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1.
Cell Host Microbe ; 31(10): 1748-1762.e8, 2023 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-37827122

RESUMO

Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonization. We combine pooled CRISPR knockout screening with dual host-microbe single-cell RNA sequencing, a method we term dual perturb-seq, to identify the molecular mediators of these transcriptional interactions. Applying dual perturb-seq to the intracellular pathogen Toxoplasma gondii, we are able to identify previously uncharacterized effector proteins and directly infer their function from the transcriptomic data. We show that TgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify an effector, TgSOS1, that is necessary for sustained host STAT6 signaling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a tool that can be broadly adapted to interrogate host-microbe transcriptional interactions and reveal mechanisms of infection and immune evasion.


Assuntos
Toxoplasma , Toxoplasma/genética , Perfilação da Expressão Gênica , Transcriptoma , Evasão da Resposta Imune , Transdução de Sinais , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
2.
PLoS Biol ; 21(7): e3002202, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37459303

RESUMO

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.


Assuntos
Parasitos , Toxoplasma , Humanos , Animais , Camundongos , Toxoplasma/metabolismo , Parasitos/metabolismo , Interferon gama , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Virulência , Vacúolos/metabolismo
3.
Nat Commun ; 14(1): 2167, 2023 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-37061511

RESUMO

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.


Assuntos
Parasitos , Toxoplasma , Animais , Parasitos/metabolismo , Toxoplasma/metabolismo , Endocitose , Proteínas de Protozoários/metabolismo
4.
PLoS Pathog ; 18(12): e1011021, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36476844

RESUMO

Toxoplasma gondii is an intracellular parasite that can infect many host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection. The vast majority of these secreted proteins have no predicted functional domains and remain uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, is essential for virulence and has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. Finally, we show that ROP1 co-immunoprecipitates with the host cell protein C1QBP, an emerging regulator of innate immune signaling. In summary, we identify putative in vivo virulence factors in the T. gondii Prugniaud strain and show that ROP1 is an important and previously overlooked effector protein that counteracts both murine and human innate immunity.


Assuntos
Imunidade Inata , Proteínas de Protozoários , Toxoplasma , Animais , Humanos , Camundongos , Proteínas de Transporte , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Virulência
5.
Cell Host Microbe ; 28(5): 752-766.e9, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33053376

RESUMO

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host's cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.


Assuntos
Proteoma , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Apicomplexa , Evolução Biológica , Epitopos , Interações Hospedeiro-Patógeno , Humanos , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Toxoplasma/genética
6.
Nat Commun ; 10(1): 3963, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481656

RESUMO

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1.


Assuntos
Sistemas CRISPR-Cas , Toxoplasma/genética , Fatores de Virulência/genética , Animais , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Inativação de Genes/métodos , Biblioteca Gênica , Genoma de Protozoário , Humanos , Camundongos Endogâmicos C57BL , RNA Guia de Cinetoplastídeos , Toxoplasma/patogenicidade , Toxoplasmose/genética , Toxoplasmose/parasitologia , Toxoplasmose/patologia
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