule permeability of the parasitophorous vacuole DOI Creative Commons
Mebratu A. Bitew, Pablo S. Gaete, Christopher Swale

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Ноя. 15, 2023

Abstract Toxoplasma gondii , a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on PVM involved in this process, but roles of additional have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with . Deleting produced similar phenotypes Δgra17 parasites, computational predictions suggested it forms pore. To understand how functions we performed immunoprecipitation experiments GRA47 an interactor GRA72. Deletion resulted aberrant ‘bubble vacuole’ morphology reduced small molecule permeability, mirroring phenotype observed knockouts. Structural indicated that form heptameric hexameric pores, respectively, conserved histidine residues lining Mutational analysis highlighted critical role these histidines for protein functionality. Validation through electrophysiology confirmed alterations conductance, corroborating their capabilities. Furthermore, Δ gra47 parasites expressing mutation had vitro proliferation attenuated virulence mice. Our findings show regulating thereby expanding repertoire potential therapeutic targets against infections. IMPORTANCE is parasite poses significant health risks those impaired immunity. It replicates inside host cells shielded by (PVM), which controls waste exchange host. GRA72, previously essential absence channel, implicated forming alternative channel. Here found associates also PVM’s permeability molecules. Removal leads distorted vacuoles impairs transport PVM, resembling effects deletions. models suggest distinct pore structures, pore-lining function. strains lacking GRA47, or mutation, growth mice, highlighting new treatments Toxoplasmosis.

Язык: Английский

CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection DOI
Christopher J. Giuliano, Kenneth Wei,

Faye M. Harling

и другие.

Nature Microbiology, Год журнала: 2024, Номер 9(9), С. 2323 - 2343

Опубликована: Июль 8, 2024

Язык: Английский

Процитировано

18
A CRISPR view on genetic screens in Toxoplasma gondii DOI Creative Commons
Franziska Hildebrandt, Ana Catarina Matias, Moritz Treeck

и другие.

Current Opinion in Microbiology, Год журнала: 2025, Номер 83, С. 102577 - 102577

Опубликована: Янв. 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.

Язык: Английский

Процитировано

1
Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole DOI Creative Commons
Mebratu A. Bitew, Pablo S. Gaete, Christopher Swale

и другие.

mBio, Год журнала: 2024, Номер 15(3)

Опубликована: Фев. 21, 2024

, a medically important intracellular parasite, uses GRA proteins secreted from dense granule organelles to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming on PVM involved in this process, but roles of additional have remained largely uncharacterized. We recently identified

Язык: Английский

Процитировано

6
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

и другие.

mBio, Год журнала: 2024, Номер 15(9)

Опубликована: Июль 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.

Язык: Английский

Процитировано

3
Metabolic adaptability and nutrient scavenging in Toxoplasma gondii : insights from ingestion pathway-deficient mutants DOI Creative Commons

Patrick A. Rimple,

Einar B. Ólafsson, Benedikt M. Markus

и другие.

mSphere, Год журнала: 2025, Номер unknown

Опубликована: Апрель 2, 2025

The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living PV, endocytoses proteins from cytosol of infected host cells via so-called ingestion pathway. pathway is initiated by dense granule protein GRA14, which binds endosomal sorting complex required for transport (ESCRT) machinery to bud vesicles into lumen PV. protein-containing are internalized and trafficked plant vacuole-like (PLVAC), where cathepsin protease L (CPL) degrades cargo, chloroquine resistance transporter (CRT) exports resulting peptides amino acids cytosol. However, although was proposed be conduit nutrients, there limited evidence this hypothesis. We reasoned if uses acquire then parasites lacking CPL, or CRT should rely more on biosynthetic pathways alternative scavenging pathways. To explore this, we conducted genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen in wild-type (WT) Δgra14, Δcpl, Δcrt mutants identify genes become fitness conferring ingestion-deficient parasites. Our revealed significant overlap potentially compared WT. Pathway analysis indicated Δcpl relied pyrimidine biosynthesis, fatty acid tricarboxylic (TCA) cycle, lysine degradation. Bulk metabolomic reduced levels glycolytic intermediates WT, highlighting pathway's potential role resource scavenging. Interestingly, an exacerbated growth defect when cultured acid-depleted media, suggesting disrupting proteolysis export proteolytic products PLVAC affects survival during nutrient scarcity. pathogen infects virtually any nucleated cell most warm-blooded animals. Infections asymptomatic cases, but people with weakened immunity can experience severe disease. For replicate host, it must efficiently essential especially as unable make several key metabolites. Understanding mechanisms scavenges nutrients crucial identifying therapeutic targets. study suggests contributes sustaining metabolites replication under acid-limiting conditions. This advances our understanding metabolic adaptability Toxoplasma.

Язык: Английский

Процитировано

0
Deciphering protein prenylation in endocytic trafficking in Toxoplasma gondii DOI Creative Commons
Vern B. Carruthers, Zhicheng Dou

mBio, Год журнала: 2024, Номер 15(4)

Опубликована: Фев. 26, 2024

Toxoplasma gondii is a widespread intracellular protozoan pathogen infecting virtually all warm-blooded animals. This parasite acquires host-derived resources to support its replication inside membrane-bound parasitophorous vacuole within infected host cells. Previous research has discovered that actively endocytoses proteins and transports them lysosome-equivalent structure for digestion. However, few molecular determinants required trafficking of material the were known. A recent study (Q.-Q. Wang, M. Sun, T. Tang, D.-H. Lai, et al., mBio 14:e01309-23, 2023, https://doi.org/10.1128/mbio.01309-23) identified critical role membrane anchoring via prenylation in endocytosed by Toxoplasma, including an essential ortholog Rab1B. The authors also found TgRab1 crucial protein rhoptry secretory organelles, indicating dual endocytic exocytic trafficking. sets stage further dissecting endomembrane along with potentially exploiting as target therapeutic development.

Язык: Английский

Процитировано

2
Dissecting EXP2 sequence requirements for protein export in malaria parasites DOI Creative Commons

Ethan L. Pitman,

Natalie A. Counihan, Joyanta K. Modak

и другие.

Frontiers in Cellular and Infection Microbiology, Год журнала: 2024, Номер 13

Опубликована: Янв. 12, 2024

Apicomplexan parasites that reside within a parasitophorous vacuole harbor conserved pore-forming protein enables small-molecule transfer across the membrane (PVM). In Plasmodium cause malaria, this nutrient pore is formed by EXP2 which can complement function of GRA17, an orthologous in Toxoplasma gondii. EXP2, however, has additional parasites, serving also as component export machinery PTEX. To examine how play role, transgenes encoded truncations hybrid GRA17-EXP2, or under transcriptional control different promoters were expressed knockdown to determine could function. This revealed unique protein, and its role P. falciparum cannot be complemented T. gondii GRA17. was despite addition assembly strand part linker helix are regions necessary for interaction with other core PTEX components. indicates body region plays critical and/or absence GRA proteins leads reduced efficiency insertion into PVM complementation potential. Altering timing abundance expression did not affect but affected parasite viability, indicating profile when compared components it serve exchange.

Язык: Английский

Процитировано

1
Whole‐genome CRISPR screens to understand Apicomplexan–host interactions DOI Creative Commons
Eva Hesping, Justin A. Boddey

Molecular Microbiology, Год журнала: 2024, Номер 121(4), С. 717 - 726

Опубликована: Янв. 15, 2024

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

Язык: Английский

Процитировано

1
In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma DOI Creative Commons
Yuta Tachibana, Miwa Sasai, Masahiro Yamamoto

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Янв. 28, 2024

ABSTRACT The research field to identify and characterize virulence genes in Toxoplasma gondii has been dramatically advanced by a series of vivo CRISPR screens. Although subcellular localizations thousands proteins were predicted the spatial proteomic method called hyperLOPIT, those more than 1000 remained unassigned their essentiality was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned performed As result, identified several fitness-conferring that previously unreported. We further characterized candidates, TgGTPase TgRimM, which are localized cytoplasm apicoplast, respectively. Both essential for 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 8000 genes; however, determine 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 fitness mice. Among top hits, genes, Our findings will contribute how apicomplexans adapt host environment cause disease.

Язык: Английский

Процитировано

1
GRA47 is important for the morphology and permeability of the parasitophorous vacuole in Toxoplasma gondii DOI
Xiao-Nan Zheng, Tingting Li, Hany M. Elsheikha

и другие.

International Journal for Parasitology, Год журнала: 2024, Номер 54(11), С. 583 - 596

Опубликована: Июнь 25, 2024

Язык: Английский

Процитировано

1