New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Gregory M. Wier, Erica M. McGreevy, Mark J. Brown, Jon P. Boyle
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/939524109c084972ba15df14d1faafa7
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:939524109c084972ba15df14d1faafa7
record_format	dspace
spelling	oai:doaj.org-article:939524109c084972ba15df14d1faafa72021-11-15T15:41:32ZNew Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell10.1128/mBio.01628-142150-7511https://doaj.org/article/939524109c084972ba15df14d1faafa72015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01628-14https://doaj.org/toc/2150-7511ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS. IMPORTANCE Studying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins during in vitro growth. It should also be applicable during in vivo infections and in other apicomplexan parasites, including Plasmodium spp.Gregory M. WierErica M. McGreevyMark J. BrownJon P. BoyleAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Gregory M. Wier Erica M. McGreevy Mark J. Brown Jon P. Boyle New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell
description	ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS. IMPORTANCE Studying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins during in vitro growth. It should also be applicable during in vivo infections and in other apicomplexan parasites, including Plasmodium spp.
format	article
author	Gregory M. Wier Erica M. McGreevy Mark J. Brown Jon P. Boyle
author_facet	Gregory M. Wier Erica M. McGreevy Mark J. Brown Jon P. Boyle
author_sort	Gregory M. Wier
title	New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell
title_short	New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell
title_full	New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell
title_fullStr	New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell
title_full_unstemmed	New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell
title_sort	new method for the orthogonal labeling and purification of <named-content content-type="genus-species">toxoplasma gondii</named-content> proteins while inside the host cell
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/939524109c084972ba15df14d1faafa7
work_keys_str_mv	AT gregorymwier newmethodfortheorthogonallabelingandpurificationofnamedcontentcontenttypegenusspeciestoxoplasmagondiinamedcontentproteinswhileinsidethehostcell AT ericammcgreevy newmethodfortheorthogonallabelingandpurificationofnamedcontentcontenttypegenusspeciestoxoplasmagondiinamedcontentproteinswhileinsidethehostcell AT markjbrown newmethodfortheorthogonallabelingandpurificationofnamedcontentcontenttypegenusspeciestoxoplasmagondiinamedcontentproteinswhileinsidethehostcell AT jonpboyle newmethodfortheorthogonallabelingandpurificationofnamedcontentcontenttypegenusspeciestoxoplasmagondiinamedcontentproteinswhileinsidethehostcell
_version_	1718427655603224576

New Method for the Orthogonal Labeling and Purification of <named-content content-type="genus-species">Toxoplasma gondii</named-content> Proteins While Inside the Host Cell

Ejemplares similares