Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host c...

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Autores principales:	Alicja M. Cygan, Terence C. Theisen, Alma G. Mendoza, Nicole D. Marino, Michael W. Panas, John C. Boothroyd
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Toxoplasma immunoprecipitation mass spectrometry parasitology protein export Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fb57c53858b645e2bbf85eb47a76cc35
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spelling	oai:doaj.org-article:fb57c53858b645e2bbf85eb47a76cc352021-11-15T15:27:53ZCoimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells10.1128/mSphere.00858-192379-5042https://doaj.org/article/fb57c53858b645e2bbf85eb47a76cc352020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00858-19https://doaj.org/toc/2379-5042ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.Alicja M. CyganTerence C. TheisenAlma G. MendozaNicole D. MarinoMichael W. PanasJohn C. BoothroydAmerican Society for MicrobiologyarticleToxoplasmaimmunoprecipitationmass spectrometryparasitologyprotein exportMicrobiologyQR1-502ENmSphere, Vol 5, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Toxoplasma immunoprecipitation mass spectrometry parasitology protein export Microbiology QR1-502
spellingShingle	Toxoplasma immunoprecipitation mass spectrometry parasitology protein export Microbiology QR1-502 Alicja M. Cygan Terence C. Theisen Alma G. Mendoza Nicole D. Marino Michael W. Panas John C. Boothroyd Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells
description	ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.
format	article
author	Alicja M. Cygan Terence C. Theisen Alma G. Mendoza Nicole D. Marino Michael W. Panas John C. Boothroyd
author_facet	Alicja M. Cygan Terence C. Theisen Alma G. Mendoza Nicole D. Marino Michael W. Panas John C. Boothroyd
author_sort	Alicja M. Cygan
title	Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells
title_short	Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells
title_full	Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells
title_fullStr	Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells
title_full_unstemmed	Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells
title_sort	coimmunoprecipitation with myr1 identifies three additional proteins within the <named-content content-type="genus-species">toxoplasma gondii</named-content> parasitophorous vacuole required for translocation of dense granule effectors into host cells
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/fb57c53858b645e2bbf85eb47a76cc35
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Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the <named-content content-type="genus-species">Toxoplasma gondii</named-content> Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

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