Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts

ABSTRACT The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is c...

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Autores principales:	Vincent Tu, Joshua Mayoral, Tatsuki Sugi, Tadakimi Tomita, Bing Han, Yan Fen Ma, Louis M. Weiss
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	bradyzoites CST1 cyst wall latency proteomics Toxoplasma gondii Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0fef660c2a504f80bd83788c140ad2ec
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spelling	oai:doaj.org-article:0fef660c2a504f80bd83788c140ad2ec2021-11-15T15:55:25ZEnrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts10.1128/mBio.00469-192150-7511https://doaj.org/article/0fef660c2a504f80bd83788c140ad2ec2019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00469-19https://doaj.org/toc/2150-7511ABSTRACT The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface. IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.Vincent TuJoshua MayoralTatsuki SugiTadakimi TomitaBing HanYan Fen MaLouis M. WeissAmerican Society for MicrobiologyarticlebradyzoitesCST1cyst walllatencyproteomicsToxoplasma gondiiMicrobiologyQR1-502ENmBio, Vol 10, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	bradyzoites CST1 cyst wall latency proteomics Toxoplasma gondii Microbiology QR1-502
spellingShingle	bradyzoites CST1 cyst wall latency proteomics Toxoplasma gondii Microbiology QR1-502 Vincent Tu Joshua Mayoral Tatsuki Sugi Tadakimi Tomita Bing Han Yan Fen Ma Louis M. Weiss Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts
description	ABSTRACT The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface. IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.
format	article
author	Vincent Tu Joshua Mayoral Tatsuki Sugi Tadakimi Tomita Bing Han Yan Fen Ma Louis M. Weiss
author_facet	Vincent Tu Joshua Mayoral Tatsuki Sugi Tadakimi Tomita Bing Han Yan Fen Ma Louis M. Weiss
author_sort	Vincent Tu
title	Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts
title_short	Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts
title_full	Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts
title_fullStr	Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts
title_full_unstemmed	Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts
title_sort	enrichment and proteomic characterization of the cyst wall from <italic toggle="yes">in vitro</italic> <named-content content-type="genus-species">toxoplasma gondii</named-content> cysts
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/0fef660c2a504f80bd83788c140ad2ec
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Enrichment and Proteomic Characterization of the Cyst Wall from <italic toggle="yes">In Vitro</italic> <named-content content-type="genus-species">Toxoplasma gondii</named-content> Cysts

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